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Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

What is the main cause of HIV-1 infection in children?

Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide.

Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

What plays the crucial role in the Mother to Child Transmission of HIV-1 and what increases the risk

DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission.

Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

How many children were infected by HIV-1 in 2008-2009, worldwide?

more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.

Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

What is the role of C-C Motif Chemokine Ligand 3 Like 1 (CCL3L1) in mother to child transmission of HIV-1?

High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants

Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

What is DC-GENR and where is it expressed?

Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells

Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

How does the presence of DC-SIGNR affect the MTCT of HIV-1?

the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation.

Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

Why do low levels of DC-SIGNR enhance Mother to Child Transmission of HIV-1?

in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1

Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

What is the percentage of Mother to Child Transmission of HIV-1, when there is no intervention?

Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45%

Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

Does C-C chemokine receptor type 5 (CCR5) affect the transmission of HIV-1?

Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans

Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

How does Mannanose Binding Lectin (MBL) affect elimination of HIV-1 pathogen?

Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis,

Functional Genetic Variants in DC-SIGNR Are Associated with Mother-to-Child Transmission of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752805/ Boily-Larouche, Geneviève; Iscache, Anne-Laure; Zijenah, Lynn S.; Humphrey, Jean H.; Mouland, Andrew J.; Ward, Brian J.; Roger, Michel 2009-10-07 DOI:10.1371/journal.pone.0007211 License:cc-by Abstract: BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45% [1] . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy (in utero, IU), delivery (intrapartum, IP) or breastfeeding (postpartum, PP). High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 [1] . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells [2] . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms [3] . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types [2] but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses [4] that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial (Harare, Zimbabwe) and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction (PCR) results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues [5] . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [6] . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously [7] . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE [8] , version 2.1.1, using single nucleotide polymorphism (SNP) with a minimum allele frequency (MAF) of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs ( Figure 1 ). Fifteen haplotype-tagged SNPs (htSNPs) were identified by the HaploBlockFinder software [9] with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously [7] . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels [10] . DNA sequences in the promoter region were analysed with the TESS interface (http//:www.cbil.upenn.edu/tess) for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream (Invitrogen Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). We cultured HeLa cells in 6 wells plates (2610 5 cells) and transfected them the following day using lipofectamine (Invitrogen) according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer (Promega) at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean (S.E.M). First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc (Montreal, Canada). Tissues from 3 H1 (associated with MTCT of HIV-1) and 3 H15 (wild-type) homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed (RT) and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues [11] . 1 mg of total RNA was reverse transcribed with Expand RT (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase (Invitrogen). Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit (Qiagen Canada inc, Mississauga, ON, Canada) and cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen). For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software (DNA Stars, Madison, WI, USA). Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer (Roche Applied Science). 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix (Invitrogen) on a Rotor Gene Realtime Rotary Analyser (Corbett Life Science, Sydney, Australia). Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair (Table S1 ). Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase (Fermantas International inc, Burlington, ON, Canada) to avoid amplification of contaminant DNA. Standard curves (50-500 000 copies per reaction) were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH (Invitrogen) plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number (crossing threshold) on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows (GraphPad Software inc, San Diego, CA, USA). Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios (OR) for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios (OR) of 3.64 (95% CI = 1.82-7.31, P = 0.0002) and 4.45 (95% CI = 1.50-13.2, P = 0.0045) for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs (htSNPs) corresponding to the 15 major DC-SIGNR haplotypes ( Figure 1 ) described among Zimbabweans [7] were genotyped in our study samples (Tables S2 and S3 ). H1 (31%) and H3 (11%) were the most frequent haplotypes observed (Figure 1 ). Being homozygous for the H1 haplotype was associated with increased risk of both IU (OR: 4.42, P = 0.022) and PP (OR: 7.31, P = 0.016) HIV-1 transmission ( Table 2) . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes (H1-H1, H1-H3 or H3-H3) had increased risk of IU (OR: 3.42, P = 0.007) and IP (OR: 5.71, P = 0.025) but not PP (P = 0.098) HIV-1 infection compared to infant noncarriers ( Table 2 ). The latter associations remained significant after adjustment was made for the maternal viral load for both IU (OR: 3.57, 95% CI = 1.30-9.82, P = 0.013) and IP (OR: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations (p-198A, int2-391C, int2-180A, ex4RPT, int5+7C) ( Figure 1 ). Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 (Table S2 ). In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU (OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively) and IP (OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively) HIV-1 infection compared to heterozygote infants or noncarriers (Table 3) . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU (OR: 3.83, 95% CI = 1.42-10.4, P = 0.008) and IP (O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025) HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires [3] . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection [11] . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type (WT) (p-198CC, int2-180GG) samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified ( Figure S1 ), of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region (exon 4) of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms [3] . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals ( Figure S1 ). The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events (Figure 2A ). We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 (E5) sequence present in all DC-SIGNR isoforms (total transcripts). We then amplified exon 3 (E3) which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR (18%) compared to that in WT individuals (36%) (P = 0.004) ( Figure 2B ) suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A (Figure 1 ). In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission (Table 3) . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Figure 3A ). The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct (p-198C/A ratio = 2, P = 0.006) ( Figure 3B ) suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants (p-577C and p-323A) observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay ( Figure S2 ). To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 (DC-SIGNR copies6S.E.M per 10 5 GAPDH copies) in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals (27816638, P = 0.011) ( Figure 3C ). As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease (H1 = 117636.2 vs WT = 9906220.6, P = 0.003) compared to a 3-fold decrease in total soluble isoforms (H1 = 5686181.9 vs WT = 19256495.3, P = 0.03) ( Figure 3C ). Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 (int2)-180 variants and intrauterine (IU), intrapartum (IP) and postpartum (PP) mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations (Figure 1 ). Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes (Figure 1) , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy (reviewed in [12] ). Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein [3] . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro [2, 10] . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo [13, 14] . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor [15] . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner [4] . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response [16, 17] . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier (BBB) endothelial cells [18, 19] . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB [20, 21] . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry (presumably ophthalmic, skin, or gastrointestinal), whereas placental insult during delivery (physical or inflammatory) may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation [22, 23] . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery [22] . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 (reviewed in [24] ). Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans [25, 26] . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 [27] , but this variant is virtually absent among African populations [28] . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants [29, 30] . Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 [31, 32] . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: doi:10.1371/journal.pone.0007211.s003 (0.05 MB DOC) Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: doi:10.1371/journal.pone.0007211.s004 (0.11 MB DOC) Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type (WT) construct (not significant). 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments.

How can CCR5's effect in HIV-1 transmission be reduced?

The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

What is IFITM?

interferon-induced transmembrane

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

How many cysteine residues are contained in the first transmembrane domain of IFITM3?

three

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

What inhibits S-palmitoylation?

2-bromopalmitic acid (2BP)

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

What interaction is inhibited by the presence of 2-bromopalmitic acid (2BP)?

IFITM5 with FKBP11

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

What is a function associated with IFITM5?

bone formation factor.

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

What regulates the antiviral activity of IFITM3?

S-palmitoylation on the protein

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

What is another name for IFITM5?

bonerestricted IFITM-like (BRIL) protein

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

Why is the expression of IFITM5 not promoted by interferons?

the region upstream of the ifitm5 gene lacks the interferon regulatory elements

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

What is the amino acid similarity between IFITM5 and the other IFITM proteins?

~ 65% similarity

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

What is the amino acid similarity between IFITM 1, IFITM 2, and IFITM 3?

~ 85% similarity

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

What amino acid might be involved in calcium binding in the C-terminal region of a protein?

aspartate

First Complete Genome Sequence of a French Bovine coronavirus Strain https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477389/ SHA: eef0ecf5b8e7b179dadaef967e65f2ab68f021e1 Authors: Kin, Nathalie; Guerard, Pauline; Diancourt, Laure; Caro, Valérie; Vabret, Astrid; Ar Gouilh, Meriadeg Date: 2017-05-25 DOI: 10.1128/genomea.00319-17 License: cc-by Abstract: We sequenced the first Bovine coronavirus (BCoV) complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus (BCoV) belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus (https://talk.ictvonline.org/ ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf). Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter (1, 2) . To date, the 19 complete BCoV genome sequences available in GenBank databases (consulted on 17 January 2017) originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR (RT-PCR) targeting the M gene (3). A cDNA library was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described (3). Sequences were assembled and annotated using the Geneious software (version 5.1.6). We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab (20kb nucleotides, located at the 5= side of the genome) gene is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F strain from the United Arab Emirates (accession no. KF906251), with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain (accession no. EU019216), with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 (accession no. KU886219), with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT (accession no. AF391541), associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 (HCoV-OC43) strains from France, was performed using the Muscle algorithm implemented in MEGA7 (4, 5) . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number(s). The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264.

What is the size of bovine coronavirus?

31 kb

First Complete Genome Sequence of a French Bovine coronavirus Strain https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477389/ SHA: eef0ecf5b8e7b179dadaef967e65f2ab68f021e1 Authors: Kin, Nathalie; Guerard, Pauline; Diancourt, Laure; Caro, Valérie; Vabret, Astrid; Ar Gouilh, Meriadeg Date: 2017-05-25 DOI: 10.1128/genomea.00319-17 License: cc-by Abstract: We sequenced the first Bovine coronavirus (BCoV) complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus (BCoV) belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus (https://talk.ictvonline.org/ ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf). Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter (1, 2) . To date, the 19 complete BCoV genome sequences available in GenBank databases (consulted on 17 January 2017) originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR (RT-PCR) targeting the M gene (3). A cDNA library was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described (3). Sequences were assembled and annotated using the Geneious software (version 5.1.6). We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab (20kb nucleotides, located at the 5= side of the genome) gene is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F strain from the United Arab Emirates (accession no. KF906251), with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain (accession no. EU019216), with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 (accession no. KU886219), with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT (accession no. AF391541), associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 (HCoV-OC43) strains from France, was performed using the Muscle algorithm implemented in MEGA7 (4, 5) . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number(s). The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264.

What is the molecular structure of bovine coronavirus?

single-stranded, linear, and nonsegmented RNA

First Complete Genome Sequence of a French Bovine coronavirus Strain https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477389/ SHA: eef0ecf5b8e7b179dadaef967e65f2ab68f021e1 Authors: Kin, Nathalie; Guerard, Pauline; Diancourt, Laure; Caro, Valérie; Vabret, Astrid; Ar Gouilh, Meriadeg Date: 2017-05-25 DOI: 10.1128/genomea.00319-17 License: cc-by Abstract: We sequenced the first Bovine coronavirus (BCoV) complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus (BCoV) belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus (https://talk.ictvonline.org/ ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf). Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter (1, 2) . To date, the 19 complete BCoV genome sequences available in GenBank databases (consulted on 17 January 2017) originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR (RT-PCR) targeting the M gene (3). A cDNA library was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described (3). Sequences were assembled and annotated using the Geneious software (version 5.1.6). We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab (20kb nucleotides, located at the 5= side of the genome) gene is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F strain from the United Arab Emirates (accession no. KF906251), with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain (accession no. EU019216), with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 (accession no. KU886219), with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT (accession no. AF391541), associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 (HCoV-OC43) strains from France, was performed using the Muscle algorithm implemented in MEGA7 (4, 5) . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number(s). The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264.

How many nucleotides does bovine coronavirus contain?

30,847 nucleotides

First Complete Genome Sequence of a French Bovine coronavirus Strain https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477389/ SHA: eef0ecf5b8e7b179dadaef967e65f2ab68f021e1 Authors: Kin, Nathalie; Guerard, Pauline; Diancourt, Laure; Caro, Valérie; Vabret, Astrid; Ar Gouilh, Meriadeg Date: 2017-05-25 DOI: 10.1128/genomea.00319-17 License: cc-by Abstract: We sequenced the first Bovine coronavirus (BCoV) complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus (BCoV) belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus (https://talk.ictvonline.org/ ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf). Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter (1, 2) . To date, the 19 complete BCoV genome sequences available in GenBank databases (consulted on 17 January 2017) originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR (RT-PCR) targeting the M gene (3). A cDNA library was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described (3). Sequences were assembled and annotated using the Geneious software (version 5.1.6). We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab (20kb nucleotides, located at the 5= side of the genome) gene is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F strain from the United Arab Emirates (accession no. KF906251), with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain (accession no. EU019216), with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 (accession no. KU886219), with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT (accession no. AF391541), associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 (HCoV-OC43) strains from France, was performed using the Muscle algorithm implemented in MEGA7 (4, 5) . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number(s). The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264.

What is the size of the orf1ab gene in bovine coronavirus?

20kb

First Complete Genome Sequence of a French Bovine coronavirus Strain https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477389/ SHA: eef0ecf5b8e7b179dadaef967e65f2ab68f021e1 Authors: Kin, Nathalie; Guerard, Pauline; Diancourt, Laure; Caro, Valérie; Vabret, Astrid; Ar Gouilh, Meriadeg Date: 2017-05-25 DOI: 10.1128/genomea.00319-17 License: cc-by Abstract: We sequenced the first Bovine coronavirus (BCoV) complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus (BCoV) belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus (https://talk.ictvonline.org/ ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf). Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter (1, 2) . To date, the 19 complete BCoV genome sequences available in GenBank databases (consulted on 17 January 2017) originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR (RT-PCR) targeting the M gene (3). A cDNA library was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described (3). Sequences were assembled and annotated using the Geneious software (version 5.1.6). We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab (20kb nucleotides, located at the 5= side of the genome) gene is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F strain from the United Arab Emirates (accession no. KF906251), with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain (accession no. EU019216), with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 (accession no. KU886219), with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT (accession no. AF391541), associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 (HCoV-OC43) strains from France, was performed using the Muscle algorithm implemented in MEGA7 (4, 5) . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number(s). The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264.

Is the orf1ab gene at the 3' or 5' end of the bovine coronavirus genome?

5= side

Species‐specific clinical characteristics of human coronavirus infection among otherwise healthy adolescents and adults https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820427/ SHA: edfe02a438fa9b667313da8f03614303fc2a4a14 Authors: Bouvier, Monique; Chen, Wei‐Ju; Arnold, John C.; Fairchok, Mary P.; Danaher, Patrick J.; Lalani, Tahaniyat; Malone, Leslie; Mor, Deepika; Ridoré, Michelande; Burgess, Timothy H.; Millar, Eugene V. Date: 2018-02-02 DOI: 10.1111/irv.12538 License: cc-by Abstract: Human coronavirus (HCoV) is a known cause of influenza‐like illness (ILI). In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 (34%), HCoV‐229E (28%), HCoV‐NL63 (22%), and HCoV‐HKU1 (16%). We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus (HCoV) infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS); case-fatality rates ranged from 14% to 45%. [1] [2] [3] By contrast, other HCoV species (HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E) are much more prevalent, much less severe, and common causes of influenza-like illness (ILI). [4] [5] [6] [7] [8] [9] [10] [11] Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, [10] [11] [12] In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness (ILI), the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions (eg, diabetes, chronic obstructive pulmonary disease, severe asthma), women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 (none) to 3 (severe). Symptom severity scores were quantified using the following five measures: (i) individual symptom score for 20 symptoms, (ii) the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, (iii) the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, (iv) the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and (Table 1) . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus (HCoV) is an important cause of influenza-like illness (ILI) ranged from 4% to 22%. [8] [9] [10] [11] 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease.

What is a significant cause of Influenze like illness among healthy adolescents and adults presenting for medical evaluation?

HCoV

Species‐specific clinical characteristics of human coronavirus infection among otherwise healthy adolescents and adults https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820427/ SHA: edfe02a438fa9b667313da8f03614303fc2a4a14 Authors: Bouvier, Monique; Chen, Wei‐Ju; Arnold, John C.; Fairchok, Mary P.; Danaher, Patrick J.; Lalani, Tahaniyat; Malone, Leslie; Mor, Deepika; Ridoré, Michelande; Burgess, Timothy H.; Millar, Eugene V. Date: 2018-02-02 DOI: 10.1111/irv.12538 License: cc-by Abstract: Human coronavirus (HCoV) is a known cause of influenza‐like illness (ILI). In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 (34%), HCoV‐229E (28%), HCoV‐NL63 (22%), and HCoV‐HKU1 (16%). We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus (HCoV) infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS); case-fatality rates ranged from 14% to 45%. [1] [2] [3] By contrast, other HCoV species (HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E) are much more prevalent, much less severe, and common causes of influenza-like illness (ILI). [4] [5] [6] [7] [8] [9] [10] [11] Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, [10] [11] [12] In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness (ILI), the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions (eg, diabetes, chronic obstructive pulmonary disease, severe asthma), women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 (none) to 3 (severe). Symptom severity scores were quantified using the following five measures: (i) individual symptom score for 20 symptoms, (ii) the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, (iii) the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, (iv) the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and (Table 1) . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus (HCoV) is an important cause of influenza-like illness (ILI) ranged from 4% to 22%. [8] [9] [10] [11] 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease.

What is the most common species of Human Coronavirus among adults?

HCoV-OC43

Species‐specific clinical characteristics of human coronavirus infection among otherwise healthy adolescents and adults https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820427/ SHA: edfe02a438fa9b667313da8f03614303fc2a4a14 Authors: Bouvier, Monique; Chen, Wei‐Ju; Arnold, John C.; Fairchok, Mary P.; Danaher, Patrick J.; Lalani, Tahaniyat; Malone, Leslie; Mor, Deepika; Ridoré, Michelande; Burgess, Timothy H.; Millar, Eugene V. Date: 2018-02-02 DOI: 10.1111/irv.12538 License: cc-by Abstract: Human coronavirus (HCoV) is a known cause of influenza‐like illness (ILI). In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 (34%), HCoV‐229E (28%), HCoV‐NL63 (22%), and HCoV‐HKU1 (16%). We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus (HCoV) infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS); case-fatality rates ranged from 14% to 45%. [1] [2] [3] By contrast, other HCoV species (HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E) are much more prevalent, much less severe, and common causes of influenza-like illness (ILI). [4] [5] [6] [7] [8] [9] [10] [11] Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, [10] [11] [12] In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness (ILI), the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions (eg, diabetes, chronic obstructive pulmonary disease, severe asthma), women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 (none) to 3 (severe). Symptom severity scores were quantified using the following five measures: (i) individual symptom score for 20 symptoms, (ii) the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, (iii) the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, (iv) the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and (Table 1) . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus (HCoV) is an important cause of influenza-like illness (ILI) ranged from 4% to 22%. [8] [9] [10] [11] 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease.

Which Human Coronavirus showed species specific clinical characteristics of its infection?

HCoV‐HKU1

Species‐specific clinical characteristics of human coronavirus infection among otherwise healthy adolescents and adults https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820427/ SHA: edfe02a438fa9b667313da8f03614303fc2a4a14 Authors: Bouvier, Monique; Chen, Wei‐Ju; Arnold, John C.; Fairchok, Mary P.; Danaher, Patrick J.; Lalani, Tahaniyat; Malone, Leslie; Mor, Deepika; Ridoré, Michelande; Burgess, Timothy H.; Millar, Eugene V. Date: 2018-02-02 DOI: 10.1111/irv.12538 License: cc-by Abstract: Human coronavirus (HCoV) is a known cause of influenza‐like illness (ILI). In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 (34%), HCoV‐229E (28%), HCoV‐NL63 (22%), and HCoV‐HKU1 (16%). We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus (HCoV) infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS); case-fatality rates ranged from 14% to 45%. [1] [2] [3] By contrast, other HCoV species (HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E) are much more prevalent, much less severe, and common causes of influenza-like illness (ILI). [4] [5] [6] [7] [8] [9] [10] [11] Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, [10] [11] [12] In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness (ILI), the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions (eg, diabetes, chronic obstructive pulmonary disease, severe asthma), women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 (none) to 3 (severe). Symptom severity scores were quantified using the following five measures: (i) individual symptom score for 20 symptoms, (ii) the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, (iii) the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, (iv) the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and (Table 1) . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus (HCoV) is an important cause of influenza-like illness (ILI) ranged from 4% to 22%. [8] [9] [10] [11] 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease.

What causes the outbreak of SARS and MERS.

Highly virulent species of HCoV

Species‐specific clinical characteristics of human coronavirus infection among otherwise healthy adolescents and adults https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820427/ SHA: edfe02a438fa9b667313da8f03614303fc2a4a14 Authors: Bouvier, Monique; Chen, Wei‐Ju; Arnold, John C.; Fairchok, Mary P.; Danaher, Patrick J.; Lalani, Tahaniyat; Malone, Leslie; Mor, Deepika; Ridoré, Michelande; Burgess, Timothy H.; Millar, Eugene V. Date: 2018-02-02 DOI: 10.1111/irv.12538 License: cc-by Abstract: Human coronavirus (HCoV) is a known cause of influenza‐like illness (ILI). In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 (34%), HCoV‐229E (28%), HCoV‐NL63 (22%), and HCoV‐HKU1 (16%). We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus (HCoV) infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS); case-fatality rates ranged from 14% to 45%. [1] [2] [3] By contrast, other HCoV species (HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E) are much more prevalent, much less severe, and common causes of influenza-like illness (ILI). [4] [5] [6] [7] [8] [9] [10] [11] Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, [10] [11] [12] In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness (ILI), the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions (eg, diabetes, chronic obstructive pulmonary disease, severe asthma), women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 (none) to 3 (severe). Symptom severity scores were quantified using the following five measures: (i) individual symptom score for 20 symptoms, (ii) the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, (iii) the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, (iv) the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and (Table 1) . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus (HCoV) is an important cause of influenza-like illness (ILI) ranged from 4% to 22%. [8] [9] [10] [11] 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease.

What is the case fatality rate of SARS and MERS?

ranged from 14% to 45%

Species‐specific clinical characteristics of human coronavirus infection among otherwise healthy adolescents and adults https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820427/ SHA: edfe02a438fa9b667313da8f03614303fc2a4a14 Authors: Bouvier, Monique; Chen, Wei‐Ju; Arnold, John C.; Fairchok, Mary P.; Danaher, Patrick J.; Lalani, Tahaniyat; Malone, Leslie; Mor, Deepika; Ridoré, Michelande; Burgess, Timothy H.; Millar, Eugene V. Date: 2018-02-02 DOI: 10.1111/irv.12538 License: cc-by Abstract: Human coronavirus (HCoV) is a known cause of influenza‐like illness (ILI). In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 (34%), HCoV‐229E (28%), HCoV‐NL63 (22%), and HCoV‐HKU1 (16%). We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus (HCoV) infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS); case-fatality rates ranged from 14% to 45%. [1] [2] [3] By contrast, other HCoV species (HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E) are much more prevalent, much less severe, and common causes of influenza-like illness (ILI). [4] [5] [6] [7] [8] [9] [10] [11] Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, [10] [11] [12] In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness (ILI), the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions (eg, diabetes, chronic obstructive pulmonary disease, severe asthma), women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 (none) to 3 (severe). Symptom severity scores were quantified using the following five measures: (i) individual symptom score for 20 symptoms, (ii) the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, (iii) the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, (iv) the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and (Table 1) . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus (HCoV) is an important cause of influenza-like illness (ILI) ranged from 4% to 22%. [8] [9] [10] [11] 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease.

What were the common HCOV strains in the 5 year USA study?

HCoV-OC43 and HCoV-229E

Species‐specific clinical characteristics of human coronavirus infection among otherwise healthy adolescents and adults https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820427/ SHA: edfe02a438fa9b667313da8f03614303fc2a4a14 Authors: Bouvier, Monique; Chen, Wei‐Ju; Arnold, John C.; Fairchok, Mary P.; Danaher, Patrick J.; Lalani, Tahaniyat; Malone, Leslie; Mor, Deepika; Ridoré, Michelande; Burgess, Timothy H.; Millar, Eugene V. Date: 2018-02-02 DOI: 10.1111/irv.12538 License: cc-by Abstract: Human coronavirus (HCoV) is a known cause of influenza‐like illness (ILI). In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 (34%), HCoV‐229E (28%), HCoV‐NL63 (22%), and HCoV‐HKU1 (16%). We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus (HCoV) infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS); case-fatality rates ranged from 14% to 45%. [1] [2] [3] By contrast, other HCoV species (HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E) are much more prevalent, much less severe, and common causes of influenza-like illness (ILI). [4] [5] [6] [7] [8] [9] [10] [11] Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, [10] [11] [12] In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness (ILI), the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions (eg, diabetes, chronic obstructive pulmonary disease, severe asthma), women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 (none) to 3 (severe). Symptom severity scores were quantified using the following five measures: (i) individual symptom score for 20 symptoms, (ii) the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, (iii) the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, (iv) the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and (Table 1) . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus (HCoV) is an important cause of influenza-like illness (ILI) ranged from 4% to 22%. [8] [9] [10] [11] 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease.

Which species are more prevalent but less severe?

HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E

One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/ SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658 Authors: Chen, Pei-Jer; Wu, T-C Date: 2013-01-11 DOI: 10.1186/2045-3701-3-2 License: cc-by Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

What is required for a Hepatitis B infection in cells?

both intracellular and cell-surface factors

One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/ SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658 Authors: Chen, Pei-Jer; Wu, T-C Date: 2013-01-11 DOI: 10.1186/2045-3701-3-2 License: cc-by Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

heparan sulfates in the membrane proteins

One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/ SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658 Authors: Chen, Pei-Jer; Wu, T-C Date: 2013-01-11 DOI: 10.1186/2045-3701-3-2 License: cc-by Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Nterminus of HBV preS1 (amino acids 1-47)

One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/ SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658 Authors: Chen, Pei-Jer; Wu, T-C Date: 2013-01-11 DOI: 10.1186/2045-3701-3-2 License: cc-by Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

Where is NTCP located in the body?

lateral surface (canalicular) of hepatocytes

One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/ SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658 Authors: Chen, Pei-Jer; Wu, T-C Date: 2013-01-11 DOI: 10.1186/2045-3701-3-2 License: cc-by Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

What does the NTCP protein mediate?

bile acid transport

One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/ SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658 Authors: Chen, Pei-Jer; Wu, T-C Date: 2013-01-11 DOI: 10.1186/2045-3701-3-2 License: cc-by Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

Is NTCP sufficient to allow HBV infection?

not sufficient

One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/ SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658 Authors: Chen, Pei-Jer; Wu, T-C Date: 2013-01-11 DOI: 10.1186/2045-3701-3-2 License: cc-by Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

Why is NTCP thought to not be sufficient for HBV infection?

the majority of HepaRG cells were found to express NPCT but not to be infected

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

What kinds of viruses are Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV)?

arboviruses

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

What are the current clinically-available methods to detect encephalitis viral antigens?

ELISA and IFA

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

What methods exist for detecting multiple antigens simultaneously in a one-sample, laboratory test?

two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

How many antigens could be detected by Liew's multiplex ELISA test?

9

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

What kind of antibodies were used in the ELISA-array assay?

monoclonal

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

How was the ELISA assay validated?

using cultured viruses and inoculated chicken eggs with patient sera

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

What capture antibodies were used in the study?

4D5, 2B5, 1F1, 2B8, 4F9, and 4E11

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

What was the spotting concentration range for the capture antibodies?

from 0.2 to 0.0125 mg/ml

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

How was the proper spotting concentration determined?

combination of minimized cross reaction and higher signal intensity

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

How was cross reaction detection determined?

by applying JEV, YF, and DV cultures

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

How was the ELISA-array assay validated?

using cultured viruses and inoculated chicken eggs with patient sera

Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734177/ SHA: ee8483f8f2cc5fe38be4e565eae3af9d0bb8220b Authors: Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A. Date: 2013-08-05 DOI: 10.1371/journal.pone.0069982 License: cc-by Abstract: Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K(i) and IC(50) values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a). Text: Cysteine-protease inhibitors (CPI) have gained considerable attention over the last couple of decades and many classes of compounds are currently in human clinical trials for a number of diseases. Interest in papain family cysteine proteases as chemotherapeutic targets is derived from the recognition that they are critical to the life cycle or pathogenicity of many microorganisms. The cysteine proteases from Streptococcus sp. (streptopain) [1] , Staphylococcus sp. (staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms. The activation of the kallikrein-kinin pathway, which could be activated by more than sixteen bacterial proteases, is a mechanism that some pathogens exploit to ensure that there is a supply of nutrients to the site of infection by increasing vascular permeability. This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp. [4] . Many bacteria secrete several nonspecific proteases e.g. Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp. have potent metallo-, cysteine and serine proteases with broad ranges of activities [5] . The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] . Recently described cystatin superfamily of proteins comprises both eukaryotic and prokaryotic cysteine protease inhibitors [7] . Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] . The key role of cysteine proteases in microbial infections, coupled with the relative lack of redundancy compared to mammalian systems has made microbial proteases attractive targets for the development of novel chemotherapeutic approaches [10, 11] . Imidazopyridine ring systems represent an important class of compounds not only for their theoretical interest but also from a pharmacological point of view. They have been shown to possess a broad range of useful pharmacological activities [12] including antigastric, antisecretory, local anesthetic, antiviral, antianxiety, antibacterial, antifungal, antihelminthic, antiprotozoal, anticonvulsant, gastrointestinal, antiulcer (Zolmidine), anxiolytic (Alpidem), hypnotic (Zolpidem) and immunomodulatory [13] . The nature and the position of the substituents on the pyridinic moiety influence these pharmacological activities. These imidazopyridine heterocyclic structures form part of the skeleton of natural alkaloids, neuromuscular blocking agents [14] , reversible inhibitors of the H + , K + -ATPase enzymes with a potent antisecretory activity, and are known to be sedative hypnotics of the nervous system [15] . In this study, we have proposed kinetically and thermodynamically characterized 1-substituted pyridylimidazo[1,5-a]pyridine derivatives as a potent and novel cysteine protease inhibitors which also acts as antibacterial agents. The crystal structure of papain was extracted from Protein Data Bank (PDB code: 1PE6) [16] . All the water molecules and heteroatoms were removed and hydrogen atoms were added to the protein. CharMm forcefield [17] was applied and the structure was subjected to energy minimization for 1000 steps using steepest descent method. The chemical structures of all the synthesized compounds were generated using chemdraw and were subsequently converted into 3D format using CORINA. A series of docking experiments were carried out with all the designed 1substituted pyridylimidazo[1,5-a]pyridine derivatives against papain using AutoDock Tools 4.0 [18] for possible cysteine-protease inhibitory activities. The compounds were selected on the basis of their binding energies and those reflecting good binding affinity were further analyzed on in silico platform. As a parameter for the molecular docking, the Lamarckian genetic algorithm, a combination between the genetic algorithm and the local search Pseudo-Solis and Wets algorithm, was employed. A grid box of 60660660 Å was generated around active site of papain making sure those inhibitors can freely rotate inside the grid. The number of docking runs was set to 10. Each docking was repeated five times, having in the end a total of 50 docking runs, to check the precision of results. The finally obtained docked complexes were subsequently visualized using PyMol [19] . The work was further authenticated in the wet lab after its detailed analysis on in silico platform. The designed derivatives were filtered by Lipinski's ''Rule of five'' that sets the criteria for drug-like properties. Drug likeness is a property that is most often used to characterize novel lead compounds [20] . According to this rule, poor absorption is expected if MW .500, log P.5, hydrogen bond donors .5, and hydrogen bond acceptors .10 [21] . In silico absorption, distribution, metabolism and excretion (ADME) properties of these derivatives were also predicted using following online bioinformatics tools. N http://www.organic-chemistry.org. N http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal. py? Form = admetox N https://secure.chemsilico.com/pages/submit.php The above study gave us an idea about the existence of possible mutagenic and tumorigenic properties in synthesized compounds. The result obtained helped us to screen out the synthesized compounds for their further usage as potent leads. Based on the results of docking studies, ten derivatives of 1pyridylimidazo[1,5-a]pyridine were synthesized according to Siddiqui et al., 2006 [22] which are named as follows: 1- The capacity of the 1-pyridylimidazo[1,5-a]pyridine derivatives to inhibit cysteine proteases was tested using papain as the model enzyme. The proteolytic activity of the reaction mixtures was determined using Bz-DL-Arg-pNA as the chromogenic substrate [23] . To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant K i was determined by Dixon method [24] and also by the Lineweaver-Burk equation. The K m value was calculated from the double-reciprocal equation by fitting the data into the computer software Origin 6.1. The Lineweaver-Burk plot was used to determine the types of inhibition. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation, Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme. The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] . In brief, the bacterial strains (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii)) were grown and diluted to 2610 5 colony-forming units (CFU)/ml in sodium phosphate buffer (SPB) containing 0.03% Luria-Bertani (LB) broth. The synthesized derivatives were dissolved in DMSO and their serial dilution was performed in 50 mL of LB medium in 96-well microtitre plate to achieve the required concentrations (0.1-10 mg/ml) with bacterial inoculums (5610 4 CFU per well). DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain. For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day. After having determined the MICs, bacterial strains from the wells of the microtitre plate with no visible bacterial growth were removed for serial sub cultivation of 2 ml into another new microtitre plate containing 100 ml of broth per well and further incubated for 24 h. The lowest concentration with no visible growth was defined as MBC [28], indicating 99.5% killing of the original inoculum. The absorbance of each well was measured at a wavelength of 620 nm by Microplate Manager 4.0 (Bio-Rad laboratories) and compared with a blank. Solvent (DMSO) was used as a negative control. Three replicates were done for each compound and experiment was repeated two times. Bacteria use their cysteine proteases for pathogenecity as could be depicted from the structure of Cif homolog in Burkholderia pseudomallei (CHBP) which reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad [29] . It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases. The similarity of this AvrPphB protein with papain includes the catalytic triad of Cys-98, His-212, and Asp-227 in the AvrPphB active site [30] . Turk et al. have proposed, on the basis of kinetic and structural studies, that papain has seven subsites at the active site but only five subsites are important which can bind to an amino acid residue of the substrate [31] . A variety of intermediates are generated when papain reacts with substrate or an inhibitor [2] . Like serine proteases, cysteine proteases tend to have relatively shallow, solvent-exposed active sites that can accommodate short substrate/inhibitor segments of protein loops (e.g. from endogenous inhibitors such as cystatins) or strands. The inhibitor Table 3 . Name, Structure, IC50 & K i of 1-substituted pyridylimidazo[1,5-a]pyridine derivatives against cysteine protease papain. Type of inhibition Ki (mM) IC 50 (mM) Non-Competitive 13.7 13.4 compound bound to protease with a combination of hydrogen bonds and hydrophobic interactions. As a part of our investigation in developing novel and efficient cysteine protease inhibitors, ten 1-substituted pyridylimidazo [1,5a] pyridine derivatives (3a-j) were primarily designed and screened on the basis of their docking energies against papain to elucidate their possible mode of action. It was found that these compounds were specific inhibitors of cysteine protease, papain and didn't show inhibition against other types of proteases like serine, aspartic or metalloproteases. They are specific for CA clan of cysteine protease and didn't show any significant inhibition against other clans of cysteine proteases. These new compounds were devised based on the knowledge of ability of a protein to alter its conformation to accommodate a binding ligand and enabled us to directly compare the relative positions of the residue in the binding pocket. Molecular docking study provided the structural insight into the binding of these compounds (3a-j) (Figure 1 ) within the active site of papain which mainly consist of a catalytic triad of Cys 25, His 159 and Asp 175 [32] . Moreover, role of other residues present in the active site of papain, playing important role in the accommodation of compounds have also been revealed. Initially, docking was performed with all the designed compounds (3a-j) against papain, a known cysteine protease enzyme and in this context, we observed very interesting results where our proposed inhibitors (3a-j) take advantage of aromatic and hydrophilic residues by making a variety of interactions with target enzyme. Although, compounds 3e-j gave significant results when docked with papain but during evaluation of antibacterial properties in wet lab experiments, they gave insignificant results (data not shown). Therefore, only four compounds were considered for discussion and further experiments like kinetic and thermodynamic studies to characterize these compounds as potent pro-inhibitors, were performed (3a-d). The findings of the above study have shown that the molecular interactions between the compounds 3a-d and papain were very similar to the interactions observed for E-64c, a derivative of naturally occurring epoxide inhibitor (E-64c) (Figure 1 ) of cysteine proteases [31, 32] , with papain; especially with regard to the hydrogen bonding and hydrophobic interactions of the ligands with conserved residues in the catalytic binding site (Figure 2 A-D). Several papain residues participated in hydrophobic interactions with compounds 3a-d, including Gln19, Cys25, Gly66 and Asp158. The pyridine moieties of compounds 3a-d interact with S2 site of papain which includes (Tyr61, Asn64, Gly65 & Tyr67) amino acids (Figure 2 A-D) . The active site residues that were found to be key player in the interaction of compounds within the active site (mostly through hydrophobic interactions) were Cys25, Tyr61, His159 and Trp177, while Trp177, Gln19 were found to me making hydrogen bonds only with compound 3a. Besides this many other residues were also found to be actively involved ( Table 1) . Furthermore, the binding energies for the compound 3a, 3b, 3c and 3d with papain were found to be 26.12, 25.76, 26.84 and 25.62 Kcal/mol respectively, which were in great agreement with our wet lab experiments; shall be discussed later ( Table 1) . This confirmed the accuracy of our docking protocol. Since, the binding energy is a direct measure of strength of interaction and our compounds 3a-d showed stronger binding within the active site of papain in comparison to the inhibitor E-64c (DG: 24.04 Kcal/mol), therefore, the results suggest that these 1-substituted pyridylimidazo[1,5-a]pyridine derivatives (3ad) could be potent inhibitors of papain like cysteine proteases. The in silico interaction of compounds 3a-d with papain, which were observed as discussed above, was validated with wet lab Table 5 . Prediction of antibacterial compounds as drugs (http://www.organic-chemistry.org). Table 2) . Interestingly, the observed in silico binding energies for the compounds 3a-d against papain were found to be in great agreement (standard error 62 Kcal/mol) with the value of free energy of binding (DG) observed during thermodynamics studies ( Table 1 and 2) . Similarly, enthalpy change (DH) of the binding was negative whereas entropy (DS) change of the binding was positive which indicated the exothermic and entropically driven nature of binding. This pattern of temperature dependence is characteristic of hydrophobic interaction [33] . As discussed earlier that all the compounds (3a-d) were found to interact with the active site residues of papain through hydrophobic interactions at most instances during in silico studies, the same was observed by the analysis of Van't Hoff plots for all the proposed inhibitors at three different temperatures (32uC, 37uC and 42uC) in wet lab experiments ( Figure 3) . This proves the importance of these types of interactions in the positioning of compounds within the active site. Hence, thermodynamics as well as in silico study reveals that hydrophobic interactions favor binding of these proposed inhibitors with papain like cysteine proteases. Further wet lab results proposed the non competitive interaction of compounds (3a, 3c & 3d) with papain except for compound 3b which showed competitive interaction. In sum up, the above results of molecular docking studies and thermodynamic analysis of compounds 3a-d with papain showed that these compounds have the potential to be novel and unique cysteine protease inhibitors. In the current study, the cysteine protease inhibitory activity of synthesized derivatives of 1-substituted pyridylimidazo[1,5-a] pyridine (3a-d)) was also performed against papain and the inhibition constants (K i ) for the above said enzyme were observed to be 13.70, 23.20, 90.00 and 99.30 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Furthermore, the calculated IC 50 values were also found to be 13.40, 21.17, 94.50 and 96.50 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Except compound 3b, rest of the compounds showed non competitive, reversible inhibitions but all the compounds irrespective of types of binding, showed hydrophobic and entropically driven interaction. These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii). Here, we are showing the data of only four compounds (3a-d) because of their significant results ( Table 4 ). All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) . Since compound 3c & 3d do not have much difference in their IC50 values (3c-94.5 mM and 3d-96.5 mM) against cysteine protease, papain and hence in antibacterial activity in all instances except one. It might be random due to so close in IC50 values. Compounds 3c & 3d are having much difference in their IC50 values (3b-21.17 mM and 3c-94.5 mM) and they showed exact pattern for their antibacterial activity for all microbes except for E. coli at one instance. Although, E. coli does contain six major cysteine proteases but none belong to the CA clan of papain. It is argued that these compounds also inhibited the cysteine proteases of other clan than papain but with low efficacy. Since, pyridylimidazo[1,5-a]pyridine derivatives is absolutely new scaffold towards antibacterial agents and hence, not any standard compound(s) of same scaffold is available for reference. So, Clotrimazole (1-[(2-chlorophenyl)(diphenyl)methyl]-1H-imidazole), an imidazole derivatives and Ceftriaxone (third-generation cephalosporin antibiotic with broad spectrum activity against Gram-positive and Gram-negative bacteria) have been used as positive control whereas DMSO has been used as negative control. All the above mentioned bacterial species have been shown to secrete certain cysteine proteases which play very important role in the pathogenecity of different diseases caused by these microorganisms. The minimum inhibitory concentration (MICs) of compounds (3a-d) ( Table 4 ) against all tested bacteria except E. coli and P. vulgaris, were observed to be in great agreement with their respective inhibition constant (K i )/IC 50 values against papain (Table 3 ) which clearly indicates that these compounds have the potential to inhibit the papain like cysteine proteases of these pathogens. The partition coefficient (logP) is a well-established measure of the compound's lipophilicity. The distribution of calculated logP (cLogP) values of a majority of drugs in the market is in the range of zero to five. All the compounds studied except 3d, showed good agreement with the criteria laid down for the prediction of a compound to be a potential drug ( Table 5 ). All the compounds do not show any threat against toxicity risk assessment except compound 3d which showed threat as tumorogenic effect due to the presence of isobutyl group. Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally. File S1 Types of inhibitions with Ki (Compounds 3a-3d). (DOC)

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

cysteine proteases

Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734177/ SHA: ee8483f8f2cc5fe38be4e565eae3af9d0bb8220b Authors: Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A. Date: 2013-08-05 DOI: 10.1371/journal.pone.0069982 License: cc-by Abstract: Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K(i) and IC(50) values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a). Text: Cysteine-protease inhibitors (CPI) have gained considerable attention over the last couple of decades and many classes of compounds are currently in human clinical trials for a number of diseases. Interest in papain family cysteine proteases as chemotherapeutic targets is derived from the recognition that they are critical to the life cycle or pathogenicity of many microorganisms. The cysteine proteases from Streptococcus sp. (streptopain) [1] , Staphylococcus sp. (staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms. The activation of the kallikrein-kinin pathway, which could be activated by more than sixteen bacterial proteases, is a mechanism that some pathogens exploit to ensure that there is a supply of nutrients to the site of infection by increasing vascular permeability. This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp. [4] . Many bacteria secrete several nonspecific proteases e.g. Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp. have potent metallo-, cysteine and serine proteases with broad ranges of activities [5] . The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] . Recently described cystatin superfamily of proteins comprises both eukaryotic and prokaryotic cysteine protease inhibitors [7] . Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] . The key role of cysteine proteases in microbial infections, coupled with the relative lack of redundancy compared to mammalian systems has made microbial proteases attractive targets for the development of novel chemotherapeutic approaches [10, 11] . Imidazopyridine ring systems represent an important class of compounds not only for their theoretical interest but also from a pharmacological point of view. They have been shown to possess a broad range of useful pharmacological activities [12] including antigastric, antisecretory, local anesthetic, antiviral, antianxiety, antibacterial, antifungal, antihelminthic, antiprotozoal, anticonvulsant, gastrointestinal, antiulcer (Zolmidine), anxiolytic (Alpidem), hypnotic (Zolpidem) and immunomodulatory [13] . The nature and the position of the substituents on the pyridinic moiety influence these pharmacological activities. These imidazopyridine heterocyclic structures form part of the skeleton of natural alkaloids, neuromuscular blocking agents [14] , reversible inhibitors of the H + , K + -ATPase enzymes with a potent antisecretory activity, and are known to be sedative hypnotics of the nervous system [15] . In this study, we have proposed kinetically and thermodynamically characterized 1-substituted pyridylimidazo[1,5-a]pyridine derivatives as a potent and novel cysteine protease inhibitors which also acts as antibacterial agents. The crystal structure of papain was extracted from Protein Data Bank (PDB code: 1PE6) [16] . All the water molecules and heteroatoms were removed and hydrogen atoms were added to the protein. CharMm forcefield [17] was applied and the structure was subjected to energy minimization for 1000 steps using steepest descent method. The chemical structures of all the synthesized compounds were generated using chemdraw and were subsequently converted into 3D format using CORINA. A series of docking experiments were carried out with all the designed 1substituted pyridylimidazo[1,5-a]pyridine derivatives against papain using AutoDock Tools 4.0 [18] for possible cysteine-protease inhibitory activities. The compounds were selected on the basis of their binding energies and those reflecting good binding affinity were further analyzed on in silico platform. As a parameter for the molecular docking, the Lamarckian genetic algorithm, a combination between the genetic algorithm and the local search Pseudo-Solis and Wets algorithm, was employed. A grid box of 60660660 Å was generated around active site of papain making sure those inhibitors can freely rotate inside the grid. The number of docking runs was set to 10. Each docking was repeated five times, having in the end a total of 50 docking runs, to check the precision of results. The finally obtained docked complexes were subsequently visualized using PyMol [19] . The work was further authenticated in the wet lab after its detailed analysis on in silico platform. The designed derivatives were filtered by Lipinski's ''Rule of five'' that sets the criteria for drug-like properties. Drug likeness is a property that is most often used to characterize novel lead compounds [20] . According to this rule, poor absorption is expected if MW .500, log P.5, hydrogen bond donors .5, and hydrogen bond acceptors .10 [21] . In silico absorption, distribution, metabolism and excretion (ADME) properties of these derivatives were also predicted using following online bioinformatics tools. N http://www.organic-chemistry.org. N http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal. py? Form = admetox N https://secure.chemsilico.com/pages/submit.php The above study gave us an idea about the existence of possible mutagenic and tumorigenic properties in synthesized compounds. The result obtained helped us to screen out the synthesized compounds for their further usage as potent leads. Based on the results of docking studies, ten derivatives of 1pyridylimidazo[1,5-a]pyridine were synthesized according to Siddiqui et al., 2006 [22] which are named as follows: 1- The capacity of the 1-pyridylimidazo[1,5-a]pyridine derivatives to inhibit cysteine proteases was tested using papain as the model enzyme. The proteolytic activity of the reaction mixtures was determined using Bz-DL-Arg-pNA as the chromogenic substrate [23] . To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant K i was determined by Dixon method [24] and also by the Lineweaver-Burk equation. The K m value was calculated from the double-reciprocal equation by fitting the data into the computer software Origin 6.1. The Lineweaver-Burk plot was used to determine the types of inhibition. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation, Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme. The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] . In brief, the bacterial strains (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii)) were grown and diluted to 2610 5 colony-forming units (CFU)/ml in sodium phosphate buffer (SPB) containing 0.03% Luria-Bertani (LB) broth. The synthesized derivatives were dissolved in DMSO and their serial dilution was performed in 50 mL of LB medium in 96-well microtitre plate to achieve the required concentrations (0.1-10 mg/ml) with bacterial inoculums (5610 4 CFU per well). DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain. For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day. After having determined the MICs, bacterial strains from the wells of the microtitre plate with no visible bacterial growth were removed for serial sub cultivation of 2 ml into another new microtitre plate containing 100 ml of broth per well and further incubated for 24 h. The lowest concentration with no visible growth was defined as MBC [28], indicating 99.5% killing of the original inoculum. The absorbance of each well was measured at a wavelength of 620 nm by Microplate Manager 4.0 (Bio-Rad laboratories) and compared with a blank. Solvent (DMSO) was used as a negative control. Three replicates were done for each compound and experiment was repeated two times. Bacteria use their cysteine proteases for pathogenecity as could be depicted from the structure of Cif homolog in Burkholderia pseudomallei (CHBP) which reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad [29] . It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases. The similarity of this AvrPphB protein with papain includes the catalytic triad of Cys-98, His-212, and Asp-227 in the AvrPphB active site [30] . Turk et al. have proposed, on the basis of kinetic and structural studies, that papain has seven subsites at the active site but only five subsites are important which can bind to an amino acid residue of the substrate [31] . A variety of intermediates are generated when papain reacts with substrate or an inhibitor [2] . Like serine proteases, cysteine proteases tend to have relatively shallow, solvent-exposed active sites that can accommodate short substrate/inhibitor segments of protein loops (e.g. from endogenous inhibitors such as cystatins) or strands. The inhibitor Table 3 . Name, Structure, IC50 & K i of 1-substituted pyridylimidazo[1,5-a]pyridine derivatives against cysteine protease papain. Type of inhibition Ki (mM) IC 50 (mM) Non-Competitive 13.7 13.4 compound bound to protease with a combination of hydrogen bonds and hydrophobic interactions. As a part of our investigation in developing novel and efficient cysteine protease inhibitors, ten 1-substituted pyridylimidazo [1,5a] pyridine derivatives (3a-j) were primarily designed and screened on the basis of their docking energies against papain to elucidate their possible mode of action. It was found that these compounds were specific inhibitors of cysteine protease, papain and didn't show inhibition against other types of proteases like serine, aspartic or metalloproteases. They are specific for CA clan of cysteine protease and didn't show any significant inhibition against other clans of cysteine proteases. These new compounds were devised based on the knowledge of ability of a protein to alter its conformation to accommodate a binding ligand and enabled us to directly compare the relative positions of the residue in the binding pocket. Molecular docking study provided the structural insight into the binding of these compounds (3a-j) (Figure 1 ) within the active site of papain which mainly consist of a catalytic triad of Cys 25, His 159 and Asp 175 [32] . Moreover, role of other residues present in the active site of papain, playing important role in the accommodation of compounds have also been revealed. Initially, docking was performed with all the designed compounds (3a-j) against papain, a known cysteine protease enzyme and in this context, we observed very interesting results where our proposed inhibitors (3a-j) take advantage of aromatic and hydrophilic residues by making a variety of interactions with target enzyme. Although, compounds 3e-j gave significant results when docked with papain but during evaluation of antibacterial properties in wet lab experiments, they gave insignificant results (data not shown). Therefore, only four compounds were considered for discussion and further experiments like kinetic and thermodynamic studies to characterize these compounds as potent pro-inhibitors, were performed (3a-d). The findings of the above study have shown that the molecular interactions between the compounds 3a-d and papain were very similar to the interactions observed for E-64c, a derivative of naturally occurring epoxide inhibitor (E-64c) (Figure 1 ) of cysteine proteases [31, 32] , with papain; especially with regard to the hydrogen bonding and hydrophobic interactions of the ligands with conserved residues in the catalytic binding site (Figure 2 A-D). Several papain residues participated in hydrophobic interactions with compounds 3a-d, including Gln19, Cys25, Gly66 and Asp158. The pyridine moieties of compounds 3a-d interact with S2 site of papain which includes (Tyr61, Asn64, Gly65 & Tyr67) amino acids (Figure 2 A-D) . The active site residues that were found to be key player in the interaction of compounds within the active site (mostly through hydrophobic interactions) were Cys25, Tyr61, His159 and Trp177, while Trp177, Gln19 were found to me making hydrogen bonds only with compound 3a. Besides this many other residues were also found to be actively involved ( Table 1) . Furthermore, the binding energies for the compound 3a, 3b, 3c and 3d with papain were found to be 26.12, 25.76, 26.84 and 25.62 Kcal/mol respectively, which were in great agreement with our wet lab experiments; shall be discussed later ( Table 1) . This confirmed the accuracy of our docking protocol. Since, the binding energy is a direct measure of strength of interaction and our compounds 3a-d showed stronger binding within the active site of papain in comparison to the inhibitor E-64c (DG: 24.04 Kcal/mol), therefore, the results suggest that these 1-substituted pyridylimidazo[1,5-a]pyridine derivatives (3ad) could be potent inhibitors of papain like cysteine proteases. The in silico interaction of compounds 3a-d with papain, which were observed as discussed above, was validated with wet lab Table 5 . Prediction of antibacterial compounds as drugs (http://www.organic-chemistry.org). Table 2) . Interestingly, the observed in silico binding energies for the compounds 3a-d against papain were found to be in great agreement (standard error 62 Kcal/mol) with the value of free energy of binding (DG) observed during thermodynamics studies ( Table 1 and 2) . Similarly, enthalpy change (DH) of the binding was negative whereas entropy (DS) change of the binding was positive which indicated the exothermic and entropically driven nature of binding. This pattern of temperature dependence is characteristic of hydrophobic interaction [33] . As discussed earlier that all the compounds (3a-d) were found to interact with the active site residues of papain through hydrophobic interactions at most instances during in silico studies, the same was observed by the analysis of Van't Hoff plots for all the proposed inhibitors at three different temperatures (32uC, 37uC and 42uC) in wet lab experiments ( Figure 3) . This proves the importance of these types of interactions in the positioning of compounds within the active site. Hence, thermodynamics as well as in silico study reveals that hydrophobic interactions favor binding of these proposed inhibitors with papain like cysteine proteases. Further wet lab results proposed the non competitive interaction of compounds (3a, 3c & 3d) with papain except for compound 3b which showed competitive interaction. In sum up, the above results of molecular docking studies and thermodynamic analysis of compounds 3a-d with papain showed that these compounds have the potential to be novel and unique cysteine protease inhibitors. In the current study, the cysteine protease inhibitory activity of synthesized derivatives of 1-substituted pyridylimidazo[1,5-a] pyridine (3a-d)) was also performed against papain and the inhibition constants (K i ) for the above said enzyme were observed to be 13.70, 23.20, 90.00 and 99.30 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Furthermore, the calculated IC 50 values were also found to be 13.40, 21.17, 94.50 and 96.50 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Except compound 3b, rest of the compounds showed non competitive, reversible inhibitions but all the compounds irrespective of types of binding, showed hydrophobic and entropically driven interaction. These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii). Here, we are showing the data of only four compounds (3a-d) because of their significant results ( Table 4 ). All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) . Since compound 3c & 3d do not have much difference in their IC50 values (3c-94.5 mM and 3d-96.5 mM) against cysteine protease, papain and hence in antibacterial activity in all instances except one. It might be random due to so close in IC50 values. Compounds 3c & 3d are having much difference in their IC50 values (3b-21.17 mM and 3c-94.5 mM) and they showed exact pattern for their antibacterial activity for all microbes except for E. coli at one instance. Although, E. coli does contain six major cysteine proteases but none belong to the CA clan of papain. It is argued that these compounds also inhibited the cysteine proteases of other clan than papain but with low efficacy. Since, pyridylimidazo[1,5-a]pyridine derivatives is absolutely new scaffold towards antibacterial agents and hence, not any standard compound(s) of same scaffold is available for reference. So, Clotrimazole (1-[(2-chlorophenyl)(diphenyl)methyl]-1H-imidazole), an imidazole derivatives and Ceftriaxone (third-generation cephalosporin antibiotic with broad spectrum activity against Gram-positive and Gram-negative bacteria) have been used as positive control whereas DMSO has been used as negative control. All the above mentioned bacterial species have been shown to secrete certain cysteine proteases which play very important role in the pathogenecity of different diseases caused by these microorganisms. The minimum inhibitory concentration (MICs) of compounds (3a-d) ( Table 4 ) against all tested bacteria except E. coli and P. vulgaris, were observed to be in great agreement with their respective inhibition constant (K i )/IC 50 values against papain (Table 3 ) which clearly indicates that these compounds have the potential to inhibit the papain like cysteine proteases of these pathogens. The partition coefficient (logP) is a well-established measure of the compound's lipophilicity. The distribution of calculated logP (cLogP) values of a majority of drugs in the market is in the range of zero to five. All the compounds studied except 3d, showed good agreement with the criteria laid down for the prediction of a compound to be a potential drug ( Table 5 ). All the compounds do not show any threat against toxicity risk assessment except compound 3d which showed threat as tumorogenic effect due to the presence of isobutyl group. Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally. File S1 Types of inhibitions with Ki (Compounds 3a-3d). (DOC)

At what temperatures was the assay completed?

32uC, 37uC, 42uC

Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734177/ SHA: ee8483f8f2cc5fe38be4e565eae3af9d0bb8220b Authors: Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A. Date: 2013-08-05 DOI: 10.1371/journal.pone.0069982 License: cc-by Abstract: Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K(i) and IC(50) values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a). Text: Cysteine-protease inhibitors (CPI) have gained considerable attention over the last couple of decades and many classes of compounds are currently in human clinical trials for a number of diseases. Interest in papain family cysteine proteases as chemotherapeutic targets is derived from the recognition that they are critical to the life cycle or pathogenicity of many microorganisms. The cysteine proteases from Streptococcus sp. (streptopain) [1] , Staphylococcus sp. (staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms. The activation of the kallikrein-kinin pathway, which could be activated by more than sixteen bacterial proteases, is a mechanism that some pathogens exploit to ensure that there is a supply of nutrients to the site of infection by increasing vascular permeability. This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp. [4] . Many bacteria secrete several nonspecific proteases e.g. Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp. have potent metallo-, cysteine and serine proteases with broad ranges of activities [5] . The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] . Recently described cystatin superfamily of proteins comprises both eukaryotic and prokaryotic cysteine protease inhibitors [7] . Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] . The key role of cysteine proteases in microbial infections, coupled with the relative lack of redundancy compared to mammalian systems has made microbial proteases attractive targets for the development of novel chemotherapeutic approaches [10, 11] . Imidazopyridine ring systems represent an important class of compounds not only for their theoretical interest but also from a pharmacological point of view. They have been shown to possess a broad range of useful pharmacological activities [12] including antigastric, antisecretory, local anesthetic, antiviral, antianxiety, antibacterial, antifungal, antihelminthic, antiprotozoal, anticonvulsant, gastrointestinal, antiulcer (Zolmidine), anxiolytic (Alpidem), hypnotic (Zolpidem) and immunomodulatory [13] . The nature and the position of the substituents on the pyridinic moiety influence these pharmacological activities. These imidazopyridine heterocyclic structures form part of the skeleton of natural alkaloids, neuromuscular blocking agents [14] , reversible inhibitors of the H + , K + -ATPase enzymes with a potent antisecretory activity, and are known to be sedative hypnotics of the nervous system [15] . In this study, we have proposed kinetically and thermodynamically characterized 1-substituted pyridylimidazo[1,5-a]pyridine derivatives as a potent and novel cysteine protease inhibitors which also acts as antibacterial agents. The crystal structure of papain was extracted from Protein Data Bank (PDB code: 1PE6) [16] . All the water molecules and heteroatoms were removed and hydrogen atoms were added to the protein. CharMm forcefield [17] was applied and the structure was subjected to energy minimization for 1000 steps using steepest descent method. The chemical structures of all the synthesized compounds were generated using chemdraw and were subsequently converted into 3D format using CORINA. A series of docking experiments were carried out with all the designed 1substituted pyridylimidazo[1,5-a]pyridine derivatives against papain using AutoDock Tools 4.0 [18] for possible cysteine-protease inhibitory activities. The compounds were selected on the basis of their binding energies and those reflecting good binding affinity were further analyzed on in silico platform. As a parameter for the molecular docking, the Lamarckian genetic algorithm, a combination between the genetic algorithm and the local search Pseudo-Solis and Wets algorithm, was employed. A grid box of 60660660 Å was generated around active site of papain making sure those inhibitors can freely rotate inside the grid. The number of docking runs was set to 10. Each docking was repeated five times, having in the end a total of 50 docking runs, to check the precision of results. The finally obtained docked complexes were subsequently visualized using PyMol [19] . The work was further authenticated in the wet lab after its detailed analysis on in silico platform. The designed derivatives were filtered by Lipinski's ''Rule of five'' that sets the criteria for drug-like properties. Drug likeness is a property that is most often used to characterize novel lead compounds [20] . According to this rule, poor absorption is expected if MW .500, log P.5, hydrogen bond donors .5, and hydrogen bond acceptors .10 [21] . In silico absorption, distribution, metabolism and excretion (ADME) properties of these derivatives were also predicted using following online bioinformatics tools. N http://www.organic-chemistry.org. N http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal. py? Form = admetox N https://secure.chemsilico.com/pages/submit.php The above study gave us an idea about the existence of possible mutagenic and tumorigenic properties in synthesized compounds. The result obtained helped us to screen out the synthesized compounds for their further usage as potent leads. Based on the results of docking studies, ten derivatives of 1pyridylimidazo[1,5-a]pyridine were synthesized according to Siddiqui et al., 2006 [22] which are named as follows: 1- The capacity of the 1-pyridylimidazo[1,5-a]pyridine derivatives to inhibit cysteine proteases was tested using papain as the model enzyme. The proteolytic activity of the reaction mixtures was determined using Bz-DL-Arg-pNA as the chromogenic substrate [23] . To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant K i was determined by Dixon method [24] and also by the Lineweaver-Burk equation. The K m value was calculated from the double-reciprocal equation by fitting the data into the computer software Origin 6.1. The Lineweaver-Burk plot was used to determine the types of inhibition. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation, Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme. The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] . In brief, the bacterial strains (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii)) were grown and diluted to 2610 5 colony-forming units (CFU)/ml in sodium phosphate buffer (SPB) containing 0.03% Luria-Bertani (LB) broth. The synthesized derivatives were dissolved in DMSO and their serial dilution was performed in 50 mL of LB medium in 96-well microtitre plate to achieve the required concentrations (0.1-10 mg/ml) with bacterial inoculums (5610 4 CFU per well). DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain. For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day. After having determined the MICs, bacterial strains from the wells of the microtitre plate with no visible bacterial growth were removed for serial sub cultivation of 2 ml into another new microtitre plate containing 100 ml of broth per well and further incubated for 24 h. The lowest concentration with no visible growth was defined as MBC [28], indicating 99.5% killing of the original inoculum. The absorbance of each well was measured at a wavelength of 620 nm by Microplate Manager 4.0 (Bio-Rad laboratories) and compared with a blank. Solvent (DMSO) was used as a negative control. Three replicates were done for each compound and experiment was repeated two times. Bacteria use their cysteine proteases for pathogenecity as could be depicted from the structure of Cif homolog in Burkholderia pseudomallei (CHBP) which reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad [29] . It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases. The similarity of this AvrPphB protein with papain includes the catalytic triad of Cys-98, His-212, and Asp-227 in the AvrPphB active site [30] . Turk et al. have proposed, on the basis of kinetic and structural studies, that papain has seven subsites at the active site but only five subsites are important which can bind to an amino acid residue of the substrate [31] . A variety of intermediates are generated when papain reacts with substrate or an inhibitor [2] . Like serine proteases, cysteine proteases tend to have relatively shallow, solvent-exposed active sites that can accommodate short substrate/inhibitor segments of protein loops (e.g. from endogenous inhibitors such as cystatins) or strands. The inhibitor Table 3 . Name, Structure, IC50 & K i of 1-substituted pyridylimidazo[1,5-a]pyridine derivatives against cysteine protease papain. Type of inhibition Ki (mM) IC 50 (mM) Non-Competitive 13.7 13.4 compound bound to protease with a combination of hydrogen bonds and hydrophobic interactions. As a part of our investigation in developing novel and efficient cysteine protease inhibitors, ten 1-substituted pyridylimidazo [1,5a] pyridine derivatives (3a-j) were primarily designed and screened on the basis of their docking energies against papain to elucidate their possible mode of action. It was found that these compounds were specific inhibitors of cysteine protease, papain and didn't show inhibition against other types of proteases like serine, aspartic or metalloproteases. They are specific for CA clan of cysteine protease and didn't show any significant inhibition against other clans of cysteine proteases. These new compounds were devised based on the knowledge of ability of a protein to alter its conformation to accommodate a binding ligand and enabled us to directly compare the relative positions of the residue in the binding pocket. Molecular docking study provided the structural insight into the binding of these compounds (3a-j) (Figure 1 ) within the active site of papain which mainly consist of a catalytic triad of Cys 25, His 159 and Asp 175 [32] . Moreover, role of other residues present in the active site of papain, playing important role in the accommodation of compounds have also been revealed. Initially, docking was performed with all the designed compounds (3a-j) against papain, a known cysteine protease enzyme and in this context, we observed very interesting results where our proposed inhibitors (3a-j) take advantage of aromatic and hydrophilic residues by making a variety of interactions with target enzyme. Although, compounds 3e-j gave significant results when docked with papain but during evaluation of antibacterial properties in wet lab experiments, they gave insignificant results (data not shown). Therefore, only four compounds were considered for discussion and further experiments like kinetic and thermodynamic studies to characterize these compounds as potent pro-inhibitors, were performed (3a-d). The findings of the above study have shown that the molecular interactions between the compounds 3a-d and papain were very similar to the interactions observed for E-64c, a derivative of naturally occurring epoxide inhibitor (E-64c) (Figure 1 ) of cysteine proteases [31, 32] , with papain; especially with regard to the hydrogen bonding and hydrophobic interactions of the ligands with conserved residues in the catalytic binding site (Figure 2 A-D). Several papain residues participated in hydrophobic interactions with compounds 3a-d, including Gln19, Cys25, Gly66 and Asp158. The pyridine moieties of compounds 3a-d interact with S2 site of papain which includes (Tyr61, Asn64, Gly65 & Tyr67) amino acids (Figure 2 A-D) . The active site residues that were found to be key player in the interaction of compounds within the active site (mostly through hydrophobic interactions) were Cys25, Tyr61, His159 and Trp177, while Trp177, Gln19 were found to me making hydrogen bonds only with compound 3a. Besides this many other residues were also found to be actively involved ( Table 1) . Furthermore, the binding energies for the compound 3a, 3b, 3c and 3d with papain were found to be 26.12, 25.76, 26.84 and 25.62 Kcal/mol respectively, which were in great agreement with our wet lab experiments; shall be discussed later ( Table 1) . This confirmed the accuracy of our docking protocol. Since, the binding energy is a direct measure of strength of interaction and our compounds 3a-d showed stronger binding within the active site of papain in comparison to the inhibitor E-64c (DG: 24.04 Kcal/mol), therefore, the results suggest that these 1-substituted pyridylimidazo[1,5-a]pyridine derivatives (3ad) could be potent inhibitors of papain like cysteine proteases. The in silico interaction of compounds 3a-d with papain, which were observed as discussed above, was validated with wet lab Table 5 . Prediction of antibacterial compounds as drugs (http://www.organic-chemistry.org). Table 2) . Interestingly, the observed in silico binding energies for the compounds 3a-d against papain were found to be in great agreement (standard error 62 Kcal/mol) with the value of free energy of binding (DG) observed during thermodynamics studies ( Table 1 and 2) . Similarly, enthalpy change (DH) of the binding was negative whereas entropy (DS) change of the binding was positive which indicated the exothermic and entropically driven nature of binding. This pattern of temperature dependence is characteristic of hydrophobic interaction [33] . As discussed earlier that all the compounds (3a-d) were found to interact with the active site residues of papain through hydrophobic interactions at most instances during in silico studies, the same was observed by the analysis of Van't Hoff plots for all the proposed inhibitors at three different temperatures (32uC, 37uC and 42uC) in wet lab experiments ( Figure 3) . This proves the importance of these types of interactions in the positioning of compounds within the active site. Hence, thermodynamics as well as in silico study reveals that hydrophobic interactions favor binding of these proposed inhibitors with papain like cysteine proteases. Further wet lab results proposed the non competitive interaction of compounds (3a, 3c & 3d) with papain except for compound 3b which showed competitive interaction. In sum up, the above results of molecular docking studies and thermodynamic analysis of compounds 3a-d with papain showed that these compounds have the potential to be novel and unique cysteine protease inhibitors. In the current study, the cysteine protease inhibitory activity of synthesized derivatives of 1-substituted pyridylimidazo[1,5-a] pyridine (3a-d)) was also performed against papain and the inhibition constants (K i ) for the above said enzyme were observed to be 13.70, 23.20, 90.00 and 99.30 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Furthermore, the calculated IC 50 values were also found to be 13.40, 21.17, 94.50 and 96.50 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Except compound 3b, rest of the compounds showed non competitive, reversible inhibitions but all the compounds irrespective of types of binding, showed hydrophobic and entropically driven interaction. These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii). Here, we are showing the data of only four compounds (3a-d) because of their significant results ( Table 4 ). All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) . Since compound 3c & 3d do not have much difference in their IC50 values (3c-94.5 mM and 3d-96.5 mM) against cysteine protease, papain and hence in antibacterial activity in all instances except one. It might be random due to so close in IC50 values. Compounds 3c & 3d are having much difference in their IC50 values (3b-21.17 mM and 3c-94.5 mM) and they showed exact pattern for their antibacterial activity for all microbes except for E. coli at one instance. Although, E. coli does contain six major cysteine proteases but none belong to the CA clan of papain. It is argued that these compounds also inhibited the cysteine proteases of other clan than papain but with low efficacy. Since, pyridylimidazo[1,5-a]pyridine derivatives is absolutely new scaffold towards antibacterial agents and hence, not any standard compound(s) of same scaffold is available for reference. So, Clotrimazole (1-[(2-chlorophenyl)(diphenyl)methyl]-1H-imidazole), an imidazole derivatives and Ceftriaxone (third-generation cephalosporin antibiotic with broad spectrum activity against Gram-positive and Gram-negative bacteria) have been used as positive control whereas DMSO has been used as negative control. All the above mentioned bacterial species have been shown to secrete certain cysteine proteases which play very important role in the pathogenecity of different diseases caused by these microorganisms. The minimum inhibitory concentration (MICs) of compounds (3a-d) ( Table 4 ) against all tested bacteria except E. coli and P. vulgaris, were observed to be in great agreement with their respective inhibition constant (K i )/IC 50 values against papain (Table 3 ) which clearly indicates that these compounds have the potential to inhibit the papain like cysteine proteases of these pathogens. The partition coefficient (logP) is a well-established measure of the compound's lipophilicity. The distribution of calculated logP (cLogP) values of a majority of drugs in the market is in the range of zero to five. All the compounds studied except 3d, showed good agreement with the criteria laid down for the prediction of a compound to be a potential drug ( Table 5 ). All the compounds do not show any threat against toxicity risk assessment except compound 3d which showed threat as tumorogenic effect due to the presence of isobutyl group. Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally. File S1 Types of inhibitions with Ki (Compounds 3a-3d). (DOC)

What criteria sets the guideline for drug-like properties?

Lipinski's ''Rule of five''

Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734177/ SHA: ee8483f8f2cc5fe38be4e565eae3af9d0bb8220b Authors: Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A. Date: 2013-08-05 DOI: 10.1371/journal.pone.0069982 License: cc-by Abstract: Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K(i) and IC(50) values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a). Text: Cysteine-protease inhibitors (CPI) have gained considerable attention over the last couple of decades and many classes of compounds are currently in human clinical trials for a number of diseases. Interest in papain family cysteine proteases as chemotherapeutic targets is derived from the recognition that they are critical to the life cycle or pathogenicity of many microorganisms. The cysteine proteases from Streptococcus sp. (streptopain) [1] , Staphylococcus sp. (staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms. The activation of the kallikrein-kinin pathway, which could be activated by more than sixteen bacterial proteases, is a mechanism that some pathogens exploit to ensure that there is a supply of nutrients to the site of infection by increasing vascular permeability. This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp. [4] . Many bacteria secrete several nonspecific proteases e.g. Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp. have potent metallo-, cysteine and serine proteases with broad ranges of activities [5] . The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] . Recently described cystatin superfamily of proteins comprises both eukaryotic and prokaryotic cysteine protease inhibitors [7] . Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] . The key role of cysteine proteases in microbial infections, coupled with the relative lack of redundancy compared to mammalian systems has made microbial proteases attractive targets for the development of novel chemotherapeutic approaches [10, 11] . Imidazopyridine ring systems represent an important class of compounds not only for their theoretical interest but also from a pharmacological point of view. They have been shown to possess a broad range of useful pharmacological activities [12] including antigastric, antisecretory, local anesthetic, antiviral, antianxiety, antibacterial, antifungal, antihelminthic, antiprotozoal, anticonvulsant, gastrointestinal, antiulcer (Zolmidine), anxiolytic (Alpidem), hypnotic (Zolpidem) and immunomodulatory [13] . The nature and the position of the substituents on the pyridinic moiety influence these pharmacological activities. These imidazopyridine heterocyclic structures form part of the skeleton of natural alkaloids, neuromuscular blocking agents [14] , reversible inhibitors of the H + , K + -ATPase enzymes with a potent antisecretory activity, and are known to be sedative hypnotics of the nervous system [15] . In this study, we have proposed kinetically and thermodynamically characterized 1-substituted pyridylimidazo[1,5-a]pyridine derivatives as a potent and novel cysteine protease inhibitors which also acts as antibacterial agents. The crystal structure of papain was extracted from Protein Data Bank (PDB code: 1PE6) [16] . All the water molecules and heteroatoms were removed and hydrogen atoms were added to the protein. CharMm forcefield [17] was applied and the structure was subjected to energy minimization for 1000 steps using steepest descent method. The chemical structures of all the synthesized compounds were generated using chemdraw and were subsequently converted into 3D format using CORINA. A series of docking experiments were carried out with all the designed 1substituted pyridylimidazo[1,5-a]pyridine derivatives against papain using AutoDock Tools 4.0 [18] for possible cysteine-protease inhibitory activities. The compounds were selected on the basis of their binding energies and those reflecting good binding affinity were further analyzed on in silico platform. As a parameter for the molecular docking, the Lamarckian genetic algorithm, a combination between the genetic algorithm and the local search Pseudo-Solis and Wets algorithm, was employed. A grid box of 60660660 Å was generated around active site of papain making sure those inhibitors can freely rotate inside the grid. The number of docking runs was set to 10. Each docking was repeated five times, having in the end a total of 50 docking runs, to check the precision of results. The finally obtained docked complexes were subsequently visualized using PyMol [19] . The work was further authenticated in the wet lab after its detailed analysis on in silico platform. The designed derivatives were filtered by Lipinski's ''Rule of five'' that sets the criteria for drug-like properties. Drug likeness is a property that is most often used to characterize novel lead compounds [20] . According to this rule, poor absorption is expected if MW .500, log P.5, hydrogen bond donors .5, and hydrogen bond acceptors .10 [21] . In silico absorption, distribution, metabolism and excretion (ADME) properties of these derivatives were also predicted using following online bioinformatics tools. N http://www.organic-chemistry.org. N http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal. py? Form = admetox N https://secure.chemsilico.com/pages/submit.php The above study gave us an idea about the existence of possible mutagenic and tumorigenic properties in synthesized compounds. The result obtained helped us to screen out the synthesized compounds for their further usage as potent leads. Based on the results of docking studies, ten derivatives of 1pyridylimidazo[1,5-a]pyridine were synthesized according to Siddiqui et al., 2006 [22] which are named as follows: 1- The capacity of the 1-pyridylimidazo[1,5-a]pyridine derivatives to inhibit cysteine proteases was tested using papain as the model enzyme. The proteolytic activity of the reaction mixtures was determined using Bz-DL-Arg-pNA as the chromogenic substrate [23] . To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant K i was determined by Dixon method [24] and also by the Lineweaver-Burk equation. The K m value was calculated from the double-reciprocal equation by fitting the data into the computer software Origin 6.1. The Lineweaver-Burk plot was used to determine the types of inhibition. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation, Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme. The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] . In brief, the bacterial strains (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii)) were grown and diluted to 2610 5 colony-forming units (CFU)/ml in sodium phosphate buffer (SPB) containing 0.03% Luria-Bertani (LB) broth. The synthesized derivatives were dissolved in DMSO and their serial dilution was performed in 50 mL of LB medium in 96-well microtitre plate to achieve the required concentrations (0.1-10 mg/ml) with bacterial inoculums (5610 4 CFU per well). DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain. For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day. After having determined the MICs, bacterial strains from the wells of the microtitre plate with no visible bacterial growth were removed for serial sub cultivation of 2 ml into another new microtitre plate containing 100 ml of broth per well and further incubated for 24 h. The lowest concentration with no visible growth was defined as MBC [28], indicating 99.5% killing of the original inoculum. The absorbance of each well was measured at a wavelength of 620 nm by Microplate Manager 4.0 (Bio-Rad laboratories) and compared with a blank. Solvent (DMSO) was used as a negative control. Three replicates were done for each compound and experiment was repeated two times. Bacteria use their cysteine proteases for pathogenecity as could be depicted from the structure of Cif homolog in Burkholderia pseudomallei (CHBP) which reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad [29] . It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases. The similarity of this AvrPphB protein with papain includes the catalytic triad of Cys-98, His-212, and Asp-227 in the AvrPphB active site [30] . Turk et al. have proposed, on the basis of kinetic and structural studies, that papain has seven subsites at the active site but only five subsites are important which can bind to an amino acid residue of the substrate [31] . A variety of intermediates are generated when papain reacts with substrate or an inhibitor [2] . Like serine proteases, cysteine proteases tend to have relatively shallow, solvent-exposed active sites that can accommodate short substrate/inhibitor segments of protein loops (e.g. from endogenous inhibitors such as cystatins) or strands. The inhibitor Table 3 . Name, Structure, IC50 & K i of 1-substituted pyridylimidazo[1,5-a]pyridine derivatives against cysteine protease papain. Type of inhibition Ki (mM) IC 50 (mM) Non-Competitive 13.7 13.4 compound bound to protease with a combination of hydrogen bonds and hydrophobic interactions. As a part of our investigation in developing novel and efficient cysteine protease inhibitors, ten 1-substituted pyridylimidazo [1,5a] pyridine derivatives (3a-j) were primarily designed and screened on the basis of their docking energies against papain to elucidate their possible mode of action. It was found that these compounds were specific inhibitors of cysteine protease, papain and didn't show inhibition against other types of proteases like serine, aspartic or metalloproteases. They are specific for CA clan of cysteine protease and didn't show any significant inhibition against other clans of cysteine proteases. These new compounds were devised based on the knowledge of ability of a protein to alter its conformation to accommodate a binding ligand and enabled us to directly compare the relative positions of the residue in the binding pocket. Molecular docking study provided the structural insight into the binding of these compounds (3a-j) (Figure 1 ) within the active site of papain which mainly consist of a catalytic triad of Cys 25, His 159 and Asp 175 [32] . Moreover, role of other residues present in the active site of papain, playing important role in the accommodation of compounds have also been revealed. Initially, docking was performed with all the designed compounds (3a-j) against papain, a known cysteine protease enzyme and in this context, we observed very interesting results where our proposed inhibitors (3a-j) take advantage of aromatic and hydrophilic residues by making a variety of interactions with target enzyme. Although, compounds 3e-j gave significant results when docked with papain but during evaluation of antibacterial properties in wet lab experiments, they gave insignificant results (data not shown). Therefore, only four compounds were considered for discussion and further experiments like kinetic and thermodynamic studies to characterize these compounds as potent pro-inhibitors, were performed (3a-d). The findings of the above study have shown that the molecular interactions between the compounds 3a-d and papain were very similar to the interactions observed for E-64c, a derivative of naturally occurring epoxide inhibitor (E-64c) (Figure 1 ) of cysteine proteases [31, 32] , with papain; especially with regard to the hydrogen bonding and hydrophobic interactions of the ligands with conserved residues in the catalytic binding site (Figure 2 A-D). Several papain residues participated in hydrophobic interactions with compounds 3a-d, including Gln19, Cys25, Gly66 and Asp158. The pyridine moieties of compounds 3a-d interact with S2 site of papain which includes (Tyr61, Asn64, Gly65 & Tyr67) amino acids (Figure 2 A-D) . The active site residues that were found to be key player in the interaction of compounds within the active site (mostly through hydrophobic interactions) were Cys25, Tyr61, His159 and Trp177, while Trp177, Gln19 were found to me making hydrogen bonds only with compound 3a. Besides this many other residues were also found to be actively involved ( Table 1) . Furthermore, the binding energies for the compound 3a, 3b, 3c and 3d with papain were found to be 26.12, 25.76, 26.84 and 25.62 Kcal/mol respectively, which were in great agreement with our wet lab experiments; shall be discussed later ( Table 1) . This confirmed the accuracy of our docking protocol. Since, the binding energy is a direct measure of strength of interaction and our compounds 3a-d showed stronger binding within the active site of papain in comparison to the inhibitor E-64c (DG: 24.04 Kcal/mol), therefore, the results suggest that these 1-substituted pyridylimidazo[1,5-a]pyridine derivatives (3ad) could be potent inhibitors of papain like cysteine proteases. The in silico interaction of compounds 3a-d with papain, which were observed as discussed above, was validated with wet lab Table 5 . Prediction of antibacterial compounds as drugs (http://www.organic-chemistry.org). Table 2) . Interestingly, the observed in silico binding energies for the compounds 3a-d against papain were found to be in great agreement (standard error 62 Kcal/mol) with the value of free energy of binding (DG) observed during thermodynamics studies ( Table 1 and 2) . Similarly, enthalpy change (DH) of the binding was negative whereas entropy (DS) change of the binding was positive which indicated the exothermic and entropically driven nature of binding. This pattern of temperature dependence is characteristic of hydrophobic interaction [33] . As discussed earlier that all the compounds (3a-d) were found to interact with the active site residues of papain through hydrophobic interactions at most instances during in silico studies, the same was observed by the analysis of Van't Hoff plots for all the proposed inhibitors at three different temperatures (32uC, 37uC and 42uC) in wet lab experiments ( Figure 3) . This proves the importance of these types of interactions in the positioning of compounds within the active site. Hence, thermodynamics as well as in silico study reveals that hydrophobic interactions favor binding of these proposed inhibitors with papain like cysteine proteases. Further wet lab results proposed the non competitive interaction of compounds (3a, 3c & 3d) with papain except for compound 3b which showed competitive interaction. In sum up, the above results of molecular docking studies and thermodynamic analysis of compounds 3a-d with papain showed that these compounds have the potential to be novel and unique cysteine protease inhibitors. In the current study, the cysteine protease inhibitory activity of synthesized derivatives of 1-substituted pyridylimidazo[1,5-a] pyridine (3a-d)) was also performed against papain and the inhibition constants (K i ) for the above said enzyme were observed to be 13.70, 23.20, 90.00 and 99.30 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Furthermore, the calculated IC 50 values were also found to be 13.40, 21.17, 94.50 and 96.50 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Except compound 3b, rest of the compounds showed non competitive, reversible inhibitions but all the compounds irrespective of types of binding, showed hydrophobic and entropically driven interaction. These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii). Here, we are showing the data of only four compounds (3a-d) because of their significant results ( Table 4 ). All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) . Since compound 3c & 3d do not have much difference in their IC50 values (3c-94.5 mM and 3d-96.5 mM) against cysteine protease, papain and hence in antibacterial activity in all instances except one. It might be random due to so close in IC50 values. Compounds 3c & 3d are having much difference in their IC50 values (3b-21.17 mM and 3c-94.5 mM) and they showed exact pattern for their antibacterial activity for all microbes except for E. coli at one instance. Although, E. coli does contain six major cysteine proteases but none belong to the CA clan of papain. It is argued that these compounds also inhibited the cysteine proteases of other clan than papain but with low efficacy. Since, pyridylimidazo[1,5-a]pyridine derivatives is absolutely new scaffold towards antibacterial agents and hence, not any standard compound(s) of same scaffold is available for reference. So, Clotrimazole (1-[(2-chlorophenyl)(diphenyl)methyl]-1H-imidazole), an imidazole derivatives and Ceftriaxone (third-generation cephalosporin antibiotic with broad spectrum activity against Gram-positive and Gram-negative bacteria) have been used as positive control whereas DMSO has been used as negative control. All the above mentioned bacterial species have been shown to secrete certain cysteine proteases which play very important role in the pathogenecity of different diseases caused by these microorganisms. The minimum inhibitory concentration (MICs) of compounds (3a-d) ( Table 4 ) against all tested bacteria except E. coli and P. vulgaris, were observed to be in great agreement with their respective inhibition constant (K i )/IC 50 values against papain (Table 3 ) which clearly indicates that these compounds have the potential to inhibit the papain like cysteine proteases of these pathogens. The partition coefficient (logP) is a well-established measure of the compound's lipophilicity. The distribution of calculated logP (cLogP) values of a majority of drugs in the market is in the range of zero to five. All the compounds studied except 3d, showed good agreement with the criteria laid down for the prediction of a compound to be a potential drug ( Table 5 ). All the compounds do not show any threat against toxicity risk assessment except compound 3d which showed threat as tumorogenic effect due to the presence of isobutyl group. Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally. File S1 Types of inhibitions with Ki (Compounds 3a-3d). (DOC)

What could be novel candidates as potent inhibitors of papain like cysteine proteases in resistant microorganisms?

1-substituted pyridylimidazo[1,5-a]pyridine derivatives

RNAi Therapeutic Platforms for Lung Diseases https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816685/ Fujita, Yu; Takeshita, Fumitaka; Kuwano, Kazuyoshi; Ochiya, Takahiro 2013-02-06 DOI:10.3390/ph6020223 License:cc-by Abstract: RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, i.e. small interfering RNAs (siRNAs) and microRNAs (miRNAs), are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field [22, 34] ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. (b) Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer (Penn-Century, Philadelphia, PA, USA) and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally [22, 35, 36 ]. An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [37] , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus (RSV) infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products [17, 38] (see Section 3.1.1. for details). Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application [31] . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers (MDIs) and dry powder inhalers (DPIs) are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (COPD), and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons (CFCs). Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin [39] and low-molecular-weight heparin [40] ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated [24, 25] . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells [41, 42] (Figure 2) . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract [23] . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance [43] . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function [44] . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers ( Figure 3 ). For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute (Ago)2/RNA-induced silencing complex (RISC), which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance [45] . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs [46] . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis [47] . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery [48, 49] . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches [50] . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model [51] and to induce RNAi in a range of animal tissues [52] . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 (NUPR1) in vivo, which resulted in inhibited tumor growth [53] . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications [54] . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem [55] . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function [56] . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers (e.g., charge and size). After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex (RISC) to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity [57] . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo [58] . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT [30] , Oligofectamine [59] , Lipofectamine [60] and TransIT-TKO [35] . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine (PEI) is widely used for siRNA delivery [13, 61] . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses [62, 63] . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses [64, 65] . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response [66] . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene [67, 68] . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs [15, 16, 20, 31] . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs [16, 35] . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used [17, 38] . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs (ALN-RSV01) that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial [69, 70] . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 [71] . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials [57, 72] . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally [73] . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics [74] . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza [13] , parainfluenza virus [35] , severe acute respiratory syndrome (SARS) [14] , and RSV [35] . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations [75] . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection [35] . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated [38] . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection [76] . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 [9] , Wilms tumor 1 (WT1) [12] , overexpressed genes such as the insulin-like growth factor receptor 1 (IGF-1R) [77] , NUPR1 [53] and EZH2 [78] . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 (PKN3), prevented lung metastasis in a phase I trial of various cancer models [79] . PKN3 is a downstream effector of the phosphoinositide 3-kinase (PI3K) signaling pathway [80] , which regulates diverse cellular responses, including development, growth, and survival [81] . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration [79] . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components [82] . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD [83] . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening [84] . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD [85] . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD [86, 87] . Polymorphisms in multiple genes have been reported to be associated with COPD [87] , such as transcription factor [e.g. nuclear factor-kappa B (NFκB)] [88] , extracellular matrix (e.g., matrix metalloproteinase-12 (MMP-12)) [89, 90] , cytokines [e.g. tumor necrosis factor (TNF)-α] [91] , chemokines [e.g. interleukins (IL)-8, IL-8 receptor and chemokine receptor (CCR)1] [92, 93] , and apoptosis (e.g., caspase-3 and vascular endothelial growth factor (VEGF)) [94, 95] . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 (IKK2) inhibitors [96, 97] , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease [98] . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines (IL-4, IL5, and IL-13), cytokine and chemokine receptors (IL-4 receptor and CCR3), and tyrosine kinases [spleen tyrosine kinase (Syk) and LCK/YES-related novel tyrosine kinase (Lyn)], as well as for transcription factors [signal transducers and activators of transcription 1 (STAT1), STAT6, GATA3, and NFκB] that are involved in asthma [19, 99, 100] . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported [101] . Currently, in a clinical trial for asthma, Excellair TM (ZaBeCor, Bala Cynwyd, PA, USA), a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation [102] . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells [103] . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region (UTR) of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs [104] . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies [105] . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD [116, 117] , pulmonary fibrosis [118] [119] [120] [121] , and asthma [122] [123] [124] [125] (Table 3) . [130] [117, 129] The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation [131] . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs [132] , and there are some reports about smoking-related miRNAs [117, 129, 130] . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories [127] . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD [126] . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history [133] . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β (TGF-β) and other cytokines produced at the lesion of active fibrosis [128] . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis (IPF) resulted in increased collagen deposition and alveolar septal thickening [119] . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis [118] . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 (Th2)-type CD4+T lymphocytes against inhaled allergens [134] . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs [123] . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 (TLR4)-induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype [124] . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling [122] . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression [135] . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer [136] . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects [137] . There are two strategies as the therapeutic applications of miRNAs for lung cancer [138] . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene [139] . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising [106, 140, 141] . Another miRNA that shows the value of miRNA replacement is provided by miR-34a [107, 142] . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 [143] . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells [138] . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated [107, 141] . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid (LNA), would increase oligo stability against nucleases [144] . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" [144, 145] . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates [145] , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth [146] . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged [147] . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis [108, 148] . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.

What method is useful in administering small molecules for systemic delivery to the body?

Intranasal

RNAi Therapeutic Platforms for Lung Diseases https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816685/ Fujita, Yu; Takeshita, Fumitaka; Kuwano, Kazuyoshi; Ochiya, Takahiro 2013-02-06 DOI:10.3390/ph6020223 License:cc-by Abstract: RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, i.e. small interfering RNAs (siRNAs) and microRNAs (miRNAs), are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field [22, 34] ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. (b) Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer (Penn-Century, Philadelphia, PA, USA) and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally [22, 35, 36 ]. An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [37] , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus (RSV) infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products [17, 38] (see Section 3.1.1. for details). Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application [31] . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers (MDIs) and dry powder inhalers (DPIs) are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (COPD), and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons (CFCs). Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin [39] and low-molecular-weight heparin [40] ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated [24, 25] . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells [41, 42] (Figure 2) . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract [23] . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance [43] . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function [44] . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers ( Figure 3 ). For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute (Ago)2/RNA-induced silencing complex (RISC), which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance [45] . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs [46] . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis [47] . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery [48, 49] . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches [50] . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model [51] and to induce RNAi in a range of animal tissues [52] . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 (NUPR1) in vivo, which resulted in inhibited tumor growth [53] . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications [54] . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem [55] . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function [56] . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers (e.g., charge and size). After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex (RISC) to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity [57] . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo [58] . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT [30] , Oligofectamine [59] , Lipofectamine [60] and TransIT-TKO [35] . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine (PEI) is widely used for siRNA delivery [13, 61] . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses [62, 63] . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses [64, 65] . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response [66] . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene [67, 68] . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs [15, 16, 20, 31] . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs [16, 35] . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used [17, 38] . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs (ALN-RSV01) that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial [69, 70] . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 [71] . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials [57, 72] . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally [73] . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics [74] . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza [13] , parainfluenza virus [35] , severe acute respiratory syndrome (SARS) [14] , and RSV [35] . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations [75] . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection [35] . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated [38] . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection [76] . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 [9] , Wilms tumor 1 (WT1) [12] , overexpressed genes such as the insulin-like growth factor receptor 1 (IGF-1R) [77] , NUPR1 [53] and EZH2 [78] . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 (PKN3), prevented lung metastasis in a phase I trial of various cancer models [79] . PKN3 is a downstream effector of the phosphoinositide 3-kinase (PI3K) signaling pathway [80] , which regulates diverse cellular responses, including development, growth, and survival [81] . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration [79] . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components [82] . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD [83] . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening [84] . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD [85] . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD [86, 87] . Polymorphisms in multiple genes have been reported to be associated with COPD [87] , such as transcription factor [e.g. nuclear factor-kappa B (NFκB)] [88] , extracellular matrix (e.g., matrix metalloproteinase-12 (MMP-12)) [89, 90] , cytokines [e.g. tumor necrosis factor (TNF)-α] [91] , chemokines [e.g. interleukins (IL)-8, IL-8 receptor and chemokine receptor (CCR)1] [92, 93] , and apoptosis (e.g., caspase-3 and vascular endothelial growth factor (VEGF)) [94, 95] . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 (IKK2) inhibitors [96, 97] , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease [98] . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines (IL-4, IL5, and IL-13), cytokine and chemokine receptors (IL-4 receptor and CCR3), and tyrosine kinases [spleen tyrosine kinase (Syk) and LCK/YES-related novel tyrosine kinase (Lyn)], as well as for transcription factors [signal transducers and activators of transcription 1 (STAT1), STAT6, GATA3, and NFκB] that are involved in asthma [19, 99, 100] . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported [101] . Currently, in a clinical trial for asthma, Excellair TM (ZaBeCor, Bala Cynwyd, PA, USA), a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation [102] . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells [103] . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region (UTR) of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs [104] . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies [105] . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD [116, 117] , pulmonary fibrosis [118] [119] [120] [121] , and asthma [122] [123] [124] [125] (Table 3) . [130] [117, 129] The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation [131] . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs [132] , and there are some reports about smoking-related miRNAs [117, 129, 130] . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories [127] . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD [126] . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history [133] . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β (TGF-β) and other cytokines produced at the lesion of active fibrosis [128] . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis (IPF) resulted in increased collagen deposition and alveolar septal thickening [119] . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis [118] . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 (Th2)-type CD4+T lymphocytes against inhaled allergens [134] . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs [123] . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 (TLR4)-induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype [124] . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling [122] . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression [135] . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer [136] . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects [137] . There are two strategies as the therapeutic applications of miRNAs for lung cancer [138] . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene [139] . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising [106, 140, 141] . Another miRNA that shows the value of miRNA replacement is provided by miR-34a [107, 142] . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 [143] . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells [138] . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated [107, 141] . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid (LNA), would increase oligo stability against nucleases [144] . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" [144, 145] . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates [145] , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth [146] . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged [147] . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis [108, 148] . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.

Why is the nasal mucosa useful in the delivery of small molecules into the body?

the surface area can result in rapid absorption of the medication into the blood

RNAi Therapeutic Platforms for Lung Diseases https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816685/ Fujita, Yu; Takeshita, Fumitaka; Kuwano, Kazuyoshi; Ochiya, Takahiro 2013-02-06 DOI:10.3390/ph6020223 License:cc-by Abstract: RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, i.e. small interfering RNAs (siRNAs) and microRNAs (miRNAs), are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field [22, 34] ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. (b) Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer (Penn-Century, Philadelphia, PA, USA) and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally [22, 35, 36 ]. An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [37] , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus (RSV) infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products [17, 38] (see Section 3.1.1. for details). Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application [31] . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers (MDIs) and dry powder inhalers (DPIs) are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (COPD), and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons (CFCs). Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin [39] and low-molecular-weight heparin [40] ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated [24, 25] . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells [41, 42] (Figure 2) . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract [23] . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance [43] . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function [44] . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers ( Figure 3 ). For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute (Ago)2/RNA-induced silencing complex (RISC), which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance [45] . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs [46] . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis [47] . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery [48, 49] . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches [50] . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model [51] and to induce RNAi in a range of animal tissues [52] . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 (NUPR1) in vivo, which resulted in inhibited tumor growth [53] . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications [54] . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem [55] . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function [56] . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers (e.g., charge and size). After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex (RISC) to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity [57] . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo [58] . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT [30] , Oligofectamine [59] , Lipofectamine [60] and TransIT-TKO [35] . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine (PEI) is widely used for siRNA delivery [13, 61] . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses [62, 63] . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses [64, 65] . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response [66] . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene [67, 68] . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs [15, 16, 20, 31] . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs [16, 35] . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used [17, 38] . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs (ALN-RSV01) that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial [69, 70] . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 [71] . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials [57, 72] . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally [73] . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics [74] . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza [13] , parainfluenza virus [35] , severe acute respiratory syndrome (SARS) [14] , and RSV [35] . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations [75] . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection [35] . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated [38] . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection [76] . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 [9] , Wilms tumor 1 (WT1) [12] , overexpressed genes such as the insulin-like growth factor receptor 1 (IGF-1R) [77] , NUPR1 [53] and EZH2 [78] . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 (PKN3), prevented lung metastasis in a phase I trial of various cancer models [79] . PKN3 is a downstream effector of the phosphoinositide 3-kinase (PI3K) signaling pathway [80] , which regulates diverse cellular responses, including development, growth, and survival [81] . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration [79] . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components [82] . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD [83] . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening [84] . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD [85] . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD [86, 87] . Polymorphisms in multiple genes have been reported to be associated with COPD [87] , such as transcription factor [e.g. nuclear factor-kappa B (NFκB)] [88] , extracellular matrix (e.g., matrix metalloproteinase-12 (MMP-12)) [89, 90] , cytokines [e.g. tumor necrosis factor (TNF)-α] [91] , chemokines [e.g. interleukins (IL)-8, IL-8 receptor and chemokine receptor (CCR)1] [92, 93] , and apoptosis (e.g., caspase-3 and vascular endothelial growth factor (VEGF)) [94, 95] . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 (IKK2) inhibitors [96, 97] , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease [98] . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines (IL-4, IL5, and IL-13), cytokine and chemokine receptors (IL-4 receptor and CCR3), and tyrosine kinases [spleen tyrosine kinase (Syk) and LCK/YES-related novel tyrosine kinase (Lyn)], as well as for transcription factors [signal transducers and activators of transcription 1 (STAT1), STAT6, GATA3, and NFκB] that are involved in asthma [19, 99, 100] . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported [101] . Currently, in a clinical trial for asthma, Excellair TM (ZaBeCor, Bala Cynwyd, PA, USA), a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation [102] . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells [103] . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region (UTR) of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs [104] . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies [105] . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD [116, 117] , pulmonary fibrosis [118] [119] [120] [121] , and asthma [122] [123] [124] [125] (Table 3) . [130] [117, 129] The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation [131] . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs [132] , and there are some reports about smoking-related miRNAs [117, 129, 130] . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories [127] . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD [126] . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history [133] . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β (TGF-β) and other cytokines produced at the lesion of active fibrosis [128] . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis (IPF) resulted in increased collagen deposition and alveolar septal thickening [119] . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis [118] . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 (Th2)-type CD4+T lymphocytes against inhaled allergens [134] . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs [123] . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 (TLR4)-induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype [124] . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling [122] . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression [135] . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer [136] . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects [137] . There are two strategies as the therapeutic applications of miRNAs for lung cancer [138] . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene [139] . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising [106, 140, 141] . Another miRNA that shows the value of miRNA replacement is provided by miR-34a [107, 142] . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 [143] . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells [138] . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated [107, 141] . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid (LNA), would increase oligo stability against nucleases [144] . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" [144, 145] . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates [145] , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth [146] . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged [147] . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis [108, 148] . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.

What are the most common methods of inhaled delivery of medications?

Metered dose inhalers (MDIs) and dry powder inhalers (DPIs)

RNAi Therapeutic Platforms for Lung Diseases https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816685/ Fujita, Yu; Takeshita, Fumitaka; Kuwano, Kazuyoshi; Ochiya, Takahiro 2013-02-06 DOI:10.3390/ph6020223 License:cc-by Abstract: RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, i.e. small interfering RNAs (siRNAs) and microRNAs (miRNAs), are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field [22, 34] ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. (b) Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer (Penn-Century, Philadelphia, PA, USA) and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally [22, 35, 36 ]. An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [37] , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus (RSV) infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products [17, 38] (see Section 3.1.1. for details). Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application [31] . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers (MDIs) and dry powder inhalers (DPIs) are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (COPD), and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons (CFCs). Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin [39] and low-molecular-weight heparin [40] ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated [24, 25] . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells [41, 42] (Figure 2) . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract [23] . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance [43] . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function [44] . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers ( Figure 3 ). For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute (Ago)2/RNA-induced silencing complex (RISC), which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance [45] . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs [46] . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis [47] . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery [48, 49] . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches [50] . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model [51] and to induce RNAi in a range of animal tissues [52] . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 (NUPR1) in vivo, which resulted in inhibited tumor growth [53] . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications [54] . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem [55] . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function [56] . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers (e.g., charge and size). After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex (RISC) to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity [57] . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo [58] . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT [30] , Oligofectamine [59] , Lipofectamine [60] and TransIT-TKO [35] . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine (PEI) is widely used for siRNA delivery [13, 61] . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses [62, 63] . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses [64, 65] . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response [66] . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene [67, 68] . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs [15, 16, 20, 31] . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs [16, 35] . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used [17, 38] . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs (ALN-RSV01) that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial [69, 70] . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 [71] . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials [57, 72] . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally [73] . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics [74] . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza [13] , parainfluenza virus [35] , severe acute respiratory syndrome (SARS) [14] , and RSV [35] . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations [75] . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection [35] . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated [38] . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection [76] . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 [9] , Wilms tumor 1 (WT1) [12] , overexpressed genes such as the insulin-like growth factor receptor 1 (IGF-1R) [77] , NUPR1 [53] and EZH2 [78] . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 (PKN3), prevented lung metastasis in a phase I trial of various cancer models [79] . PKN3 is a downstream effector of the phosphoinositide 3-kinase (PI3K) signaling pathway [80] , which regulates diverse cellular responses, including development, growth, and survival [81] . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration [79] . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components [82] . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD [83] . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening [84] . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD [85] . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD [86, 87] . Polymorphisms in multiple genes have been reported to be associated with COPD [87] , such as transcription factor [e.g. nuclear factor-kappa B (NFκB)] [88] , extracellular matrix (e.g., matrix metalloproteinase-12 (MMP-12)) [89, 90] , cytokines [e.g. tumor necrosis factor (TNF)-α] [91] , chemokines [e.g. interleukins (IL)-8, IL-8 receptor and chemokine receptor (CCR)1] [92, 93] , and apoptosis (e.g., caspase-3 and vascular endothelial growth factor (VEGF)) [94, 95] . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 (IKK2) inhibitors [96, 97] , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease [98] . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines (IL-4, IL5, and IL-13), cytokine and chemokine receptors (IL-4 receptor and CCR3), and tyrosine kinases [spleen tyrosine kinase (Syk) and LCK/YES-related novel tyrosine kinase (Lyn)], as well as for transcription factors [signal transducers and activators of transcription 1 (STAT1), STAT6, GATA3, and NFκB] that are involved in asthma [19, 99, 100] . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported [101] . Currently, in a clinical trial for asthma, Excellair TM (ZaBeCor, Bala Cynwyd, PA, USA), a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation [102] . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells [103] . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region (UTR) of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs [104] . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies [105] . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD [116, 117] , pulmonary fibrosis [118] [119] [120] [121] , and asthma [122] [123] [124] [125] (Table 3) . [130] [117, 129] The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation [131] . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs [132] , and there are some reports about smoking-related miRNAs [117, 129, 130] . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories [127] . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD [126] . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history [133] . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β (TGF-β) and other cytokines produced at the lesion of active fibrosis [128] . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis (IPF) resulted in increased collagen deposition and alveolar septal thickening [119] . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis [118] . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 (Th2)-type CD4+T lymphocytes against inhaled allergens [134] . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs [123] . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 (TLR4)-induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype [124] . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling [122] . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression [135] . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer [136] . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects [137] . There are two strategies as the therapeutic applications of miRNAs for lung cancer [138] . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene [139] . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising [106, 140, 141] . Another miRNA that shows the value of miRNA replacement is provided by miR-34a [107, 142] . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 [143] . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells [138] . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated [107, 141] . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid (LNA), would increase oligo stability against nucleases [144] . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" [144, 145] . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates [145] , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth [146] . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged [147] . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis [108, 148] . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.

What medications have shown good promise to in vivo delivery via dry powder inhalers?

insulin [39] and low-molecular-weight heparin [40]

RNAi Therapeutic Platforms for Lung Diseases https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816685/ Fujita, Yu; Takeshita, Fumitaka; Kuwano, Kazuyoshi; Ochiya, Takahiro 2013-02-06 DOI:10.3390/ph6020223 License:cc-by Abstract: RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, i.e. small interfering RNAs (siRNAs) and microRNAs (miRNAs), are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field [22, 34] ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. (b) Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer (Penn-Century, Philadelphia, PA, USA) and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally [22, 35, 36 ]. An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [37] , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus (RSV) infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products [17, 38] (see Section 3.1.1. for details). Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application [31] . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers (MDIs) and dry powder inhalers (DPIs) are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (COPD), and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons (CFCs). Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin [39] and low-molecular-weight heparin [40] ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated [24, 25] . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells [41, 42] (Figure 2) . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract [23] . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance [43] . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function [44] . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers ( Figure 3 ). For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute (Ago)2/RNA-induced silencing complex (RISC), which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance [45] . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs [46] . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis [47] . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery [48, 49] . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches [50] . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model [51] and to induce RNAi in a range of animal tissues [52] . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 (NUPR1) in vivo, which resulted in inhibited tumor growth [53] . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications [54] . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem [55] . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function [56] . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers (e.g., charge and size). After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex (RISC) to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity [57] . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo [58] . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT [30] , Oligofectamine [59] , Lipofectamine [60] and TransIT-TKO [35] . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine (PEI) is widely used for siRNA delivery [13, 61] . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses [62, 63] . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses [64, 65] . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response [66] . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene [67, 68] . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs [15, 16, 20, 31] . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs [16, 35] . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used [17, 38] . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs (ALN-RSV01) that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial [69, 70] . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 [71] . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials [57, 72] . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally [73] . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics [74] . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza [13] , parainfluenza virus [35] , severe acute respiratory syndrome (SARS) [14] , and RSV [35] . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations [75] . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection [35] . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated [38] . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection [76] . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 [9] , Wilms tumor 1 (WT1) [12] , overexpressed genes such as the insulin-like growth factor receptor 1 (IGF-1R) [77] , NUPR1 [53] and EZH2 [78] . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 (PKN3), prevented lung metastasis in a phase I trial of various cancer models [79] . PKN3 is a downstream effector of the phosphoinositide 3-kinase (PI3K) signaling pathway [80] , which regulates diverse cellular responses, including development, growth, and survival [81] . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration [79] . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components [82] . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD [83] . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening [84] . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD [85] . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD [86, 87] . Polymorphisms in multiple genes have been reported to be associated with COPD [87] , such as transcription factor [e.g. nuclear factor-kappa B (NFκB)] [88] , extracellular matrix (e.g., matrix metalloproteinase-12 (MMP-12)) [89, 90] , cytokines [e.g. tumor necrosis factor (TNF)-α] [91] , chemokines [e.g. interleukins (IL)-8, IL-8 receptor and chemokine receptor (CCR)1] [92, 93] , and apoptosis (e.g., caspase-3 and vascular endothelial growth factor (VEGF)) [94, 95] . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 (IKK2) inhibitors [96, 97] , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease [98] . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines (IL-4, IL5, and IL-13), cytokine and chemokine receptors (IL-4 receptor and CCR3), and tyrosine kinases [spleen tyrosine kinase (Syk) and LCK/YES-related novel tyrosine kinase (Lyn)], as well as for transcription factors [signal transducers and activators of transcription 1 (STAT1), STAT6, GATA3, and NFκB] that are involved in asthma [19, 99, 100] . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported [101] . Currently, in a clinical trial for asthma, Excellair TM (ZaBeCor, Bala Cynwyd, PA, USA), a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation [102] . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells [103] . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region (UTR) of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs [104] . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies [105] . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD [116, 117] , pulmonary fibrosis [118] [119] [120] [121] , and asthma [122] [123] [124] [125] (Table 3) . [130] [117, 129] The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation [131] . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs [132] , and there are some reports about smoking-related miRNAs [117, 129, 130] . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories [127] . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD [126] . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history [133] . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β (TGF-β) and other cytokines produced at the lesion of active fibrosis [128] . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis (IPF) resulted in increased collagen deposition and alveolar septal thickening [119] . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis [118] . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 (Th2)-type CD4+T lymphocytes against inhaled allergens [134] . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs [123] . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 (TLR4)-induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype [124] . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling [122] . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression [135] . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer [136] . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects [137] . There are two strategies as the therapeutic applications of miRNAs for lung cancer [138] . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene [139] . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising [106, 140, 141] . Another miRNA that shows the value of miRNA replacement is provided by miR-34a [107, 142] . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 [143] . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells [138] . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated [107, 141] . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid (LNA), would increase oligo stability against nucleases [144] . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" [144, 145] . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates [145] , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth [146] . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged [147] . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis [108, 148] . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.

How are siRNAs typically delivered for systemic effect?

intratracheal or intranasal delivery

RNAi Therapeutic Platforms for Lung Diseases https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816685/ Fujita, Yu; Takeshita, Fumitaka; Kuwano, Kazuyoshi; Ochiya, Takahiro 2013-02-06 DOI:10.3390/ph6020223 License:cc-by Abstract: RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, i.e. small interfering RNAs (siRNAs) and microRNAs (miRNAs), are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field [22, 34] ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. (b) Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer (Penn-Century, Philadelphia, PA, USA) and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally [22, 35, 36 ]. An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [37] , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus (RSV) infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products [17, 38] (see Section 3.1.1. for details). Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application [31] . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers (MDIs) and dry powder inhalers (DPIs) are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (COPD), and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons (CFCs). Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin [39] and low-molecular-weight heparin [40] ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated [24, 25] . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells [41, 42] (Figure 2) . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract [23] . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance [43] . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function [44] . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers ( Figure 3 ). For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute (Ago)2/RNA-induced silencing complex (RISC), which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance [45] . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs [46] . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis [47] . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery [48, 49] . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches [50] . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model [51] and to induce RNAi in a range of animal tissues [52] . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 (NUPR1) in vivo, which resulted in inhibited tumor growth [53] . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications [54] . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem [55] . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function [56] . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers (e.g., charge and size). After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex (RISC) to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity [57] . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo [58] . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT [30] , Oligofectamine [59] , Lipofectamine [60] and TransIT-TKO [35] . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine (PEI) is widely used for siRNA delivery [13, 61] . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses [62, 63] . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses [64, 65] . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response [66] . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene [67, 68] . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs [15, 16, 20, 31] . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs [16, 35] . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used [17, 38] . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs (ALN-RSV01) that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial [69, 70] . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 [71] . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials [57, 72] . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally [73] . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics [74] . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza [13] , parainfluenza virus [35] , severe acute respiratory syndrome (SARS) [14] , and RSV [35] . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations [75] . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection [35] . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated [38] . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection [76] . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 [9] , Wilms tumor 1 (WT1) [12] , overexpressed genes such as the insulin-like growth factor receptor 1 (IGF-1R) [77] , NUPR1 [53] and EZH2 [78] . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 (PKN3), prevented lung metastasis in a phase I trial of various cancer models [79] . PKN3 is a downstream effector of the phosphoinositide 3-kinase (PI3K) signaling pathway [80] , which regulates diverse cellular responses, including development, growth, and survival [81] . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration [79] . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components [82] . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD [83] . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening [84] . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD [85] . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD [86, 87] . Polymorphisms in multiple genes have been reported to be associated with COPD [87] , such as transcription factor [e.g. nuclear factor-kappa B (NFκB)] [88] , extracellular matrix (e.g., matrix metalloproteinase-12 (MMP-12)) [89, 90] , cytokines [e.g. tumor necrosis factor (TNF)-α] [91] , chemokines [e.g. interleukins (IL)-8, IL-8 receptor and chemokine receptor (CCR)1] [92, 93] , and apoptosis (e.g., caspase-3 and vascular endothelial growth factor (VEGF)) [94, 95] . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 (IKK2) inhibitors [96, 97] , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease [98] . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines (IL-4, IL5, and IL-13), cytokine and chemokine receptors (IL-4 receptor and CCR3), and tyrosine kinases [spleen tyrosine kinase (Syk) and LCK/YES-related novel tyrosine kinase (Lyn)], as well as for transcription factors [signal transducers and activators of transcription 1 (STAT1), STAT6, GATA3, and NFκB] that are involved in asthma [19, 99, 100] . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported [101] . Currently, in a clinical trial for asthma, Excellair TM (ZaBeCor, Bala Cynwyd, PA, USA), a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation [102] . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells [103] . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region (UTR) of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs [104] . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies [105] . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD [116, 117] , pulmonary fibrosis [118] [119] [120] [121] , and asthma [122] [123] [124] [125] (Table 3) . [130] [117, 129] The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation [131] . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs [132] , and there are some reports about smoking-related miRNAs [117, 129, 130] . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories [127] . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD [126] . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history [133] . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β (TGF-β) and other cytokines produced at the lesion of active fibrosis [128] . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis (IPF) resulted in increased collagen deposition and alveolar septal thickening [119] . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis [118] . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 (Th2)-type CD4+T lymphocytes against inhaled allergens [134] . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs [123] . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 (TLR4)-induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype [124] . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling [122] . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression [135] . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer [136] . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects [137] . There are two strategies as the therapeutic applications of miRNAs for lung cancer [138] . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene [139] . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising [106, 140, 141] . Another miRNA that shows the value of miRNA replacement is provided by miR-34a [107, 142] . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 [143] . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells [138] . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated [107, 141] . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid (LNA), would increase oligo stability against nucleases [144] . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" [144, 145] . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates [145] , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth [146] . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged [147] . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis [108, 148] . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.

What structures form the human airway?

respiratory bronchioles, alveolar ducts, and alveolar sacs

RNAi Therapeutic Platforms for Lung Diseases https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816685/ Fujita, Yu; Takeshita, Fumitaka; Kuwano, Kazuyoshi; Ochiya, Takahiro 2013-02-06 DOI:10.3390/ph6020223 License:cc-by Abstract: RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, i.e. small interfering RNAs (siRNAs) and microRNAs (miRNAs), are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field [22, 34] ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. (b) Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer (Penn-Century, Philadelphia, PA, USA) and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally [22, 35, 36 ]. An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [37] , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus (RSV) infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products [17, 38] (see Section 3.1.1. for details). Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application [31] . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers (MDIs) and dry powder inhalers (DPIs) are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (COPD), and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons (CFCs). Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin [39] and low-molecular-weight heparin [40] ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated [24, 25] . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells [41, 42] (Figure 2) . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract [23] . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance [43] . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function [44] . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers ( Figure 3 ). For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute (Ago)2/RNA-induced silencing complex (RISC), which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance [45] . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs [46] . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis [47] . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery [48, 49] . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches [50] . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model [51] and to induce RNAi in a range of animal tissues [52] . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 (NUPR1) in vivo, which resulted in inhibited tumor growth [53] . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications [54] . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem [55] . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function [56] . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers (e.g., charge and size). After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex (RISC) to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity [57] . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo [58] . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT [30] , Oligofectamine [59] , Lipofectamine [60] and TransIT-TKO [35] . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine (PEI) is widely used for siRNA delivery [13, 61] . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses [62, 63] . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses [64, 65] . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response [66] . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene [67, 68] . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs [15, 16, 20, 31] . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs [16, 35] . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used [17, 38] . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs (ALN-RSV01) that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial [69, 70] . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 [71] . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials [57, 72] . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally [73] . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics [74] . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza [13] , parainfluenza virus [35] , severe acute respiratory syndrome (SARS) [14] , and RSV [35] . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations [75] . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection [35] . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated [38] . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection [76] . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 [9] , Wilms tumor 1 (WT1) [12] , overexpressed genes such as the insulin-like growth factor receptor 1 (IGF-1R) [77] , NUPR1 [53] and EZH2 [78] . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 (PKN3), prevented lung metastasis in a phase I trial of various cancer models [79] . PKN3 is a downstream effector of the phosphoinositide 3-kinase (PI3K) signaling pathway [80] , which regulates diverse cellular responses, including development, growth, and survival [81] . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration [79] . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components [82] . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD [83] . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening [84] . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD [85] . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD [86, 87] . Polymorphisms in multiple genes have been reported to be associated with COPD [87] , such as transcription factor [e.g. nuclear factor-kappa B (NFκB)] [88] , extracellular matrix (e.g., matrix metalloproteinase-12 (MMP-12)) [89, 90] , cytokines [e.g. tumor necrosis factor (TNF)-α] [91] , chemokines [e.g. interleukins (IL)-8, IL-8 receptor and chemokine receptor (CCR)1] [92, 93] , and apoptosis (e.g., caspase-3 and vascular endothelial growth factor (VEGF)) [94, 95] . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 (IKK2) inhibitors [96, 97] , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease [98] . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines (IL-4, IL5, and IL-13), cytokine and chemokine receptors (IL-4 receptor and CCR3), and tyrosine kinases [spleen tyrosine kinase (Syk) and LCK/YES-related novel tyrosine kinase (Lyn)], as well as for transcription factors [signal transducers and activators of transcription 1 (STAT1), STAT6, GATA3, and NFκB] that are involved in asthma [19, 99, 100] . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported [101] . Currently, in a clinical trial for asthma, Excellair TM (ZaBeCor, Bala Cynwyd, PA, USA), a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation [102] . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells [103] . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region (UTR) of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs [104] . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies [105] . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD [116, 117] , pulmonary fibrosis [118] [119] [120] [121] , and asthma [122] [123] [124] [125] (Table 3) . [130] [117, 129] The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation [131] . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs [132] , and there are some reports about smoking-related miRNAs [117, 129, 130] . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories [127] . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD [126] . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history [133] . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β (TGF-β) and other cytokines produced at the lesion of active fibrosis [128] . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis (IPF) resulted in increased collagen deposition and alveolar septal thickening [119] . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis [118] . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 (Th2)-type CD4+T lymphocytes against inhaled allergens [134] . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs [123] . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 (TLR4)-induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype [124] . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling [122] . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression [135] . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer [136] . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects [137] . There are two strategies as the therapeutic applications of miRNAs for lung cancer [138] . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene [139] . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising [106, 140, 141] . Another miRNA that shows the value of miRNA replacement is provided by miR-34a [107, 142] . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 [143] . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells [138] . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated [107, 141] . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid (LNA), would increase oligo stability against nucleases [144] . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" [144, 145] . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates [145] , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth [146] . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged [147] . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis [108, 148] . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.

What size of particle has been shown to be most effective in the delivery to the lower airway?

1-5 μm

RNAi Therapeutic Platforms for Lung Diseases https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816685/ Fujita, Yu; Takeshita, Fumitaka; Kuwano, Kazuyoshi; Ochiya, Takahiro 2013-02-06 DOI:10.3390/ph6020223 License:cc-by Abstract: RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, i.e. small interfering RNAs (siRNAs) and microRNAs (miRNAs), are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field [22, 34] ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. (b) Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer (Penn-Century, Philadelphia, PA, USA) and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally [22, 35, 36 ]. An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [37] , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus (RSV) infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products [17, 38] (see Section 3.1.1. for details). Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application [31] . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers (MDIs) and dry powder inhalers (DPIs) are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (COPD), and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons (CFCs). Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin [39] and low-molecular-weight heparin [40] ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated [24, 25] . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells [41, 42] (Figure 2) . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract [23] . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance [43] . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function [44] . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers ( Figure 3 ). For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute (Ago)2/RNA-induced silencing complex (RISC), which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance [45] . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs [46] . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis [47] . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery [48, 49] . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches [50] . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model [51] and to induce RNAi in a range of animal tissues [52] . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 (NUPR1) in vivo, which resulted in inhibited tumor growth [53] . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications [54] . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem [55] . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function [56] . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers (e.g., charge and size). After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex (RISC) to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity [57] . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo [58] . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT [30] , Oligofectamine [59] , Lipofectamine [60] and TransIT-TKO [35] . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine (PEI) is widely used for siRNA delivery [13, 61] . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses [62, 63] . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses [64, 65] . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response [66] . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene [67, 68] . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs [15, 16, 20, 31] . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs [16, 35] . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used [17, 38] . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs (ALN-RSV01) that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial [69, 70] . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 [71] . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials [57, 72] . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally [73] . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics [74] . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza [13] , parainfluenza virus [35] , severe acute respiratory syndrome (SARS) [14] , and RSV [35] . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations [75] . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection [35] . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated [38] . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection [76] . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 [9] , Wilms tumor 1 (WT1) [12] , overexpressed genes such as the insulin-like growth factor receptor 1 (IGF-1R) [77] , NUPR1 [53] and EZH2 [78] . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 (PKN3), prevented lung metastasis in a phase I trial of various cancer models [79] . PKN3 is a downstream effector of the phosphoinositide 3-kinase (PI3K) signaling pathway [80] , which regulates diverse cellular responses, including development, growth, and survival [81] . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration [79] . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components [82] . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD [83] . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening [84] . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD [85] . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD [86, 87] . Polymorphisms in multiple genes have been reported to be associated with COPD [87] , such as transcription factor [e.g. nuclear factor-kappa B (NFκB)] [88] , extracellular matrix (e.g., matrix metalloproteinase-12 (MMP-12)) [89, 90] , cytokines [e.g. tumor necrosis factor (TNF)-α] [91] , chemokines [e.g. interleukins (IL)-8, IL-8 receptor and chemokine receptor (CCR)1] [92, 93] , and apoptosis (e.g., caspase-3 and vascular endothelial growth factor (VEGF)) [94, 95] . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 (IKK2) inhibitors [96, 97] , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease [98] . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines (IL-4, IL5, and IL-13), cytokine and chemokine receptors (IL-4 receptor and CCR3), and tyrosine kinases [spleen tyrosine kinase (Syk) and LCK/YES-related novel tyrosine kinase (Lyn)], as well as for transcription factors [signal transducers and activators of transcription 1 (STAT1), STAT6, GATA3, and NFκB] that are involved in asthma [19, 99, 100] . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported [101] . Currently, in a clinical trial for asthma, Excellair TM (ZaBeCor, Bala Cynwyd, PA, USA), a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation [102] . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells [103] . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region (UTR) of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs [104] . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies [105] . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD [116, 117] , pulmonary fibrosis [118] [119] [120] [121] , and asthma [122] [123] [124] [125] (Table 3) . [130] [117, 129] The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation [131] . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs [132] , and there are some reports about smoking-related miRNAs [117, 129, 130] . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories [127] . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD [126] . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history [133] . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β (TGF-β) and other cytokines produced at the lesion of active fibrosis [128] . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis (IPF) resulted in increased collagen deposition and alveolar septal thickening [119] . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis [118] . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 (Th2)-type CD4+T lymphocytes against inhaled allergens [134] . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs [123] . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 (TLR4)-induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype [124] . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling [122] . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression [135] . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer [136] . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects [137] . There are two strategies as the therapeutic applications of miRNAs for lung cancer [138] . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene [139] . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising [106, 140, 141] . Another miRNA that shows the value of miRNA replacement is provided by miR-34a [107, 142] . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 [143] . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells [138] . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated [107, 141] . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid (LNA), would increase oligo stability against nucleases [144] . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" [144, 145] . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates [145] , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth [146] . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged [147] . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis [108, 148] . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration

Complete Genome Sequence of a Nephropathogenic Infectious Bronchitis Virus Strain Isolated in China https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795213/ SHA: f2df4fc3c60338755fd23da3d7e01c0455e20745 Authors: Yang, Jing-tian; Ma, Bing-cun Date: 2013-10-10 DOI: 10.1128/genomea.00815-13 License: cc-by Abstract: Infectious bronchitis virus (IBV) causes tremendous economic losses to the poultry industry. Here, we report the complete genome analysis results for a new natural recombination nephropathogenic IBV strain named SAIBK, which was isolated in the Sichuan province of China in 2005. Text: tagious and acute disease in domestic chickens, belongs to group III of the genus Coronavirus in the family Coronaviridae (1) . It is an enveloped, unsegmented, positive-sense, single-stranded RNA (ssRNA) virus and has a genome of approximately 27.6 kb (2) . Recently, many epidemiological analysis reports have suggested that nephropathogenic IBVs have become increasingly prevalent (3) (4) (5) (6) in China. In this work, the complete genome sequence of an isolate named SAIBK was analyzed and recombination was detected between SAIBK and some previously reported IBVs. A rapid amplification of cDNA ends (RACE) kit (TaKaRa, Japan) was used to obtain the 5= and 3= ends of the genome. Other parts were amplified by 19 primers with overlap between each fragment and were cloned into the pMD19-T vector (TaKaRa, Japan). All fragments were sequenced three times by Sangon Biotech (Shanghai, China). The sequenced fragments were assembled using the SeqMan software program (DNAStar, Inc.). Sequence alignment was conducted and a phylogenetic tree was constructed using the software program MEGA5 (7). Recombination analysis was performed using the RDP 4.14 (8) and SimPlot 3.5.1 (9) software programs. The complete genome of the SAIBK strain is 27,534 nucleotides (nt) in length, including the poly(A) tail. It has a classical IBV genome organization with 10 open reading frames (ORFs): The genome sequence of SAIBK shows the highest identity (94.3%) to the Chinese IBV strain SC021202 (GenBank accession no. EU714029) and the lowest identity (85.8%) to two Chinese IBV strains, BJ (GenBank accession no. AY319651) and DY07 (GenBank accession no. HM245923). It has lower nucleotide identities of 88.1%, 87.9%, and 87.7% to the most popularly used IBV vaccine strains, H120, H52, and M41, respectively. Phylogenetic analysis of the complete genome results indicated that the SAIBK strain clusters into the same branch as the IBV YN strain (GenBank accession no. JF893452) and the SC021202 strain (GenBank accession no. EU714029). The S1 subunit of the IBV genome is the major determinant of serotype (10) (11) (12) (13) , and S1 analysis indicated that the SAIBK strain has a 4/91-like serotype. The employed recombination detection methods revealed that SAIBK is a chimera virus, with recombination by the SC021202 strain as a major parent and the H120 vaccine strain as a minor parent. The first and second recombination regions were located at positions 7231 to 9126 and 13437 to 14473 in genes 1a and 1b, respectively. There were two other recombination regions detected at positions 951 to 1067 and 5393 to 5605 of SAIBK, which were recombined with the SC021202 strain as a major parent and the H52 vaccine strain as a minor parent. The recombination detection results suggested that SAIBK is possibly a chimera virus derived from the popularly used vaccine strains H120 and H52 and the field strain SC021202, and the SC021202 strain was isolated from chickens vaccinated with H120 in the Sichuan province of China in 2003 (14) . This result revealed that the field IBVs in Sichuan Province have undergone genetic recombination and are possibly emerging as new mutant strains, such as SAIBK. Nucleotide sequence accession number. The complete genome sequence of the SAIBK isolate was submitted to GenBank and assigned the accession no. DQ288927.

How long is the SAIBK gene?

27,534 nucleotides

Complete Genome Sequence of a Nephropathogenic Infectious Bronchitis Virus Strain Isolated in China https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795213/ SHA: f2df4fc3c60338755fd23da3d7e01c0455e20745 Authors: Yang, Jing-tian; Ma, Bing-cun Date: 2013-10-10 DOI: 10.1128/genomea.00815-13 License: cc-by Abstract: Infectious bronchitis virus (IBV) causes tremendous economic losses to the poultry industry. Here, we report the complete genome analysis results for a new natural recombination nephropathogenic IBV strain named SAIBK, which was isolated in the Sichuan province of China in 2005. Text: tagious and acute disease in domestic chickens, belongs to group III of the genus Coronavirus in the family Coronaviridae (1) . It is an enveloped, unsegmented, positive-sense, single-stranded RNA (ssRNA) virus and has a genome of approximately 27.6 kb (2) . Recently, many epidemiological analysis reports have suggested that nephropathogenic IBVs have become increasingly prevalent (3) (4) (5) (6) in China. In this work, the complete genome sequence of an isolate named SAIBK was analyzed and recombination was detected between SAIBK and some previously reported IBVs. A rapid amplification of cDNA ends (RACE) kit (TaKaRa, Japan) was used to obtain the 5= and 3= ends of the genome. Other parts were amplified by 19 primers with overlap between each fragment and were cloned into the pMD19-T vector (TaKaRa, Japan). All fragments were sequenced three times by Sangon Biotech (Shanghai, China). The sequenced fragments were assembled using the SeqMan software program (DNAStar, Inc.). Sequence alignment was conducted and a phylogenetic tree was constructed using the software program MEGA5 (7). Recombination analysis was performed using the RDP 4.14 (8) and SimPlot 3.5.1 (9) software programs. The complete genome of the SAIBK strain is 27,534 nucleotides (nt) in length, including the poly(A) tail. It has a classical IBV genome organization with 10 open reading frames (ORFs): The genome sequence of SAIBK shows the highest identity (94.3%) to the Chinese IBV strain SC021202 (GenBank accession no. EU714029) and the lowest identity (85.8%) to two Chinese IBV strains, BJ (GenBank accession no. AY319651) and DY07 (GenBank accession no. HM245923). It has lower nucleotide identities of 88.1%, 87.9%, and 87.7% to the most popularly used IBV vaccine strains, H120, H52, and M41, respectively. Phylogenetic analysis of the complete genome results indicated that the SAIBK strain clusters into the same branch as the IBV YN strain (GenBank accession no. JF893452) and the SC021202 strain (GenBank accession no. EU714029). The S1 subunit of the IBV genome is the major determinant of serotype (10) (11) (12) (13) , and S1 analysis indicated that the SAIBK strain has a 4/91-like serotype. The employed recombination detection methods revealed that SAIBK is a chimera virus, with recombination by the SC021202 strain as a major parent and the H120 vaccine strain as a minor parent. The first and second recombination regions were located at positions 7231 to 9126 and 13437 to 14473 in genes 1a and 1b, respectively. There were two other recombination regions detected at positions 951 to 1067 and 5393 to 5605 of SAIBK, which were recombined with the SC021202 strain as a major parent and the H52 vaccine strain as a minor parent. The recombination detection results suggested that SAIBK is possibly a chimera virus derived from the popularly used vaccine strains H120 and H52 and the field strain SC021202, and the SC021202 strain was isolated from chickens vaccinated with H120 in the Sichuan province of China in 2003 (14) . This result revealed that the field IBVs in Sichuan Province have undergone genetic recombination and are possibly emerging as new mutant strains, such as SAIBK. Nucleotide sequence accession number. The complete genome sequence of the SAIBK isolate was submitted to GenBank and assigned the accession no. DQ288927.

How many open reading frames are in the SAIBK gene?

10

Complete Genome Sequence of a Nephropathogenic Infectious Bronchitis Virus Strain Isolated in China https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795213/ SHA: f2df4fc3c60338755fd23da3d7e01c0455e20745 Authors: Yang, Jing-tian; Ma, Bing-cun Date: 2013-10-10 DOI: 10.1128/genomea.00815-13 License: cc-by Abstract: Infectious bronchitis virus (IBV) causes tremendous economic losses to the poultry industry. Here, we report the complete genome analysis results for a new natural recombination nephropathogenic IBV strain named SAIBK, which was isolated in the Sichuan province of China in 2005. Text: tagious and acute disease in domestic chickens, belongs to group III of the genus Coronavirus in the family Coronaviridae (1) . It is an enveloped, unsegmented, positive-sense, single-stranded RNA (ssRNA) virus and has a genome of approximately 27.6 kb (2) . Recently, many epidemiological analysis reports have suggested that nephropathogenic IBVs have become increasingly prevalent (3) (4) (5) (6) in China. In this work, the complete genome sequence of an isolate named SAIBK was analyzed and recombination was detected between SAIBK and some previously reported IBVs. A rapid amplification of cDNA ends (RACE) kit (TaKaRa, Japan) was used to obtain the 5= and 3= ends of the genome. Other parts were amplified by 19 primers with overlap between each fragment and were cloned into the pMD19-T vector (TaKaRa, Japan). All fragments were sequenced three times by Sangon Biotech (Shanghai, China). The sequenced fragments were assembled using the SeqMan software program (DNAStar, Inc.). Sequence alignment was conducted and a phylogenetic tree was constructed using the software program MEGA5 (7). Recombination analysis was performed using the RDP 4.14 (8) and SimPlot 3.5.1 (9) software programs. The complete genome of the SAIBK strain is 27,534 nucleotides (nt) in length, including the poly(A) tail. It has a classical IBV genome organization with 10 open reading frames (ORFs): The genome sequence of SAIBK shows the highest identity (94.3%) to the Chinese IBV strain SC021202 (GenBank accession no. EU714029) and the lowest identity (85.8%) to two Chinese IBV strains, BJ (GenBank accession no. AY319651) and DY07 (GenBank accession no. HM245923). It has lower nucleotide identities of 88.1%, 87.9%, and 87.7% to the most popularly used IBV vaccine strains, H120, H52, and M41, respectively. Phylogenetic analysis of the complete genome results indicated that the SAIBK strain clusters into the same branch as the IBV YN strain (GenBank accession no. JF893452) and the SC021202 strain (GenBank accession no. EU714029). The S1 subunit of the IBV genome is the major determinant of serotype (10) (11) (12) (13) , and S1 analysis indicated that the SAIBK strain has a 4/91-like serotype. The employed recombination detection methods revealed that SAIBK is a chimera virus, with recombination by the SC021202 strain as a major parent and the H120 vaccine strain as a minor parent. The first and second recombination regions were located at positions 7231 to 9126 and 13437 to 14473 in genes 1a and 1b, respectively. There were two other recombination regions detected at positions 951 to 1067 and 5393 to 5605 of SAIBK, which were recombined with the SC021202 strain as a major parent and the H52 vaccine strain as a minor parent. The recombination detection results suggested that SAIBK is possibly a chimera virus derived from the popularly used vaccine strains H120 and H52 and the field strain SC021202, and the SC021202 strain was isolated from chickens vaccinated with H120 in the Sichuan province of China in 2003 (14) . This result revealed that the field IBVs in Sichuan Province have undergone genetic recombination and are possibly emerging as new mutant strains, such as SAIBK. Nucleotide sequence accession number. The complete genome sequence of the SAIBK isolate was submitted to GenBank and assigned the accession no. DQ288927.

What virus has the closest genetic identity with the SAIBK gene?

Chinese IBV strain SC021202

Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics https://jamanetwork.com/journals/jama/fullarticle/2761285 February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323(12):1129-1130. doi:10.1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview (25:53) COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A(H1N1) virus ([H1N1]pdm09) pandemic or the severe acute respiratory syndrome coronavirus (SARS-CoV) or the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A(H1N1)pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A(H1N1)pdm09 had spread to 41 US states and 21 countries.1 While influenza A(H1N1)pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A(H1N1)pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A(H1N1)pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics (estimated by the World Health Organization [WHO] to be 250 000 to 500 000 per year, although estimation methods differ).3 Influenza A(H1N1)pdm09 was highly transmissible but not severe. SARS-CoV (2003) and MERS-CoV (2012-current) cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV (indicating sustained and even worldwide transmission could occur), and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths (case-fatality rate, 34%) in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical (border and school closing, infection control measures) and pharmaceutical (antiviral prophylaxis, vaccines) interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A(H1N1)pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention (CDC) distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 (9%) health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate (an important measure of severity), MERS cases can be asymptomatic and mild (25% in one outbreak). Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates (the proportion of individuals who become ill with or die from a disease in a population initially uninfected) of 20% to 30% (for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic), depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus (2019-nCoV) epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 ([email protected]). Published Online: February 11, 2020. doi:10.1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52(suppl 1):S1-S3. doi:10.1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7(45). doi:10.1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-695. doi:10.1016/S1473-3099(12)70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10(7):1258-1263. doi:10.3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26(2):191-198. doi:10.3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome (MERS). Microbiol Spectr. 2016;4(3). doi:10.1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A(H7N9) as an example. Clin Infect Dis. 2015;60(suppl):S1-S63. https://academic.oup.com/cid/issue/60/suppl_1.Google Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. doi:10.1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. doi:10.1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus (2019-nCoV) situation reports. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled "because a high proportion of cases were severe." While that helped , it was because cases were not infectious before some days after symptom onset (usually in the second week of illness). This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1) the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever (influenza-like illness).Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses (HuCoV: 229E,NL 63,0C43 and HKU1) can however be found in up to 30% ofpatients negative for influenza viruses (1). Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a "novel" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 (the source). Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al.:Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, https://doi.org/10.1007/s15010-019-01388-1).CONFLICT OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus (2019-nCoV) Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry (1919-1959) SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo

How many surgical masks or respirators have past studies projected will be required for a pandemic in the United States?

an estimated 7.3 billion

Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics https://jamanetwork.com/journals/jama/fullarticle/2761285 February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323(12):1129-1130. doi:10.1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview (25:53) COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A(H1N1) virus ([H1N1]pdm09) pandemic or the severe acute respiratory syndrome coronavirus (SARS-CoV) or the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A(H1N1)pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A(H1N1)pdm09 had spread to 41 US states and 21 countries.1 While influenza A(H1N1)pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A(H1N1)pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A(H1N1)pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics (estimated by the World Health Organization [WHO] to be 250 000 to 500 000 per year, although estimation methods differ).3 Influenza A(H1N1)pdm09 was highly transmissible but not severe. SARS-CoV (2003) and MERS-CoV (2012-current) cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV (indicating sustained and even worldwide transmission could occur), and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths (case-fatality rate, 34%) in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical (border and school closing, infection control measures) and pharmaceutical (antiviral prophylaxis, vaccines) interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A(H1N1)pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention (CDC) distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 (9%) health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate (an important measure of severity), MERS cases can be asymptomatic and mild (25% in one outbreak). Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates (the proportion of individuals who become ill with or die from a disease in a population initially uninfected) of 20% to 30% (for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic), depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus (2019-nCoV) epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 ([email protected]). Published Online: February 11, 2020. doi:10.1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52(suppl 1):S1-S3. doi:10.1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7(45). doi:10.1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-695. doi:10.1016/S1473-3099(12)70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10(7):1258-1263. doi:10.3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26(2):191-198. doi:10.3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome (MERS). Microbiol Spectr. 2016;4(3). doi:10.1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A(H7N9) as an example. Clin Infect Dis. 2015;60(suppl):S1-S63. https://academic.oup.com/cid/issue/60/suppl_1.Google Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. doi:10.1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. doi:10.1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus (2019-nCoV) situation reports. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled "because a high proportion of cases were severe." While that helped , it was because cases were not infectious before some days after symptom onset (usually in the second week of illness). This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1) the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever (influenza-like illness).Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses (HuCoV: 229E,NL 63,0C43 and HKU1) can however be found in up to 30% ofpatients negative for influenza viruses (1). Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a "novel" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 (the source). Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al.:Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, https://doi.org/10.1007/s15010-019-01388-1).CONFLICT OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus (2019-nCoV) Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry (1919-1959) SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo

What is the acronym MERS-CoV?

Middle East respiratory syndrome coronavirus

Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics https://jamanetwork.com/journals/jama/fullarticle/2761285 February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323(12):1129-1130. doi:10.1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview (25:53) COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A(H1N1) virus ([H1N1]pdm09) pandemic or the severe acute respiratory syndrome coronavirus (SARS-CoV) or the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A(H1N1)pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A(H1N1)pdm09 had spread to 41 US states and 21 countries.1 While influenza A(H1N1)pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A(H1N1)pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A(H1N1)pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics (estimated by the World Health Organization [WHO] to be 250 000 to 500 000 per year, although estimation methods differ).3 Influenza A(H1N1)pdm09 was highly transmissible but not severe. SARS-CoV (2003) and MERS-CoV (2012-current) cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV (indicating sustained and even worldwide transmission could occur), and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths (case-fatality rate, 34%) in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical (border and school closing, infection control measures) and pharmaceutical (antiviral prophylaxis, vaccines) interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A(H1N1)pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention (CDC) distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 (9%) health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate (an important measure of severity), MERS cases can be asymptomatic and mild (25% in one outbreak). Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates (the proportion of individuals who become ill with or die from a disease in a population initially uninfected) of 20% to 30% (for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic), depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus (2019-nCoV) epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 ([email protected]). Published Online: February 11, 2020. doi:10.1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52(suppl 1):S1-S3. doi:10.1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7(45). doi:10.1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-695. doi:10.1016/S1473-3099(12)70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10(7):1258-1263. doi:10.3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26(2):191-198. doi:10.3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome (MERS). Microbiol Spectr. 2016;4(3). doi:10.1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A(H7N9) as an example. Clin Infect Dis. 2015;60(suppl):S1-S63. https://academic.oup.com/cid/issue/60/suppl_1.Google Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. doi:10.1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. doi:10.1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus (2019-nCoV) situation reports. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled "because a high proportion of cases were severe." While that helped , it was because cases were not infectious before some days after symptom onset (usually in the second week of illness). This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1) the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever (influenza-like illness).Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses (HuCoV: 229E,NL 63,0C43 and HKU1) can however be found in up to 30% ofpatients negative for influenza viruses (1). Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a "novel" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 (the source). Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al.:Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, https://doi.org/10.1007/s15010-019-01388-1).CONFLICT OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus (2019-nCoV) Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry (1919-1959) SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. 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What are the critical factors that determine the effect of an epidemic?

Transmissibility and severity

Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics https://jamanetwork.com/journals/jama/fullarticle/2761285 February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323(12):1129-1130. doi:10.1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview (25:53) COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A(H1N1) virus ([H1N1]pdm09) pandemic or the severe acute respiratory syndrome coronavirus (SARS-CoV) or the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A(H1N1)pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A(H1N1)pdm09 had spread to 41 US states and 21 countries.1 While influenza A(H1N1)pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A(H1N1)pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A(H1N1)pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics (estimated by the World Health Organization [WHO] to be 250 000 to 500 000 per year, although estimation methods differ).3 Influenza A(H1N1)pdm09 was highly transmissible but not severe. SARS-CoV (2003) and MERS-CoV (2012-current) cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV (indicating sustained and even worldwide transmission could occur), and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths (case-fatality rate, 34%) in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical (border and school closing, infection control measures) and pharmaceutical (antiviral prophylaxis, vaccines) interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A(H1N1)pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention (CDC) distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 (9%) health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate (an important measure of severity), MERS cases can be asymptomatic and mild (25% in one outbreak). Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates (the proportion of individuals who become ill with or die from a disease in a population initially uninfected) of 20% to 30% (for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic), depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus (2019-nCoV) epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 ([email protected]). Published Online: February 11, 2020. doi:10.1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52(suppl 1):S1-S3. doi:10.1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7(45). doi:10.1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-695. doi:10.1016/S1473-3099(12)70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10(7):1258-1263. doi:10.3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26(2):191-198. doi:10.3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome (MERS). Microbiol Spectr. 2016;4(3). doi:10.1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A(H7N9) as an example. Clin Infect Dis. 2015;60(suppl):S1-S63. https://academic.oup.com/cid/issue/60/suppl_1.Google Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. doi:10.1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. doi:10.1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus (2019-nCoV) situation reports. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled "because a high proportion of cases were severe." While that helped , it was because cases were not infectious before some days after symptom onset (usually in the second week of illness). This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1) the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever (influenza-like illness).Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses (HuCoV: 229E,NL 63,0C43 and HKU1) can however be found in up to 30% ofpatients negative for influenza viruses (1). Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a "novel" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 (the source). Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al.:Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, https://doi.org/10.1007/s15010-019-01388-1).CONFLICT OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus (2019-nCoV) Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry (1919-1959) SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo

When did the World Health Organization (WHO) officially declare the 2019-nCoV epidemic as a Public Health Emergency of International Concern?

January 30, 2020

Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics https://jamanetwork.com/journals/jama/fullarticle/2761285 February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323(12):1129-1130. doi:10.1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview (25:53) COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A(H1N1) virus ([H1N1]pdm09) pandemic or the severe acute respiratory syndrome coronavirus (SARS-CoV) or the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A(H1N1)pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A(H1N1)pdm09 had spread to 41 US states and 21 countries.1 While influenza A(H1N1)pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A(H1N1)pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A(H1N1)pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics (estimated by the World Health Organization [WHO] to be 250 000 to 500 000 per year, although estimation methods differ).3 Influenza A(H1N1)pdm09 was highly transmissible but not severe. SARS-CoV (2003) and MERS-CoV (2012-current) cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV (indicating sustained and even worldwide transmission could occur), and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths (case-fatality rate, 34%) in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical (border and school closing, infection control measures) and pharmaceutical (antiviral prophylaxis, vaccines) interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A(H1N1)pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention (CDC) distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 (9%) health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate (an important measure of severity), MERS cases can be asymptomatic and mild (25% in one outbreak). Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates (the proportion of individuals who become ill with or die from a disease in a population initially uninfected) of 20% to 30% (for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic), depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus (2019-nCoV) epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 ([email protected]). Published Online: February 11, 2020. doi:10.1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52(suppl 1):S1-S3. doi:10.1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7(45). doi:10.1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-695. doi:10.1016/S1473-3099(12)70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10(7):1258-1263. doi:10.3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26(2):191-198. doi:10.3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome (MERS). Microbiol Spectr. 2016;4(3). doi:10.1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A(H7N9) as an example. Clin Infect Dis. 2015;60(suppl):S1-S63. https://academic.oup.com/cid/issue/60/suppl_1.Google Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. doi:10.1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. doi:10.1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus (2019-nCoV) situation reports. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled "because a high proportion of cases were severe." While that helped , it was because cases were not infectious before some days after symptom onset (usually in the second week of illness). This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1) the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever (influenza-like illness).Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses (HuCoV: 229E,NL 63,0C43 and HKU1) can however be found in up to 30% ofpatients negative for influenza viruses (1). Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a "novel" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 (the source). Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al.:Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, https://doi.org/10.1007/s15010-019-01388-1).CONFLICT OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus (2019-nCoV) Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry (1919-1959) SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo

What influenza virus was identified in China in 2013?

H7N9

Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics https://jamanetwork.com/journals/jama/fullarticle/2761285 February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323(12):1129-1130. doi:10.1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview (25:53) COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A(H1N1) virus ([H1N1]pdm09) pandemic or the severe acute respiratory syndrome coronavirus (SARS-CoV) or the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A(H1N1)pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A(H1N1)pdm09 had spread to 41 US states and 21 countries.1 While influenza A(H1N1)pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A(H1N1)pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A(H1N1)pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics (estimated by the World Health Organization [WHO] to be 250 000 to 500 000 per year, although estimation methods differ).3 Influenza A(H1N1)pdm09 was highly transmissible but not severe. SARS-CoV (2003) and MERS-CoV (2012-current) cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV (indicating sustained and even worldwide transmission could occur), and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths (case-fatality rate, 34%) in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical (border and school closing, infection control measures) and pharmaceutical (antiviral prophylaxis, vaccines) interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A(H1N1)pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention (CDC) distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 (9%) health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate (an important measure of severity), MERS cases can be asymptomatic and mild (25% in one outbreak). Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates (the proportion of individuals who become ill with or die from a disease in a population initially uninfected) of 20% to 30% (for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic), depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus (2019-nCoV) epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 ([email protected]). Published Online: February 11, 2020. doi:10.1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52(suppl 1):S1-S3. doi:10.1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7(45). doi:10.1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-695. doi:10.1016/S1473-3099(12)70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10(7):1258-1263. doi:10.3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26(2):191-198. doi:10.3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome (MERS). Microbiol Spectr. 2016;4(3). doi:10.1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A(H7N9) as an example. Clin Infect Dis. 2015;60(suppl):S1-S63. https://academic.oup.com/cid/issue/60/suppl_1.Google Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. doi:10.1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. doi:10.1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus (2019-nCoV) situation reports. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled "because a high proportion of cases were severe." While that helped , it was because cases were not infectious before some days after symptom onset (usually in the second week of illness). This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1) the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever (influenza-like illness).Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses (HuCoV: 229E,NL 63,0C43 and HKU1) can however be found in up to 30% ofpatients negative for influenza viruses (1). Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a "novel" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 (the source). Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al.:Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, https://doi.org/10.1007/s15010-019-01388-1).CONFLICT OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus (2019-nCoV) Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry (1919-1959) SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo

What past research has been done on severe, single-wave pandemics?

After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.

Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics https://jamanetwork.com/journals/jama/fullarticle/2761285 February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323(12):1129-1130. doi:10.1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview (25:53) COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A(H1N1) virus ([H1N1]pdm09) pandemic or the severe acute respiratory syndrome coronavirus (SARS-CoV) or the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A(H1N1)pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A(H1N1)pdm09 had spread to 41 US states and 21 countries.1 While influenza A(H1N1)pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A(H1N1)pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A(H1N1)pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics (estimated by the World Health Organization [WHO] to be 250 000 to 500 000 per year, although estimation methods differ).3 Influenza A(H1N1)pdm09 was highly transmissible but not severe. SARS-CoV (2003) and MERS-CoV (2012-current) cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV (indicating sustained and even worldwide transmission could occur), and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths (case-fatality rate, 34%) in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical (border and school closing, infection control measures) and pharmaceutical (antiviral prophylaxis, vaccines) interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A(H1N1)pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention (CDC) distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 (9%) health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate (an important measure of severity), MERS cases can be asymptomatic and mild (25% in one outbreak). Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates (the proportion of individuals who become ill with or die from a disease in a population initially uninfected) of 20% to 30% (for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic), depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus (2019-nCoV) epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 ([email protected]). Published Online: February 11, 2020. doi:10.1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52(suppl 1):S1-S3. doi:10.1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7(45). doi:10.1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-695. doi:10.1016/S1473-3099(12)70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10(7):1258-1263. doi:10.3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26(2):191-198. doi:10.3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome (MERS). Microbiol Spectr. 2016;4(3). doi:10.1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A(H7N9) as an example. Clin Infect Dis. 2015;60(suppl):S1-S63. https://academic.oup.com/cid/issue/60/suppl_1.Google Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. doi:10.1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. doi:10.1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus (2019-nCoV) situation reports. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled "because a high proportion of cases were severe." While that helped , it was because cases were not infectious before some days after symptom onset (usually in the second week of illness). This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1) the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever (influenza-like illness).Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses (HuCoV: 229E,NL 63,0C43 and HKU1) can however be found in up to 30% ofpatients negative for influenza viruses (1). Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a "novel" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 (the source). Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al.:Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, https://doi.org/10.1007/s15010-019-01388-1).CONFLICT OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus (2019-nCoV) Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry (1919-1959) SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo

What is a clinical attack rate?

the proportion of individuals who become ill with or die from a disease in a population initially uninfected

Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics https://jamanetwork.com/journals/jama/fullarticle/2761285 February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323(12):1129-1130. doi:10.1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview (25:53) COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A(H1N1) virus ([H1N1]pdm09) pandemic or the severe acute respiratory syndrome coronavirus (SARS-CoV) or the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A(H1N1)pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A(H1N1)pdm09 had spread to 41 US states and 21 countries.1 While influenza A(H1N1)pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A(H1N1)pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A(H1N1)pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics (estimated by the World Health Organization [WHO] to be 250 000 to 500 000 per year, although estimation methods differ).3 Influenza A(H1N1)pdm09 was highly transmissible but not severe. SARS-CoV (2003) and MERS-CoV (2012-current) cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV (indicating sustained and even worldwide transmission could occur), and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths (case-fatality rate, 34%) in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical (border and school closing, infection control measures) and pharmaceutical (antiviral prophylaxis, vaccines) interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A(H1N1)pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention (CDC) distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 (9%) health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate (an important measure of severity), MERS cases can be asymptomatic and mild (25% in one outbreak). Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates (the proportion of individuals who become ill with or die from a disease in a population initially uninfected) of 20% to 30% (for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic), depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus (2019-nCoV) epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 ([email protected]). Published Online: February 11, 2020. doi:10.1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52(suppl 1):S1-S3. doi:10.1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7(45). doi:10.1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-695. doi:10.1016/S1473-3099(12)70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10(7):1258-1263. doi:10.3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26(2):191-198. doi:10.3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome (MERS). Microbiol Spectr. 2016;4(3). doi:10.1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A(H7N9) as an example. Clin Infect Dis. 2015;60(suppl):S1-S63. https://academic.oup.com/cid/issue/60/suppl_1.Google Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. doi:10.1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. doi:10.1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus (2019-nCoV) situation reports. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled "because a high proportion of cases were severe." While that helped , it was because cases were not infectious before some days after symptom onset (usually in the second week of illness). This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1) the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever (influenza-like illness).Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses (HuCoV: 229E,NL 63,0C43 and HKU1) can however be found in up to 30% ofpatients negative for influenza viruses (1). Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a "novel" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 (the source). Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al.:Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, https://doi.org/10.1007/s15010-019-01388-1).CONFLICT OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus (2019-nCoV) Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry (1919-1959) SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. 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What was the clinical attack rate in the 2009 H1N1 pandemic?

20%

Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics https://jamanetwork.com/journals/jama/fullarticle/2761285 February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323(12):1129-1130. doi:10.1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview (25:53) COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A(H1N1) virus ([H1N1]pdm09) pandemic or the severe acute respiratory syndrome coronavirus (SARS-CoV) or the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A(H1N1)pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A(H1N1)pdm09 had spread to 41 US states and 21 countries.1 While influenza A(H1N1)pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A(H1N1)pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A(H1N1)pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics (estimated by the World Health Organization [WHO] to be 250 000 to 500 000 per year, although estimation methods differ).3 Influenza A(H1N1)pdm09 was highly transmissible but not severe. SARS-CoV (2003) and MERS-CoV (2012-current) cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV (indicating sustained and even worldwide transmission could occur), and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths (case-fatality rate, 34%) in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical (border and school closing, infection control measures) and pharmaceutical (antiviral prophylaxis, vaccines) interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A(H1N1)pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention (CDC) distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 (9%) health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate (an important measure of severity), MERS cases can be asymptomatic and mild (25% in one outbreak). Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates (the proportion of individuals who become ill with or die from a disease in a population initially uninfected) of 20% to 30% (for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic), depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus (2019-nCoV) epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 ([email protected]). Published Online: February 11, 2020. doi:10.1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52(suppl 1):S1-S3. doi:10.1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7(45). doi:10.1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-695. doi:10.1016/S1473-3099(12)70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10(7):1258-1263. doi:10.3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26(2):191-198. doi:10.3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome (MERS). Microbiol Spectr. 2016;4(3). doi:10.1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A(H7N9) as an example. Clin Infect Dis. 2015;60(suppl):S1-S63. https://academic.oup.com/cid/issue/60/suppl_1.Google Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. doi:10.1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. doi:10.1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus (2019-nCoV) situation reports. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled "because a high proportion of cases were severe." While that helped , it was because cases were not infectious before some days after symptom onset (usually in the second week of illness). This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1) the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever (influenza-like illness).Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses (HuCoV: 229E,NL 63,0C43 and HKU1) can however be found in up to 30% ofpatients negative for influenza viruses (1). Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a "novel" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 (the source). Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al.:Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, https://doi.org/10.1007/s15010-019-01388-1).CONFLICT OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus (2019-nCoV) Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry (1919-1959) SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. 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What is the estimated R0 of COVID-19?

2.2

Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics https://jamanetwork.com/journals/jama/fullarticle/2761285 February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323(12):1129-1130. doi:10.1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview (25:53) COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A(H1N1) virus ([H1N1]pdm09) pandemic or the severe acute respiratory syndrome coronavirus (SARS-CoV) or the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A(H1N1)pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A(H1N1)pdm09 had spread to 41 US states and 21 countries.1 While influenza A(H1N1)pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A(H1N1)pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A(H1N1)pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics (estimated by the World Health Organization [WHO] to be 250 000 to 500 000 per year, although estimation methods differ).3 Influenza A(H1N1)pdm09 was highly transmissible but not severe. SARS-CoV (2003) and MERS-CoV (2012-current) cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV (indicating sustained and even worldwide transmission could occur), and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths (case-fatality rate, 34%) in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical (border and school closing, infection control measures) and pharmaceutical (antiviral prophylaxis, vaccines) interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A(H1N1)pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention (CDC) distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 (9%) health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate (an important measure of severity), MERS cases can be asymptomatic and mild (25% in one outbreak). Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates (the proportion of individuals who become ill with or die from a disease in a population initially uninfected) of 20% to 30% (for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic), depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus (2019-nCoV) epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 ([email protected]). Published Online: February 11, 2020. doi:10.1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52(suppl 1):S1-S3. doi:10.1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7(45). doi:10.1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-695. doi:10.1016/S1473-3099(12)70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10(7):1258-1263. doi:10.3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26(2):191-198. doi:10.3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome (MERS). Microbiol Spectr. 2016;4(3). doi:10.1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A(H7N9) as an example. Clin Infect Dis. 2015;60(suppl):S1-S63. https://academic.oup.com/cid/issue/60/suppl_1.Google Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. doi:10.1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. doi:10.1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus (2019-nCoV) situation reports. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled "because a high proportion of cases were severe." While that helped , it was because cases were not infectious before some days after symptom onset (usually in the second week of illness). This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1) the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever (influenza-like illness).Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses (HuCoV: 229E,NL 63,0C43 and HKU1) can however be found in up to 30% ofpatients negative for influenza viruses (1). Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a "novel" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 (the source). Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al.:Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, https://doi.org/10.1007/s15010-019-01388-1).CONFLICT OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus (2019-nCoV) Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry (1919-1959) SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo

How many ventilators have past studies projected will be required for a pandemic in the United States?

35 000 to 60 000

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

How is exhaled breath condensate used in viral research?

the isolation of respiratory viruses

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

How many patients were i this study?

102

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

What was the conclusion of this study?

EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

How long did the patient breath into the RTube?

10 minutes

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

What followed the reverse transcription step in the analysis?

denaturation step

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

What was the last step in the analysis?

amplification step

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

What percentage of the patients were between 20 and 30 years old in this study?

52%

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

Why is EBC an attractive method for screening?

easy to perform and can be conducted in a home environment

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the leading cause of death among children after the age of 1 month?

Pneumonia remains the leading cause of death in children outside the neonatal period,

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How has the number of childhood pneumonia been reduced?

New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What percentage of childhood deaths are due to pneumonia?

approximately 900,000 of the estimated 6.3 million child deaths in 2013

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How has the childhood population grown in the last two decades?

global childhood population from 605 million in 2000 to 664 million in 2015

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the reduction in the number of childhood pneumonia cases?

Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How much is the reduction in the childhood pneumonia deaths?

Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

Childhood pneumonia rate for high income countries vs low and middle income countries.

The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries.

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What percentage of childhood pneumonia deaths occur outside hospital in low and middle income countries?

up to 81% of severe pneumonia deaths occur outside a hospital

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

Case Fatality Rates for Childhood Pneumonia in high income vs low and middle income countries.

the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How can childhood pneumonia affect the subsequent health of a person?

Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the increase in the risk of respiratory disease after having childhood pneumonia.

The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

Which is the best method to identify pneumonia in a person?

Chest radiologic changes have been considered the gold standard for defining a pneumonia event

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is responsible for the reduction of radiologic pneumonia?

Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia.

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the percentage reduction in pneumonia cases due to vaccination?

Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the revised WHO definition of Bacterial Pneumonia?

A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L