Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination.RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type  N. benthamiana plant (p19syn) capable of sustaining the ectopic expression of the Cymbidium ringspot virus (CymRSV) p19 upon infection with a suppressor-deficient CymRSV (Cym19stop). By using wt and p19syn plants in combination with CymRSV and Cym19stop, we were able to analyze the effects of p19 provided “in trans” and “in cis” during the viral invasion of the plant. We have shown that p19 can efficiently sequester endogenous small RNAs (sRNAs) in mock-inoculated p19syn plants while it does not bind these sRNAs upon Cym19stop infection. Also, the presence of p19 in virus infection did not alter the expression of miRNAs significantly. These findings do not support the widely accepted assumption that viral symptoms are the direct consequence of the impact of VSRs on endogenous silencing pathways. We demonstrated that p19 preferentially sequesters positive:negative viral short interfering RNAs (vsiRNAs) pairs and that the binding by p19 is independent of vsiRNA sequence or the type of the 5’-end nucleotide. We have also found that 3’ truncation is induced on p19 bound sRNAs. Finally using AGO1- and AGO2- immunoprecipitation experiments we observed that p19 specifically compromises vsiRNAs’ loading into AGO1 but not AGO2. Since antiviral silencing is strongly inhibited by p19, this suggests that AGO1 is the main effector protein against CymRSV tombusvirus.To better understand the specific effect of p19 viral suppressor of RNA silencing (VSR) on antiviral silencing and endogenous small RNA pathways, we generated a  Viruses are among the most important plant pathogens that cause huge economic losses in many agriculturally important crops worldwide. The invasion of the host by viruses deeply alters the physiology of the plants at cellular and tissue levels due to the interaction of the virus with the cellular pathways, which ultimately leads to viral symptom development. During evolution, plants have developed diverse strategies to combat virus infections. Amongst others, RNA silencing is one of the most important mechanisms that serve to fight against viruses ,2.RNA silencing is a conserved eukaryotic pathway involved in almost all cellular processes like development, stress responses and antiviral defense. RNA silencing relays on the 21–24 nt short interfering RNAs (siRNAs) or micro RNAs (miRNAs) the hallmark molecules of silencing . The siRA. thaliana [N. benthamiana [A. thaliana [N. benthamiana, the important model organism for plant virology studies, AGO2 was proposed to protect against Potato virus X [Tomato bushy stunt virus (TBSV) [Players of the antiviral silencing overlap with those of the endogenous silencing pathway ,26. Antithaliana –36, N. bthamiana ,38 and ithamiana . AGO2 wathaliana ,36,40–45 virus X  and the s (TBSV) ,48. Howes (TBSV) . As AGO1Viruses, to counteract host defense, have evolved viral suppressors of RNA silencing (VSRs) providing strong evidence for the antiviral nature of silencing ,50,51. MCymbidium ringspot virus (CymRSV) infections miRNA sequestration by p19 is very poor and may depend on spatial and temporal co-expression of miRNA duplex and the VSR [The p19 protein of tombusviruses is one of the best-known VSR. Crystallographic studies have shown that p19 tail-to-tail homodimer acts as a molecular caliper to size-select and sequester siRNA duplexes in a sequence-independent manner –58 preve the VSR . vsiRNAs the VSR ,67. Thes the VSR ,68. ThusN. benthamiana plant line (p19syn) or wild type plant as control in combination with its wild-type (CymRSV) or suppressor deficient virus (Cym19stop) infection: (i) wt virus infection of wt N. benthamiana (p19 in “cis”), (ii) Cym19stop-infection in wt N. benthamiana , (iii) Cym19stop-infection in p19syn plants (p19 in “trans”) and (iv) p19syn plants (p19 only). In this way we were able to compare the impact of p19 on its own or in the genuine virus-infected background.To better understand the impact of p19 on silencing and its role in viral symptom development we employed a synthetic p19-expressing transgenic N. benthamiana) or “in trans” . In line with our previous findings, p19 efficiently bound endogenous sRNA duplexes only in the absence of the virus infection, suggesting that p19 impact on endogenous pathways is restricted. Analyzing the siRNA pool immunoprecipitated by p19 through high-throughput sequencing, we found that p19 changes the bias of positive vsiRNAs towards a more equilibrated positive/negative strand ratio, suggesting a preference for perfect ds-vsiRNAs. We also showed that p19 prevents mainly AGO1 but not AGO2 loading with vsiRNAs. This finding suggests a key role of AGO1 opposed to AGO2 during the antiviral response.We analyzed p19 ability to sequester vsiRNAs and plant endogenous sRNAs, with and without viral background. Consistently with our previous results, we have found that p19 can bind vsiRNAs when expressed either “in cis” , we modified the p19 transgene introducing all possible silent nucleotide changes. In this way, we reduced the nucleotide sequence similarity between the transgene and the challenging virus to 68% while keeping the amino acid identity at 100% (Cucumber mosaic virus + yellow satellite RNA (CMV + Y-satRNA). CMV + Y-satRNA was reported to induce bright yellow symptoms on N. benthamiana through targeting the tobacco magnesium protoporphyrin chelatase subunit I (ChlI) gene involved in chlorophyll biosynthesis by Y-satRNA derived siRNA [N. benthamiana plants developed the bright yellow symptoms while the infected p19syn plants failed to show the typical yellowing  and Cym19stop-infected p19syn plants (“trans-p19“) have shown a different picture: p19 bound almost exclusively vsiRNAs but not endogenous sRNAs . Similarly, in the CymRSV-infected wt plant 12% N. benthamiana reads in the input sample has dropped to 1% (p19 enriched the 88% vsiRNAs of the input to 99% in the p19-IP). We concluded therefore that p19 ability to sequester endogenous sRNAs is strongly decreased by the virus infection and p19 preference to vsiRNAs does not depend on the expressional origin of p19 protein .Analysis of p19-bound sRNAs from mock-inoculated p19syn plants revealed that p19 binds efficiently endogenous sRNAs , includius sRNAs . This suus sRNAs . We alsous sRNAs . The inpN. benthamiana, N. benthamiana). This was likely the consequence of the inhibition of the cleavage of TAS3 transcripts by p19-captured miR390. Indeed, miR390 is efficiently enriched in p19 IP  . These dThe polarity analysis of p19-immunoprecipitated vsiRNAs revealed a more equilibrated positive/negative strand ratio  in the CymRSV-infected plants . In Cym1Arabidopsis miR171a, miR171b, miR171c all containing mismatches) and a perfect artificial siR171 duplex  . The relTo better understand the p19 protein effects on vsiRNAs we analyzed the size distribution of these during infection. Upon CymRSV infection vsiRNAs produced are predominantly of 21nt and 22nt in length . In addiin vitro bound p19:siRNA complexes further confirmed that the p19 protein can protect the double-stranded stretch of sRNA duplexes  derived vsiRNA when p19 was provided “in trans“. The length shift to 1- or 2-nucleotide shorter vsiRNA forms was confirmed . Shortenduplexes . The exoA. thaliana and N. benthamiana AGO1 and AGO2 were described as the most important effectors while others such as AGO5, 7 and 10 to have minor roles during antiviral silencing [Multiple AGOs were shown to have antiviral functions. In ilencing ,34,36,44ilencing , the virilencing  and S2 FN. benthamiana by co-immunoprecipitations : we observed relatively high “background” of vsiRNAs without 5’ sorting preference in AGO1 compare . ConversThe AGO1 IP derived from CymRSV-infected plants contained a similar miRNA profile as the mock inoculated plants, in contrast to AGO2 IP in which the levels of analyzed miRNAs were reduced  Figs. ImThe specific impact of p19 on vsiRNA AGO1-loading found in the deep sequencing analysis was also confirmed by Northern blot analysis. vsiRNAs loading into AGO1 was less efficient than AGO2 in CymRSV-infected plants when the p19 was provided in “cis”  or in “tWe also analyzed the distribution of AGO1- and AGO2-bound vsiRNA along the viral genome. This generally followed the biogenesis of vsiRNAs and we could not define any sequence preference or specific hotspots of AGO1- or AGO2-loading . The strMost of the identified VSRs are multifunctional, and this nature of VSRs often causes serious difficulties in the exploration of their action during the natural virus infection. The inactivation of VSRs often leads to loss of viability of the given virus, due to their multifunctional nature. It is hard therefore to separate the suppressor’s impact and virus infection effects. Either transgenic or transient expression of VSRs without the parental virus background might not reflect the natural interaction with the host cellular machinery. As a consequence of these difficulties, the molecular mechanisms of the action of VSRs and their impact on the host often remain elusive. To overcome this pitfall, we combined the transgenic expression of the VSR (p19) suppressor with its authentic suppressor mutant virus (Cym19stop)-infection in a novel experimental setup.During virus infection, high amounts of vsiRNAs are produced. These vsiRNAs are efficiently sequestered by p19 suppressor inhibiting their incorporation into RISC . The conIn addition to the previous findings, we have also shown that sRNA binding by p19 happens without 5’-end nucleotide selection including vsiRNAs or endogenous miRNAs.trans, it can be observed. Shortening also happens on miRNAs in p19syn plants without virus infection. In summary, the truncation is likely induced directly by p19-binding and is not due to the virus infection or restricted to a specific class of sRNAs. Why shortening does not happen in the absence of p19, on the free vsiRNAs, which theoretically would be more accessible? In cells endogenous free sRNA duplexes  [Previous studies ,77 have 168star)  and free168star) ,67. The 168star) . The cry168star) ,58. Furt168star)  therefor168star) .N. benthamiana leads to the development of a strong phenotype that is quite different from symptoms observed during parental viral infection  [Regardless of the reason of how AGO1-loading is compromised by p19, it seems that AGO2 is not enough to fight off the virus and help the plant to recover in the absence of AGO1-loading/activity. It has been suggested previously that AGO2 but not AGO1 plays role in the antiviral response against tombusvirus infections, including s (TBSV) ,48. We hN. tabacum codon usage. The resulting nucleotide sequence was further modified to avoid the presence of cryptic splicing and polyadenylation signals using Net2gene splicing prediction (http://www.cbs.dtu.dk/services/NetGene2/) and HCpolyA (http://bioinfo4.itb.cnr.it/~webgene/wwwHC_polya_ex.html) software, respectively. The synthetic ORF5 (KpnI and BglII and cloned in KpnI-BamHI of pBin19 (pBinCymRSVp19syn)The synthetic CymRSV ORF5 (p19) was essentially constructed following the previously described antivirus-induced transgene silencing strategy . As the tic ORF5  was syntN. benthamiana was transformed with the recombinant Agrobacterium tumefaciens strain C58C1 (pGV2260) harboring the plasmid pBinCymRSVp19syn, and kanamycin-resistant plants were regenerated as previously described [p19syn transgene by PCR and for the expression of the p19 protein by Western blotting with the anti-CymRSV-p19 antibody as previously described [escribed . The priescribed .N. benthamiana plants were grown at 22°C. At six-leaves stage plants were infiltrated with A. tumefaciens C58C1 harboring the appropriate constructs in the pBIN61 plasmid. pBIN61-Cymp19 and pBIN61-GFP were grown on selective media overnight, resuspended in the infiltration buffer  kept on 25°C for 4h, and subsequently infiltrated into wild-type or p19syn plant leaves at OD600 = 0.4.In vitro transcription of CymRSV, Cym19stop, TBSV-PDS-GFP and TBSV-PDS-AGO1-1 RNAs from linearized template plasmids and inoculation of RNA transcripts onto N. benthamiana plants were performed as described previously [eviously . CMV Y-seviously .Total RNA was extracted from 100 mg of leaf tissue. The homogenized plant materials were resuspended in 600 μl of extraction buffer  and mixed with an equal volume of phenol. The aqueous phase was treated with equal volumes of phenol-chloroform and chloroform, precipitated with ethanol, and finally resuspended in sterile water. RNA gel blot analysis of higher molecular weight RNAs was performed as described previously .http://www.exiqon.com).RNA gel blot analysis of 21–24 nt RNAs was performed as follows. Approximately 5 μg of total RNA was separated by 15% PAGE with 8.6 M urea and 1xTris-borate-EDTA. RNA was electro-blotted onto Hybond-NX membranes and fixed by chemical crosslinking at 60°C for 1 hr . Small Rg at 4°C for 10 min. The supernatants were resolved on 12% SDS-PAGE, transferred to Hybond PVDF membranes  and subjected to Western blot analysis. For detection anti-p19 [http://www.genscript.com/). The proteins were visualized by chemiluminescence  according to the manufacturer’s instructions.Mock- or virus-infected systemical leaf tissues were homogenized in extraction buffer . Samples were boiled, and cell debris was removed by centrifugation at 18,000 x anti-p19 , NbAGO1 anti-p19  and NbAGN. benthamiana leaves showing systemic symptoms (or leaves at the same stage and positions from mock-inoculated plants) were collected, ground in 1:3 (w/v) amount of immunoprecipitation buffer , and cleared by centrifugation . Cleared lysates were kept on ice until immunoprecipitation with antibody-coated protein A-Sepharose . Beads were washed before adding the antibodies (described earlier). For mock immunoprecipitation preimmune serum was used. Antibodies coated beads were incubated with the relevant cleared lysates for 4h at 4°C. After immunoprecipitations the beads were washed five times with ice-cold immunoprecipitation buffer for 2 min each. Input extracts and eluates of immunoprecipitations were used for Western and Northern blot analysis.For immunoprecipitation, 1–5 grams of mock-, CymRSV- or Cym19stop-infected The library preparation was described previously , shortlyRNA-seq library preparation was done according to the manufacturer protocol (TruSeqStranded mRNA Library Prep Kit). The libraries were sequenced on Illumina HiScanSQ. 135 M 100 bp paired-end reads were produced per lane. 3 samples were pooled together on a lane. The libraries were submitted to GEO and can be accessed through series accession number GSE77070.32P isotope. After the reaction was stopped, 10 ng was saved for further process and the rest was mixed with 500 ng of unlabeled synthetic RNA with the sequence of 5’-UAUUGGCGCGGCUCAAUCAGA-3’. The mixture was heated to 95°C for 2 minutes in a thermocycler and was cooled to 5°C (2°/2 min) to gain 19 nt perfectly matched double strand with 2 nt protruding at the 3’ end. The sufficient amount of DNA loading dye was added to the mixture and to the saved labeled single stranded RNA. Both samples were run on a 8% acrylamide:bis-acrylamide 19:1 1xTBE gel. Gel was directly exposed. The dsRNA region was cut from the gel. The gel piece was shredded using a 0.5 ml tube with several holes in the bottom in a 2 ml tube via centrifugation. The shredded gel pieces were shaken overnight in 500 μl of 300 mM NaCl at 4°C. Gel pieces were filtered out by using Spin-X column (Corning). 400 μl of dsRNA solution was precipitated by adding 20 μg glycogen (Fermentas) and 1 ml ethanol. The precipitated ds-RNA was resolved in IP buffer described before.One hundred nanograms of synthetic sRNA (5’-UGAUUGAGCCGCGCCAAUAUC-3’) was 5’ end labeled by T4 polynucleotide kinase (Fermentas) with Purified p19 described earlier was used for the assay . A dilutE. coli as described previously [http://dharmacon.gelifesciences.com) for sequence see For band shift assays wild-type p19 protein was purified from eviously ,67. Custin vitro transcription from the linearized CymRSV construct.In situ hybridization was performed as previously described in . DetectiAfter demultiplexing of the raw data we used the UEA workbench version 3.0  for adapN.benthamiana genome matching reads. In all other cases reads were normalized to 1 million total reads.In After demultiplexing we used FastQC 0.10.1 to check general attributes. Trim_galore 0.4.1 and FASTX Toolkit 0.0.13 were used to remove adaptor sequences, low quality bases, reads under 20 nt and unpaired reads. Bowtie2  was usedS1 Figp19syn). Start and stop codons are in bold, modified nucleotides are in red lowercase letters, red and green highlights are restriction sites used for cloning. (B) Alignment of CymRSV p19 ORF with the synthetic p19 ORF (p19syn). Nucleotide sequences show 68% similarity. (C) Amino acid alignment of p19 translated from p19syn and CymRSV p19 ORF shows 100% identity.(A) Nucleotide sequence of CymRSV p19 synthetic ORF used for plant transformation ((DOCX)Click here for additional data file.S2 FigIn situ hybridization of Cym19stop-infected wt N. benthamiana and p19syn plants or CymRSV-infected wt N. benthamiana leaf cross sections showing distribution of virus within the leaf tissue. Mock-infected wt N. benthamiana cross section shown as negative control (on the right).(A) Characteristic phenotype of p19syn (1–57 line)(blue arrow) and virus-induced sytemic symptoms on wt or p19syn plants (red arrow) are shown. Pictures were taken at 10 dpi. (B) (PDF)Click here for additional data file.S3 Fig6N. benthamiana genome matching read counts. (B) Ratio of miRNA-targeted mRNAs in mock-infected p19syn plants (relative to mock-infected wt N. benthamiana) and Cym19stop-infected p19syn plants (relative to mock-infected p19syn plants). Heatmap legend is shown on the right on a log2 scale. (C) Accumulation of miR390 and TAS3-derived D7 tasiRNAs in mock-infected or Cym19stop-infected p19syn plants compared to mock-infected wt N. benthamiana plants. vsiRNAs, miR159, and nucleolar small RNA U6 are shown as controls.(A) Abundance of p19-bound miRNAs (listed on the left) in mock- or Cym19stop-infected p19syn plants and in CymRSV-infected wild type plants. The normalized total reads are shown on a log10 scale, values under threshold (ut). Heatmap legend is shown on the right. Read counts were normalized to 10(PDF)Click here for additional data file.S4 Fig6 “trimmed” read counts.Location of vsiRNAs on the viral genome is presented on the x-axis, read counts are shown on the y-axis. Y-axis positive and negative values represent read counts derived from the positive or negative viral strand, respectively. 21nt and 22nt vsiRNAs are indicated in blue and red, respectively. Read counts were normalized to 10(TIF)Click here for additional data file.S5 Fig5’ labeled siRNAs were incubated for 10 minutes with different amout of p19 and then exposed to RNase A-digestion for 10 and 30 minutes, upper and lower panel, respectively. The protected p19-bound RNA duplexes were analyzed on 16% denaturing acrylamide gel. Size marker is shown on the left.(TIF)Click here for additional data file.S6 Fig6N. benthamiana genome matching read counts.miRNAs analyzed are listed on the left. The normalized read counts values are shown on a log10 scale, values under threshold (ut). Read counts were normalized to 10(TIF)Click here for additional data file.S7 FigAbundance of miRNAs (listed on the left) in IP fraction were calculated relative to their inputs. The normalized values are shown on a log2 scale. Heatmap is shown on the right.(TIF)Click here for additional data file.S8 Fig6 total read counts.Location of vsiRNAs on the viral genome is presented on the x-axis, read counts are shown on the y-axis. Y-axis positive and negative values represent read counts derived from the positive or negative viral strand, respectively. 21nt and 22nt vsiRNAs are indicated in blue and red, as shown. vsiRNAs in AGO1-IP derived from CymRSV-infected plants is likely due to the nonspecific background as discussed in the text (black asterisk). Read counts were normalized to 10(TIF)Click here for additional data file.S9 FigN. benthamiana specific reads from mock-inoculated (A), CymRSV- (C) and Cym19stop-infected wild-type plants (E). The vsiRNA reads of the same samples are presented in (B), (D) and (F), respectively. 5’ nucleotides of vsiRNAs are indicated by color code on the right. Size classes of sRNAs are indicated by numbers. Read counts were normalized to 106 total reads. Note the different scale in panel A.(TIF)Click here for additional data file.S10 FigFor detailed data processing information, please see (TIF)Click here for additional data file.S11 Fig(A) Genomic organization of pTBSV-PDS-GFP and pTBSV-PDS-NbAGO1-1 VIGS constructs. (B) TBSV virus accumulation in NbAGO1-silenced, GFP-silenced and mock-infected (negative control) plants. (C) GFP-silenced but not NbAGO1-silenced plants show fast recovery phenotype.(TIFF)Click here for additional data file.


In December 2013, the World Health Organization reported the first local transmission of chikungunya virus in the Western Hemisphere, with autochthonous cases identified in Saint Martin  with positive tests for recent chikungunya virus infection from one of the four U.S. laboratories that perform testing. All were travelers visiting or returning to the United States from affected areas, mostly in Asia . Only 23Chikungunya virus infection should be considered in patients with acute onset of fever and polyarthralgia, especially travelers who recently returned from areas with known virus transmission. Chikungunya virus diagnostic testing currently is performed at CDC, three state health departments , and one commercial laboratory (Focus Diagnostics).No specific treatment, vaccine, or preventive drug is available for chikungunya virus infection. Treatment is palliative and can include rest, fluids, and use of analgesics and antipyretics ,3. Most Aedes species mosquitoes are currently active. State health departments are encouraged to report confirmed chikungunya virus infections to CDC through ArboNET (Health-care providers are encouraged to report suspected chikungunya cases to their state or local health department to facilitate diagnostic testing and mitigate the risk for local transmission. CDC and the Council of State and Territorial Epidemiologists urge health departments to perform surveillance for chikungunya cases in returning travelers and be aware of the risk for possible local transmission in areas where  ArboNET .


Ixodes ricinus) in Belgium. The genome consists of two segments, L and S, and is most similar to the tick-borne South Bay virus, with amino acid identities ranging from 60 to 64%.We report here the draft genome of a novel nairovirus, Grotenhout virus, isolated from deer ticks ( Nairovirus forms one of five genera within the family Bunyaviridae, a large group of enveloped, negative-sense, single-stranded RNA viruses with a trisegmented genome organization known to infect a wide variety of hosts, ranging from different types of mammals to a plethora of insects  originating from Belgium.The genus  insects . Membersortality . We repode novo assembly was generated using the CLC Genomics Workbench  by coassembling both paired and unpaired reads. A tBLASTx search of the generated contigs identified 13 contigs, composed of 3,783 reads, that displayed significant similarity to South Bay virus, an unclassified nairovirus . Next, the RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen). The extracted RNA was amplified by whole-transcriptome amplification  and subsequently subjected to Illumina NextSeq 500 sequencing (Illumina). A Bunyaviridae, based on the coding sequence of the L segment, groups this virus within the genus Nairovirus, with South Bay virus being the most closely related virus. The L segment contains a 4,812-amino acid (aa) polymerase gene displaying 64% amino acid similarity with South Bay virus, while the S segment contains a 551-aa nucleocapsid gene (60% similarity with South Bay virus). Members of the family Bunyaviridae, including those belonging to the genus Nairovirus, typically have a third genomic segment, the M segment, which encodes two or more structural glycoproteins. Despite the use of extensive bioinformatics-based search strategies, a putative M segment for Grotenhout virus could not be identified, something also observed for South Bay virus  and KY700684 .The genome sequence of Grotenhout virus has been deposited in NCBI GenBank under the accession numbers


Micromonas. We furthermore assessed how temperature influenced viral infectivity and production. Phylogenetic analysis indicated that the putative double-stranded DNA (dsDNA) Micromonas polaris viruses (MpoVs) are prasinoviruses (Phycodnaviridae) of approximately 120 nm in particle size. One MpoV showed intrinsic differences to the other three viruses, i.e., larger genome size (205 ± 2 vs. 191 ± 3 Kb), broader host range, and longer latent period (39 vs. 18 h). Temperature increase shortened the latent periods (up to 50%), increased the burst size (up to 40%), and affected viral infectivity. However, the variability in response to temperature was high for the different viruses and host strains assessed, likely affecting the Arctic picoeukaryote community structure both in the short term  and long term .Global climate change-induced warming of the Artic seas is predicted to shift the phytoplankton community towards dominance of smaller-sized species due to global warming. Yet, little is known about their viral mortality agents despite the ecological importance of viruses regulating phytoplankton host dynamics and diversity. Here we report the isolation and basic characterization of four prasinoviruses infectious to the common Arctic picophytoplankter  Micromonas pusilla MicromonaMicromonas species and M. polaris strains were tested for susceptibility to infection by the four MpoVs  were taken at several time points within the latent period. Samples were prefixed with glutaraldehyde  for 30 min on ice. Algal cells were concentrated by low-speed centrifugation , after which the supernatant was decanted and the pellets were transferred to 1.5 mL Eppendorf tubes (three per tube) using a Pasteur pipet. These samples were further concentrated by centrifugation , followed by the transfer of two of the pellets into one Eppendorf tube and another round of centrifugation . Finally, these samples were fixed with glutaraldehyde  in 1 mL citrate-phosphate buffer  containing 2.5 mM CaCl2 on ice.For ultrastructural analysis by TEM, thin sectioned samples were prepared. Briefly, exponentially growing algal cells of 4 in water, and washed again in distilled water with centrifugation steps in between to spin down the algae. After the last spin down, the supernatant was removed and the algae were re-suspended in the remaining volume. An equal volume of 12% gelatin was added to the algal sample and centrifuged again to a non-compact pellet. The gelatin was solidified on ice and after 20 min a fixative  was added to let the gelatin fixate overnight. The gelatin containing the algae was cut into small blocks of 1–2 mm2 and dehydrated through a series of ethanols . As a last dehydration step, propylene oxide was used before the samples were embedded in LX-112 resin. After polymerization at 60 °C, ultrathin sections of 90 nm were cut on a Reichert EM UC6 with a diamond knife, collected on Formvar coated grids and stained with uranyl acetate and lead citrate. Sections were examined with a FEI Tecnai-12 G2 Spirit Biotwin electron microscope , and images were taken with a Veleta camera using Radius software . The data analysis program within Radius was used to perform measurements of the virus particle size. The capsid diameter was measured for 100–400 virus particles per infection, discriminating intracellular and extracellular particles.After fixation, the algae were washed in distilled water, osmicated for 60 min in 1% OsOMicromonas viruses has been reported to possess a lipid membrane [v/v) chloroform for 10 min, after which the chloroform was separated by centrifugation  and the aqueous phase containing the viruses was recovered (in new 1.5 mL Eppendorf tubes). Tubes were left overnight at 3 °C to allow any remaining chloroform to evaporate. Treated lysates were added to exponentially growing cultures in 5 mL borosilicate tubes  and incubated at standard light conditions and 3 °C. Non-infected negative controls received the same volume of media. Tubes were screened for lysis twice a week for three weeks. Recently, a new group of membrane . To testmembrane ,52. Aliqg for 30 min at 4 °C using a fixed angle rotor (type F 34-6-38) with conical adapters to fit the 30 mL Nalgene Oak Ridge centrifuge tubes in a Eppendorf 5810R centrifuge . Viral genome sizes were determined by Pulse Field Gel Electrophoresis (PFGE) according to Baudoux and Brussaard [g for 2 h at 8 °C, using a fixed-angle rotor Beckman Coulter type 50.2Ti, in a Beckman Coulter Optima XPN-80 ultracentrifuge) . Pellets were resuspended in 150 μL SM buffer  glycerin), after which agarose plugs were prepared by mixing equal volumes of molten 1.5% (w/v) agarose  with the virus concentrate in plastic molds. Plugs were incubated overnight at 30 °C in lysis buffer with proteinase K, followed by washing in TE buffer  and storage in TE buffer  at 4 °C until analysis. Plugged samples were loaded onto 1% SeaKem GTG agarose gels  prepared in 1 × TBE gel buffer  and run in a PFGE Bio-Rad CHEF DR-II cell unit , and corresponding CHEF DR-II chiller system, filled with 2 L 0.5 × TBE buffer , pre-cooled at 15 °C. Plugs were loaded with 0.5 × TBE buffer , at 6 V cm−1 with pulse ramps of 20 to 45 s at 14 °C for 22 h. Molecular size markers were included: DNA Lambda ladder plugs (Bio-Rad) and Saccharomyces cerevisiae DNA ladder plugs (Bio-Rad). Gels were visualized in a FluorS imager (Bio-Rad Instrument) after staining with SYBR Green I . Viral genome sizes were estimated in comparison to a molecular size marker (n ≥ 2). Mean ± standard deviation were determined and the differences were tested by ANOVA (significance level p = 0.05) and Holm-Šidák multiple comparisons.MpoV lysates (~25 mL) were partially purified from cell debris and bacteria by centrifugation at 10,000× russaard . In shorMicromonas viruses, we amplified a part of the DNA polymerase B gene (polB) using the primer pair AVS1/AVS2 [2), 0.25 mM of each dNTP, 0.8 µM of each primer, and 0.4 mg/mL BSA. Negative controls contained all reagents except the template. PCR cycling included an initial denaturation at 94 °C (4 min) followed by 37 cycles of denaturation at 94 °C (30 s), annealing at 45 °C (30 s), and extension at 72 °C (1 min), followed by a final extension at 72 °C (7 min). Sequencing was performed by BaseClear Ltd. . Based on 178 amino acid positions, a Maximum-Likelihood dendrogram was constructed with RAxML [polB sequences of Bathycoccus viruses . These, and the polB sequences of Ostreococcus viruses  were grouped to obtain a more compact tree. For comparison we added published polB sequences of the Micromonas virus isolates and contigs of an Arctic metagenome [polB fragment that we analyzed. Contig-95-10186 and contig-79-31207 were shorter  and were later added to the dendrogram via ARB Parsimony.To determine the phylogenetic relationship between our new Arctic virus isolates and other VS1/AVS2 . The virth RAxML  implemenWe studied the effect of different temperatures on virus growth characteristics as well as on virus stability (loss of infectivity). To determine how the virus-host interaction might be affected over a range of different ecologically relevant temperatures, we used our model system of TX-01 with MpoV-45T, as both the host and virus were isolated in the same location and same period. Temperature sensitivity of the viral latent period and burst size were examined by one-step viral growth experiments at a range of growth temperatures, i.e., 0.5, 2.5, 3.5, and 7 °C. This range of temperatures represents natural water temperatures during the Arctic growth season ,24,25. An = 5; 12 × 10-fold dilutions). In each MPN rack one additional row of tubes containing non-infected culture  served as a negative control. The MPN cultures were incubated at 3 °C under standard light conditions and were inspected at least once a week for 3 weeks for lysis. The titers were determined with the MPN Assay Analyzer [8, 4.6 × 109, 2.1 × 101, and 4.6 × 109 mL−1 for MpoV-44T, 45T, 46T, and 47T, respectively. Statistics were carried out in SigmaPlot 13.0 . Differences between the viruses and temperature treatments were tested by ANOVA  and Holm-Šidák multiple comparisons, either directly or after log transformation.Viral infectivity was determined after exposure at −196, −80, −20, 0, 3, 7, and 15 °C and determined using the Most Probable Number (MPN) assay on the host strain RCC2258. This host strain was used instead of TX-01, because the latter did not grow well in the 5 mL tubes that we used for the assays . Aliquots of virus lysates (3 mL) were exposed to the different temperatures for 24 h when the exposure temperature was below zero and for 1 h when exposure temperature was above zero. Following exposure, viral lysates were added to the algal host  and a thick outer layer surrounding an electron-dense inner core  using AVS1/AVS2 primers that were originally designed for dsDNA prasinoviruses [polB phylogeny based on inferred amino acid sequences indeed grouped the newly isolated MpoVs with other Micromonas viruses, but did not show a close match  to any of the other Arctic sequences  but were significantly smaller (p < 0.008) than the genome of MpoV-44T which displayed a genome size of 205 ± 2 Kb . No relationship could be established between virus infectivity or host susceptibility to infection based on the time of isolation, geographical origin, or host culture temperature.The virus isolates were specific for the genus cromonas , but werp = 0.317) and the averages varied between 233 and 296 viruses produced per lysed host cell . The latent period of MpoV-45T did not change with temperature  and declined with lower temperatures by 15% and 28% for 2.5 and 0.5 °C, respectively, compared to 3.5 °C , the virus growth characteristics revealed host-specific effects. MpoV-45T showed a shorter latent period at higher temperature when RCC2257 was the host (from 12–18 h to 6–12 h), whereas no such effects were observed on hosts TX-01 and RCC2258; p = 0.044), which was also smaller than that for MpoV-45T (115% compared to 150%). Irrespective of temperature, on host RCC2258 the viral burst sizes of MpoV-45T were higher than those of MpoV-44T, but for host RCC2257 no such difference was observed .There were also virus-specific responses to temperature, as an increase from 3 °C to 7 °C showed a stronger effect on the latent periods of MpoV-44T rather than that of MpoV-45T, reducing it by roughly 50% from >30 h to 12–18 h on both hosts. Moreover, the viral burst sizes of MpoV-44T showed only a significant increase at 7 °C on host RCC2257  their sensitivity to infection of LAC38 or CCMP1545, (ii) genome size (206 ± 6 Kb (n = 12) vs. 191 ± 4 Kb (n = 8)), and (iii) presence of a lipid membrane. Strikingly, all LAC38-infecting viruses with larger genomes contained a lipid membrane, whereas the smaller genome sized CCMP1545-infecting ones did not. Our data show a similar larger genome size for MpoV-44T which infects LAC38 , but in our case all MpoVs contained a lipid membrane. To our knowledge this is the first report of the isolation and characterization of phycoviruses from polar marine waters. Similar to other reports of st cells . The parst cells . These aMicromonas viruses belonging to the genus Prasinovirus. The genus Prasinovirus infects Ostreococccus and Micromonas species and is one of the six virus genera belonging to the Phycodnaviridae family; eukaryotic algal viruses with large dsDNA genomes (100–560 Kbp) [polB that the Arctic Micromonas viruses do not form a separate cluster. Screening the Tara Oceans’ contigs and the KEGG Environmental database for homologs of the amplified nucleotide region of our MpoV isolates revealed a worldwide distribution and high diversity on thermal stability  ,66. A re560 Kbp) . The newMicromonas strains, but MpoV-44T could be distinguished from the other Arctic MpoV isolates based on its capability to virally infect M. commoda LAC38. Although LAC38 was being cultured at low temperature (3 °C) at the time of virus isolation, it is originally a temperate Micromonoas strain  [Micromonas host strain. These differences may also be due to MpoV-44T having been isolated during midwinter and years before the other MpoVs (isolated in summertime during two consecutive years). Successional patterns for marine virus communities with associations to temperature and host dynamics have been demonstrated  and low light  . We cannd . Even t0.2 d−1, ), photosCMP2099; ) that coCMP2099; ). Yet, oCMP2099;  was respCMP2099; . Additiothe host ,72. The Micromonas host and virus succession. The infectivity loss at higher temperature (7 °C) for MpoV-45T and MpoV-46T shortens their window of optimal activity during the warmer summer months. Considering that the range of temperatures we tested are ecologically relevant for the Artic seas  was highly variable and strain-specific. Two of the four virus isolates MpoV-45 and 46T) even showed a loss of infectivity above 0 °C. All of the virus isolates, except for MpoV-44T, were able to maintain over 25% of their infectivity after being frozen at –20 °C. This suggests that these viruses are well able to withstand the freezing process during ice formation, a property which would maintain high titers during winter. Similar findings have been reported for Arctic marine bacteriophages  and dsDN and 46T nd 7 °C; ), our reM. polaris strain TX-01, MpoV-44T displayed a much longer median latent period than the other Arctic MpoVs . A comparably long latent period had, until recently, only been described for the dsRNA virus MpRV infecting M. commoda LAC38 . Although Baudoux et al. [2 = 0.952, p = 0.018). Virus MpoV-45T did not show a dependency on host growth rate , but instead showed a shortened latent period on host RCC2257 at the highest temperature (7 °C compared to 3 °C). Furthermore, the latent period of MpoV-44T was strongly affected by temperature whereas increasing temperature only shortened the MpoV-45T latent period on RCC2257. On host TX-01, the latent period was unaffected over the whole range of 0.5 to 7 °C, however, we found a steeper increase of viruses with increasing temperature, i.e., virus production rates of 0.18, 0.37, 1.4, and 1.6 × 105 viruses h−1 between 16 and 30 h for 0.5, 2.5, 3.5, and 7.0 °C, respectively. While the data confirm our second hypothesis that the temperature increase stimulates MpoV production , there is nonetheless a virus-specific response for the range of temperatures tested. Instead we found a high variability in response for the different virus isolates and host strains. When looking at a temperature increase from 3 to 7 °C, most virus-host combinations in our study showed enhanced viral burst sizes with a temperature-regulated increase in the host growth rates (0.53–0.59 d−1 at 3 °C and approximately 1.2-fold higher at 7 °C). However, MpoV-45T infecting TX-01 did not show this increase from 3 to 7 °C, but did exhibit an increasing burst size with temperatures from 0.5 up to 3.5 °C . Hence, the optimum temperature for virus production was not the same as the optimum host growth temperature. Wells and Deming [Colwellia psychrerythraea strain 34H, had a burst size optimum at −1 °C while the host’s growth rate optimum was at 8 °C. These authors suggested that specific virally encoded enzymes have their own optimum temperatures. When propagated on the putative 8 . Howeve8 . By the end of May, the temperatures and consequently the growth rates had increased to 2–3 °C and 1.1 d−1, respectively. This 20–50% growth rate increase is similar to TX-01 in the present study over the same temperature range. Our results indicate that over an Arctic growing season with increasing temperatures and host growth rates, viral activity can be expected to increase as a result of the decreasing latent periods and increasing burst sizes. Our data imply that temperature could affect host and virus diversity (strain dynamics), as for MpoV-45T with host TX-01 the latent period and viral burst size did not further change above 3 °C, but with other hosts (RCC2257 and 2258) and other viruses (MpoV-44T) the latent periods shortened and/or burst sizes increased to several extents. The different susceptibilities of viral infectivity to temperature, with a tolerance for the highest temperatures for MpoV-44T, strengthened this idea even more. Tarutani et al. [Heterosigma akashiwo and the lytic virus HaV, a successional shift in clonal composition occurred due to differences in susceptibility/resistance of the host to the viruses. Based on our data a similar shift in strain and clonal composition of Micromonas and MpoV may occur, not only due to differences in susceptibility to the viruses but also because of differences in virus proliferation success at different temperatures [Temperature strongly regulates Arctic s study; ,29). Thes study; ,25. At ti et al.  showed heratures . Micromonas which belongs to the picophytoplankton fraction and is expected to be favored under future Arctic conditions  isolation, characterization of virus-host dynamics, and responses to changing ecologically relevant environmental factors are fundamentally essential to understanding the role of algal viruses in (Arctic) marine waters. The newly isolated viruses make it possible to comprehensively investigate the interactions of these unique virus-host combinations under climate change relevant environmental variables. Joli et al. [Micromonas viruses by metagenome sequencing. It would be interesting to investigate the ecological relevance of the strains tested in our study using molecular approaches. In the natural environment, selective effects of temperature may drive (intra)species diversity, potentially affecting the ability of Micromonas to respond to the changing conditions of the vulnerable Arctic. Modeling studies could help to comprehend (and predict) the extent to which the Arctic phytoplankton community would be influenced by changes in infection dynamics associated with temperature changes. In summary, the present study describes the first isolation and characterization of viruses infecting a cold-adapted polar phytoplankter. The relevance seems high, as it concerns the ubiquitous genus ication; ,27,28,29ication; ,24 and cication;  have beei et al.  showed t


Varroa destructor has been well documented. However, DWV is just one of many insect RNA viruses which infect a wide range of hosts. Here we report the full genome sequence of a novel Iflavirus named Moku virus (MV), discovered in the social wasp Vespula pensylvanica collected in Hawaii. The novel genome is 10,056 nucleotides long and encodes a polyprotein of 3050 amino acids. Phylogenetic analysis showed that MV is most closely related to Slow bee paralysis virus (SBPV), which is highly virulent in honey bees but rarely detected. Worryingly, MV sequences were also detected in honey bees and Varroa from the same location, suggesting that MV can also infect other hymenopteran and Acari hosts.There is an increasing global trend of emerging infectious diseases (EIDs) affecting a wide range of species, including honey bees. The global epidemic of the single stranded RNA Deformed wing virus (DWV), driven by the spread of  Emerging and re-emerging diseases affecting a diverse range of organisms pose an ongoing threat to global health and food security. The transmission of DWV around the world in conjunction with Varroa is a well-studied example of an emerging insect pathogen1RNA viruses, such as Deformed wing virus, are of particular interest due to their lack of host specificity and capacity to jump between hosts345It is becoming apparent that insects which interact with honey bees can act as viral reservoirs and infect honey bees via spillover events311Vespula pensylvanica is a common species native to the western half of temperate North America. In the Hawaiian archipelago, it was first recorded in Kauai in 1919, but was not recorded on Maui and the Big Island until 1978V. pensylvanica is a general predator that feeds on a wide range of arthropods including the honey bee, Apis melliferaV. pensylvanica as an invasive species with widespread geographic distribution, abundant numbers in some areas and generalist feeding preferences including its use of floral resourcesThe predatory social wasp, V. pensylvanica. We named the novel virus “Moku”, which means Island in Hawaiian.Here we report a novel RNA virus, genetically dissimilar to any other virus at the nucleotide level, detected in Iflavirus  and phylogenetic analysis confirmed that Moku virus (MV) lies within the genus flavirus . The genflavirus . The genflavirus . In addig Island .Iflavirus genome structure; structural proteins in the N-terminal region and non-structural proteins in the C-terminal region   . No consSince we were unable to assemble a full-length MV genome from both the honey bee and Varroa samples, pairwise comparison between these genomes was not possible. Taken together, genomic variants of MV were observed in both the honey bee and Varroa samples. The amino acid identities between MV variants were highly conserved , therefoV. pensylvanica, virus reads can make up to 91.5% of total Illumina reads (average of 54.6%)  preference not to use host species names to assign virus speciesAdvancements in NGS technologies have enabled an exponential increase in RNA virus discoveryf 54.6%) . Due to f 54.6%)  and fullf 54.6%)  observedBombus spp.) rather than honey beesV. pensylvanica in Hawaii and Vespa velutina (Asian hornet) in Europe could act as a new transmission route and source of emerging viruses in honey bees.Moku virus is most closely related to SBPV . SBPV isVespula pensylvanica to honey bees (or vice versa as V. pensylvanica is known to predate on honey bees) and then onto Varroa. Once in Varroa, transmission at epidemic proportions within honey bee populations is a possible outcomeV. pensylvanica supplements its diet by feeding on floral nectarDetection of MV in Varroa is of concern, as Varroa is known to facilitate the spread and amplification of some RNA virusesThe dominance of the DWV type A master-variant in honey bees and Varroa is the result of the arrival of Varroa on Big Island, which facilitated the spread of this variantin vitro that DWV can cause premature death of adult honey beesV. pensylvanica  contained several orders of magnitude less MV reads than the other wasp samples  to confirm the quality of the raw read data. An in-house contamination-screening pipeline called Kontaminant (http://www.tgac.ac.uk/kontaminant/) was used to check for any contamination in the raw reads. The wasp libraries showed less than 5% of host mRNA. Even with very low host contamination, kmer filtering was performed to remove any host RNA. There was no viral mapping/filtering done, so we carried out a metagenomic study to assemble all the non-host RNA.QC was done using FastQC  were assembled together in a single assembly run using MetaCortex . MetaCortex is a recently developed variant of Cortex27Iflavirus amino acid sequences obtained from Genbank. The alignment was carried out using the Muscle aligner with 8 iterations. The phylogenetic tree was built by the Geneious tree builder using a neighbour joining method and the Jukes-Cantor genetic distance model based on the conserved RdRP region of picorna-like virusesOne contig in particular was translated within Geneious (Biomatters) and aligned with other Individual reads were aligned against the novel Moku virus genome to create coverage plots for each Illumina sample . From th15The insect viromes  were creHow to cite this article: Mordecai, G. J. et al. Moku virus; a new Iflavirus found in wasps, honey bees and Varroa. Sci. Rep.6, 34983; doi: 10.1038/srep34983 (2016).


Aedes genus in the past decades has been associated with major Alphavirus-associated outbreaks. Climate changes and intensification of air travels have favored vector expansion and virus dissemination in new territories leading to virus emergence not only in tropical areas but also in temperate regions. The detection of emergence is based upon surveillance networks with epidemiological and laboratory investigation.Alphaviruses are arthropod borne RNA viruses of medical importance. Geographical expansion of mosquitoes of the Alphavirus is critically required. To address this issue, we developed a new molecular assay targeting nsP4 gene and using a TaqMan® real time RT-PCR method for the specific detection of all major Alphavirus genus members.A specific, sensitive and rapid screening test for genus-specific Alphavirus species. We also tested successfully clinical sensitivity using patient’s samples collected during the Chikungunya outbreak of 2005–2006 in the Indian Ocean.This assay was tested for specificity using several Alphavirus molecular diagnostic tool offers great potential for exclusion diagnosis and emergence detection given its broad specificity restricted to Alphavirus genus.This new pan- Alphavirus belongs to the family Togaviridae and comprises 29 virus species. Alphaviruses have a positive single stranded RNA. All of them are arthropod-borne except salmon pancreatic disease virus (SPDV) and southern elephant seal virus (SESV). Transmission involves intermediate host from avian or mammal origins for virus replication and mosquitoes as vectors for spillover infection in humans. Alphaviruses form icosahedral structures of 70 nm in diameter that contain a 5′ capped- and 3′ polyadenylated genome. Virus genome is ranging from 10 to 12 kb in length and comprises 2 open reading frames (ORF) separated by an intergenic sequence. 5’ORF encodes a polyprotein comprising 4 non-structural proteins (nsP) 1 to 4 and is involved in a replicative complex. The subgenomic RNA encodes for instance 3 structural proteins, E1, E2 and Capsid that interact to form virion envelope [Alphaviruses form a small subset among all reported arboviruses. In the international arbovirus catalogue maintained by the Centers for Disease Control and Prevention (CDC – Atlanta), 537 viruses are actually reported; 9 of them are known to be Alphaviruses of medical relevance, Chikungunya virus (CHIKV), Ross river virus (RRV), O’nyong nyong virus (ONNV), Sindbis virus (SINV), Mayaro virus (MAYV), Barmah forest virus (BFV), Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) [The genus Large outbreaks of CHIKV disease were documented in Gabon 1999), Indian Ocean (2006), India and Southern Asia 2006, 2007) –7. ONNV  –7. ONNV99, IndiaAedes and Culex genus are effective contributors of virus to human transmission. In the case of Aedes, 2 species Aedes aegypti and Aedes albopictus are involved. Aedes mosquitoes contribute to Alphavirus circulation inside the tropical and subtropical belt but Aedes albopictus is prone to geographical expansion to North countries from the late 1970s and spreading from East European countries to Mediterranean countries [Alphavirus epidemiology is intrinsically related to homophilic arthropod vectors distribution. Among these vectors, mosquitoes of ountries . This isountries .Alphavirus infections may be related to arthritogenic (Old World) alphaviruses or encephalitic (New World) alphaviruses. Classically, Old World alphaviruses like CHIKV cause an infection including a 4–7 days incubation period followed with the apparition of clinical signs including strong fever, headache, myalgia, arthralgia, maculopapular rash, edemas, abdominal pains and encephalitis in 5% of the [Alphavirus infections remain sometimes asymptomatic or can lead to chronic arthritic features over time [Human % of the . New Wor% of the , 16. Morver time .Since 2006, imported CHIKV human cases were annually reported in France during the summer with limited autochthonous virus circulation in some cases. This was related to recreational activities and air travels over the world in CHIKV endemic regions –20. ThisAlphavirus emergence is subjected to many determinants. CHIKV outbreak in Indian Ocean in 2005–2006 provided an example of adaptation of virus to vector. The adaptive CHIKV E1-A226V mutation was responsible for higher infectivity and virus transmission by Aedes albopictus [bopictus –24. The bopictus . Virus aAlphavirus infections may result in a non-specific syndrome it is difficult to accurately diagnose these infections. Other arboviruses including Flavivirus, Bunyavirus, Rhabdovirus and Reovirus can lead to the apparition of the same symptoms in humans and this is also reported for infection by pathogenic leptospira and Plasmodium. Detection of virus emergence is sometimes complicated by the cocirculation of 2 distinct arboviruses circulating concomitantly in the same geographical area or by successive outbreaks involving different viruses of Alphavirus or Flavivirus genus. In French Polynesia, successive outbreaks of DENV, CHIKV, and ZIKA virus were reported in the past five years. Moreover, RRV circulation was serologically reported supporting autochthonous RRV circulation in a silent manner [Given that human t manner .Alphavirus genus [Alphavirus and good clinical sensitivity [Alphavirus nsP4 sequences with high sensitivity. Moreover, the format used for the assay design is compatible with routinely implemented molecular tests in clinical laboratories and will simplify diagnosis exclusion.Differential diagnosis is often necessary to identify the infectious causative agent. Moreover, in case of a new emergence, an extensive panel of pathogen-specific detection tests can be difficult to set up. To overcome this constraint a genus-specific test is required to identify clinical samples positive for alphaviruses. In the acute phase of infection with high levels of viremia, RT-PCR is best suited for virus detection. In the last two decades, several nested or hemi-nested RT-PCR assays were designed to detect members of us genus –30. For sitivity . UnfortuComité Consultatif de Protection de Personnes se prêtant à des Recherche Biomédicales’, Bordeaux France, ref. 2008-A00151–54).Written informed consent was obtained from healthy subjects and patients undergoing arbovirus infection screening and attending Reunion Island University Hospital. The study was approved by the Human Ethics Committee of University of Bordeaux . RRV and SINV were purchased from the National Collection of Pathogenic Viruses . CHIKV strain clone #4.2 was isolated from a clinical sample in our laboratory during the CHIKV outbreak that occurred in Reunion Island in 2005 . Other vAdditionally we used RNA standards for quantification of WEEV, EEEV, VEEV, BFV and CHIKV. RNA standards were purchased from Eurogentec and designed with RNA nucleotides using specific viral sequences selected at the location corresponding to the expected amplicon generated by our RT-PCR assay. Accession number of selected sequences was AM258995 (CHIKV), NC001786 (BFV), GQ287640 (WEEV), KC344475 (VEEV), KP282670 (EEEV).Total nucleic acids were extracted from 200 μL aliquots of plasma human samples or supernatant from cell cultures using Nuclisens® reagents and EasyMAG® nucleic acid isolation platform according to Biomerieux’s recommendations. Final elution was done in 50 μL.BioEdit Sequence Alignment Editor v7.1.3. Sequence alignment was performed with a Clustal tool provided as a plugin in BioEdit software [Alphavirus genomic sequences were available from Virus Pathogen Database and Analysis Resource . Sequencsoftware . Meltingsoftware . LNA™ prTo efficiently prime RT-PCR for pan-Alphavirus detection, we applied a degenerate primer design according to Li  and consAlignment of consensus sequences was a prerequisite to select a nucleotide sequence with high similarity between viruses tested in our study. As mentioned by Grywna, nsP4 gene appeared to be highly conserved and thus acted as a good candidate for amplicon design . Using GAlphavirus genus. We designed within the amplified region two probes with a length ranging from 14 to 17 nucleotides and harboring LNA™ modifications to fit thermal requirement of a TaqMan® probe.Degeneracy of a primer is the total number of sequence combinations it contains. For this purpose, inosine was used as an alternative for N nucleotide into forward and reverse primers with ambiguous nucleotides. To take into account nucleotide variation inside the reverse primer, we designed a mix of 3 similar primers harboring different degenerated nucleotide at ambiguous positions. This allowed us to select for primers with 3′ extremity that will match with viral targeted sequences. Primers’ combinations with or without inosine were ultimately compared .RNA standards corresponding to designed amplicons were diluted at the concentration of 1.35 10Alphavirus genus and 15 unrelated viruses from different lineages .Tests comparison was assayed for statistical significance using two-tailed Student test with α-risk set at 0.05 using GraphPad Prism v5.0  obtained from 6 CHIKV RNA samples or 6 RRV RNA samples (data not shown). Averaged CHIKV ∆Ct was set at +0.27 and did not account for differences between probes. In contrast, in our pan-Alphavirus assay, averaged RRV ∆Ct at −1.80 clearly indicated a better performance with P1 probe, ie, the shorter LNA® probe. Therefore, we decided to select inosine-enriched primers in association with a short LNA® probe and use mix2 for the remaining validation studies of our assay.We further investigated the performance of these two probes by calculating ∆CtAlphavirus. We successfully detected genomic sequence of CHIKV, ONNV, RRV, SINV and SFV  and the limit of detection was ranging from 26 to 111 copies/run depending on which RNA standard was used. This was also concordant with the different LODs ranging from 5 to 100 copies per reaction obtained with Grywna’s pan-Alphavirus nested RT-PCR assay [Linearity of the assay was optimal for each specific RNA standard tested with RCR assay .Alphavirus detection. A couple of RT-PCR methods were already available but were difficult to use in clinical laboratories because of their endpoint PCR format involving amplicon detection. Gel electrophoresis, amplicon sequencing even mass spectrophotometry were used for PCR product visualization. All these methods involved the need to work with open tubes thus raising the risk of cross-over contamination. Our real time RT PCR assay addressed and solved this issue by generating and detecting amplicon at the same time in closed tubes.The aim of our study was to implement a new molecular method for Our approach took benefits from careful alignments of available genomic sequences prior to virus-specific consensus sequence generation. In silico analysis was crucial to select for virus consensus sequences, allowing accurate determination of ambiguous nucleotide positions in primers and probes. We especially took into account primers’ nucleotide variation in the 3′ extremity. Primers’ 3′ ends are important for primer extension as unfortunate mismatch in the 5 last 3′ nucleotides usually compromise efficient priming of the PCR reaction. To avoid mismatches between primers and template, 3 strategies were developed: we used degenerated primers; we chose to blast our reverse primer into 3 distinct primers harboring different ambiguous nucleotide combination and to mix them; we used inosine enriched primers instead A or T or C or G containing primers. The latter approach allowed us to select for primers with limited degeneracy . We obseWe also checked for the performance of two Taqman® probe designs incorporating LNA modified nucleotides; the former consisted of a short 14 nucleotide stretch with ambiguous position, the latter was longer with inosine enrichment. The two probes performed equally well but in some cases a better result was obtained with the shorter probe despite of its residual degeneracy. Classically shorter LNA® probes behave better and our design was not affected by a low level of degeneracy.Alphaviruses using this test. These Alphaviruses include members of the SFV antigenic complex, Getah virus and Bebaru virus; members of the VEEV complex, Fort Morgan virus, Highlands J virus; Ndumu virus and Middelburg virus that are related to 2 distinct antigenic complexes and responsible for human illness [Getah virus infects horses, pigs and goats and Highland J virus is found in horses and poultry [Hendra virus, a member of Henipavirus genus is not an arbovirus. It is hosted in frugivorous bats causing a zoonosis affecting horses and incidentally humans. Due to increased exposure to natural host’s reservoir, human could contract infection with different and multiple Alphaviruses that usually target animals. Risk assessment leading to cross species’ barrier is hard to establish but can result in new virus emergence. The potential of our test to detect unsuspected Alphavirus in human related disease may be of great clinical interest.Our test was validated using clinical samples for CHIKV, ONNV, SINV, SFV and RRV. We also used synthetic RNA standards to detect WEEV, EEEV, VEEV and BFV sequences. Alignments of genomic sequences indicated a potential for the detection of other  illness . Interes poultry , 41. TheAlphavirus infections are known to cause high viremia in humans and should be easily detectable by RT-PCR. Serological assays could be used in complement at a later stage of the disease [Alphavirus in disease etiology.TaqMan® assays perfectly suit constraints for virus emergence detection at early stage of disease. In the acute phase of the disease,  disease . Our tes


Estimating health care costs, either in the context of understanding resource utilization in the implementation of a health plan, or in the context of economic evaluation, has become a common activity of health planners, health technology assessment agencies and academic groups. However, data sources for costs outside of direct service delivery are often scarce. WHO-CHOICE produces global price databases and guidance on quantity assumptions to support country level costing exercises. This paper presents updates to the WHO-CHOICE methodology and price databases for programme costs.We collated publicly available databases for 14 non-traded cost variables, as well as a set of traded items used within health systems . Within each of the variables, missing data was present for some proportion of the WHO member states. For each variables statistical or econometric models were used to model prices for each of the 194 WHO member states in 2010 International Dollars. Literature reviews were used to update quantity assumptions associated with each variable to contribute to the support costs of disease control programmes.A full database of prices for disease control programme support costs is available for country-specific costing purposes. Human resources are the largest driver of disease control programme support costs, followed by supervision costs.Despite major advances in the availability of data since the previous version of this work, there are still some limitations in data availability to respond to the needs of those wishing to develop cost and cost-effectiveness estimates. Greater attention to programme support costs in cost data collection activities would contribute to an understanding of how these costs contribute to quality of health service delivery and should be encouraged. Estimating health care costs, either in the context of understanding resource utilization in the implementation of a health plan, or in the context of economic evaluation, has become a common activity of health planners, health technology assessment agencies and academic groups. A critical component of health care costing is the estimation of resource utilization linked to activities of a disease control programme aimed at supporting the quality of delivery or encouraging the uptake of a specific set of services, but which do not occur as part of direct service provision. Johns et al.  defined We define programme support costs are those which occur outside of direct service delivery, such as the drugs and tests associated with an intervention, but are not shared health system costs such as supply chain and infrastructure. Programme support costs are for those activities taking place at the national, district and province level which are directed towards enhancing the quality of a programme, for example training, supervision visits and monitoring and evaluation. A programme is considered a set of interventions which contribute to the prevention and control of a common health outcome—for example, HIV, maternal and newborn health, non-communicable diseases.q and assigning a price to each resource p. The multiplication of p and q then gives us the cost.Since its conception in 1998, the WHO-CHOICE programme has advocated the use of an ingredients based approach identifying all resources required to deliver a health care intervention, quantifying the resource requirements WHO-CHOICE takes the costing perspective of “the health system”, by which is meant the ensemble of actions and actors whose primary intent is to improve human health. CHOICE therefore includes all direct, market-valued costs, whether public or private, that are required to deliver the intervention, regardless of payer. WHO-CHOICE does not account for non-monetary patient contributions such as travel time, time off work, or lost income—nor do we account for costs outside of the health system, such as the cost of social services whose aim is not primarily health oriented. So the costing perspective of CHOICE is broader than the health sector per se, and is health system focused according to accepted international definitions of the health system. Other sector costs (e.g. legislation) are included to the extent that they are a direct component of the intervention that is intended to improve human health.p and q. WHO-CHOICE GCEA includes patient level costs incurred at the point of delivery, i.e., medicines, tests and health facility visits (including human resources); as well as programme support costs which reflect the additional activities required to run a disease control programme, such as administration, monitoring and evaluation, supervision, legislation, training and law enforcement , but it is also used for other purposes such as the production of global resource needs assessments and investment cases, both of which require processing data for multiple interventions using a standardised format for nt Table . The scoWithin WHO CHOICE, programme costs are included in GCEA as the comparison of single interventions is only the starting point. In constructing an expansion path, the programme costs will change as more interventions are added to the pathway, creating a difference in marginal costs between each additional intervention. The only time when programme costs would not be relevant is in a marginal decision between two interventions which act within the same programme area—for example a choice between metformin and sulfonylurea for treatment of diabetes, where only the costs of the drugs differ. Programme costs are important as they are part of the full cost of delivering the intervention. Without their inclusion the cost-effectiveness ratio calculated could be misleading.A disease control programme within this context is a set of interventions addressing a common need . Disease control programmes may differ significantly in their cost drivers, with those aiming for behaviour change requiring more programme-level costs and less direct patient-level resources, e.g., tobacco control programmes. Similarly, interventions introducing new technologies may require more intense upfront investments in specific components such as training and supervision. Examining programme-level costs is therefore essential to understand the full resource need. In 2005 WHO-CHOICE produced a global price database to facilitate the estimation of programme costs, with a reference year of 2000, along with generic costing templates with quantity assumptions. While quantity assumptions are standardised, the database provides prices at the level of WHO regionIn 2013 a process to update the WHO-CHOICE price database, to a reference year of 2010, along with a review of the quantity assumptions, was started. This paper reports the data sources and methodologies used in the update of the price databases, the new quantity assumptions and suggested scaling factors for programme support costs based on the number and coverage of interventions delivered by a programme.This paper seeks to achieve two aims: (i) to describe the methods used to derive updated estimates for prices of inputs that go into programme costs; and (ii) to define the scope of a set of programme costs that go with a WHO-CHOICE CEA application, and describe new quantity assumptions for the typical WHO-CHOICE CEA application, including newly developed scaling factors.The starting point of the 2010 update of the price database was a review of the 2000 database, including data sources and methodology. Table For each data point, we searched the internet to identify global price databases covering as many WHO member states as possible. This section reports, for each data point, the data reported in the WHO-CHOICE price database and the methodology used to extract data and estimate missing values.WHO region.Population.Total GDP.World Bank income level classification.USD exchange rate and GDP deflators.Number of administrative divisions within each member state.Number of certain types of healthcare facilities including health posts and hospitals.Density of health care facilities per 100,000 people.The database contains the following general information on each WHO member state:Statoids: Administrative Divisions of Countries [Statoids: Administrative Divisions of Countries, uses the most recent national survey and usually dates to sometime in the past 5–6 years. Missing data were imputed using iterative robust model based imputation [The general information including population, GDP and price deflators for each WHO member state is sourced from WHO’s Global Health Expenditure Database . The numountries . The terountries , and mayThe database contains for each WHO Member State a daily subsistence allowance and accommodation rate in USD at 2010 prices for the capital city and a lower and upper range for other parts of the country where available. The average daily cost of living rates for a number of cities was calculated from an online database of cost of living values on numbeo.com . These dex-stock prices were selected over ex-factory prices since they are closer to the final price paid by the buyer. Specifically, they include free carrier charge to move the vehicle to a port ready for export. Freight, insurance and customs duties to export the vehicle are not included in the price because they are likely to vary significantly between destination countries. Operating cost per kilometre is estimated as the sum of the following three expenses.Fuel economy = fuel price/L × fuel consumption/km.Tyre cost/km = cost of four new tyres/average lifespan of tyre in km.Cost of basic maintenance/km .The database contains new vehicle costs for several modes of transport used in the field for health interventions, including sedans, four-wheel drives, utility trucks, motorcycles, mini-buses and refrigerator vehicles. The database also includes their approximate per kilometre operating costs. Prices and vehicle specifications for a set range of vehicles were sourced from UNOPS procurement website, UN Web Buy . These pFuel price was sourced from the World Bank Indicator database , fuel coWHO (2002) Guidelines for Estimating Costs of Introducing New Vaccines into the National Immunization System .WHO’s Performance, Quality and Safety (PQS) Catalogue  and,UNICEF’s Supply Catalogue .The database contains the costs of an assortment of cold-chain equipment and devices relevant to health interventions. The list of cold-chain equipment and devices was compiled based on the general requirements of immunisation programmes, which use cold-chain storage and distribution extensively. As such, these products can also be applied to other disease control programmes requiring cold-chain management. The list was prepared in consultation with the WHO prequalification team (PQT) and with reference to:These cold-chain products are relevant in a variety of contexts, such as different climates and varying health care infrastructure quality. Where a product, such as a cold room or cold box, might have several models varying in characteristics such as size, a single representative model, which is of high quality and value for money, is listed for simplification. This choice was made in consultation with WHO PQT. As of December 2013, all of these products were pre-qualified under WHO’s PQS system. The prices of the specified models were collected from WHO’s PQS Catalogue and UNICEF’s Supply Catalogue. The prices in these catalogues are listed in principal international currencies such as USD, EUR and CHF. In most cases, a price year was also included which varied from 2007 to 2013. Non-USD prices were converted into USD using the WHO’s Global Health Expenditure Database exchange rate for years up to 2011 [The database contains the costs associated with power generation and general utilities for electricity  and water.Energy Prices and Taxes, 3rd quarter 2011 [Data on the price of electricity supplied by mains power was sourced from the International Energy Agency’s (IEA) quarterly publication ter 2011 . Given tter 2011 . Data onter 2011 .The electricity dataset contained data for only 26% of WHO Member States, thus it was not suitable to be included in the Iterative Robust Model Based Imputation. Instead, a simple linear regression using GDP/capita as the explanatory variable was used to estimate missing values.3 in the local currency is calculated based on the use of 15 m3 of water/month and converted to USD. For further information see the GWI survey’s methodology. A water price for each country in the GWI survey was compiled based on the average price of water in its surveyed cities. This national price for 2011 given in USD was converted into a 2010 price using the survey’s available information about the previous year’s price increases. The dataset covered 52% of WHO Member States, and the Iterative Robust Model Based Imputation was used to estimate missing values.Data on water prices was sourced from the Global Water Intelligence (GWI) Survey, conducted in August, 2011 . The GWIFixed (wired) broadband connection charge in USD.Fixed (wired) broadband monthly subscription charge.Monthly subscription for business telephone service (USD).Installation fee for business telephone service.Price of a 3-min local call to a fixed-telephone line (peak rate),Price of a 3-min local call to a fixed-telephone line (off-peak rate),Mobile prepaid—1-min local call .Mobile-prepaid—1-min local call .Mobile prepaid—1-min local call .Mobile prepaid—1-min local call .Mobile prepaid—price of SMS (on network).Mobile prepaid—1 min local call .Mobile prepaid—1 min local call .Mobile prepaid—SMS (off-net).The database contains 14 telecommunications variables:Data was obtained from the International Telecommunications Union (ITU) Information, Communication and Technology (ICT) indicators database , with daPrices are based on a competitive procurement process and economies of scale.Reliable supply of goods at stated price is guaranteed.Large range of goods are costed from a single, up-to-date source.Prices listed included freight and insurance to destination port.The database contains costs associated with office furniture and supplies such as stationery and basic ICT hardware. The list of office supplies, ICT equipment and furniture was prepared by considering the basic resources required to operate a small office. If necessary, these resources were treated as tradable goods that could be imported at a cheaper price than what is locally available. The costs of the office supplies and furniture were sourced from the 2014 Global Supply Catalogue of the US General Services Administration (GSA), which is accessible to US government agencies and eligible NGOs and International Organisations including the United Nations . Using tThe average costs of advertising could not be found for any set of countries despite an extensive search. Most information publicly available pertains to estimates of the value of different segments of the advertising industry. That is, the revenue that it raises. The key publication on this is the PricewaterhouseCoopers (PWC) annual report, Global Entertainment and Media Outlook .In 2000 an econometric model estimating advertising prices  as a proportion of GDP/capita was created. No new data was available to contribute to an updated econometric model, thus this model was applied to 2010 GDP/capita by country to estimate updated media prices.This section outlines standard quantities of inputs assumed to be required to implement a programme with ten health interventions at full coverage .Despite recent strong focus on planning for Human Resources for Health, there is scant quantitative evidence on staffing requirements. At a minimum, WHO has estimated 4.45 “health workers”  per 1000 population will be required by the end of the SDG period . HoweverMaterials and supplies (e.g. consumables such as stationary) build off of previous WHO-CHOICE estimates with some important updates. Assumptions around computer storage devices have been updated in line with current technology. However, computer and printing equipment assumptions have remained constant, including useful life of devices, ratio of printers and photocopiers required per person. Also important to note is that computer prices have not increased in line with inflation. Again, there were no quantitative references available to base the quantity assumptions for this section of the programme support costs; therefore expert assumption and consensus was the basis for the quantity assumptions.3/office of 3 people/year. Telephone calls are assumed as 2 calls/person/work day [In line with the previous WHO CHOICE methodology, quantity assumptions for electricity are based on 3 people occupying a work space using an average of 64 kWh/month. Water consumption is assumed at 10 mRadios are present in at least 75% of households in developing countries . Radios In order to use this data for our quantity assumptions, we require data on listenership for each of the regions included in our analysis, or average listenership in low and middle income countries.Radio ownership and listenership was found for a selection of African countries. Similar data have not been identified for other global regions, so the assumptions are based on Africa alone. The amount of time/day that each person listens, on average, was not identified. Calculations for the number of advertising minutes required are based on the assumption that 56–78% of people listen to the radio daily . We assuThus a radio advertisement of 30 s, played 82 times/week would achieve the required listenership over the 30 day schedule as recommended in the marketing literature.The same information applies to television as radio advertising—individuals need to see an advertisement five times to remember it. Television ownership in low and middle income countries is lower than radio ownership; however, this statistic has large variation across regions. We assume the same intensity is required but that the proportion of the population reached is lower than for radio advertisements due to lower ownership.Supervision is carried out for the programme as a whole. The staff carrying out supervision visits are those whose wages are already accounted for in the human resources segment. We assume each province, district and facility is visited twice per year for 2 days at a time.At the programme level the model assumes that each programme conducts one in-service/refresher training at the provincial level per year, and one per district every 3 years. A training of trainers workshop is required each year. Specific interventions, such as newly implemented interventions, may require more intense training but we assume these costs to be analysed as part of the intervention implementation cost.At the national level we assume an all-terrain vehicle is required for each programme. We assume each province requires one heavy-duty 4 × 4 per programme, and at the district level 3 motorcycles are needed. These assumptions were validated with national planners in a non-random, purposive selection of countries.We make the assumption that a programme running at full capacity can support the implementation of ten interventions at full coverage. This assumption is based on the experience of the authors working in countries developing costed national health plans. No literature was found in support of such an assumption, thus country users of the methodology are strongly encouraged to evaluate the appropriateness of this assumption in their setting. If running at a lower capacity, the human resources, vehicles and office running costs are reduced. We assume that to implement a single intervention, 30% of these resources would be required, with a linear increase to 100% resource requirement for ten interventions. Following the 10th intervention a marginal 3% increase is added for each additional intervention. Costs for training and supervision are assumed to be constant regardless of the number of interventions delivered.We assume that human resource and office running costs remain constant regardless of the coverage level of interventions delivered by the programme however are influenced by the quantity of interventions delivered by the programme. In-service training and training of trainers are scaled to coverage. This assumes clients seeking care will be seen by the right trained health worker when they access health services—e.g., as in a geographically targeted scale-up. However, development of training programmes and materials remains a fixed investment at 100% regardless of coverage level. Supervision visits to staff are scaled to coverage . All other costs are assumed to remain at 100% regardless of the coverage level.health systems costs as part of this economic evaluation. We assume a constant supply chain multiplier regardless of coverage level, as we were not able to find adequate evidence of increasing logistical costs as coverage increases. We apply the multipliers below based on data reported for countries which are deemed to have a fairly well functioning systems [ITNs: apply 26% mark-up (as they are more bulky).Cold chain costs: apply an additional 6%.Supply chain costs per se are not theoretically covered under programme support costs, but the WHO-CHOICE model for GCEA applies a mark-up ratio in order to capture these  systems . Overallhttp://www.who.int/choice/costs/en. The data is presented at the country level for 194 WHO member states.Table The main imputation dataset contained 24 variables, of which 9 had complete data available and 15 had varying levels of missingness. The first step in the analysis was to correlate all variables with missing data against the 9 complete observations, to test the relationship of the missing variable against the complete variables. This indicated that there was a lack of consistent correlation against the 9 complete variables, such as GDP/capita, population size and area. This implied that the use of linear regression or conditional mean to impute variables was not an ideal estimation method. Of the options available related to missingness, missing completely at random was considered too strong an assumption as this is rarely the case in real world data, and missing not at random was not felt to be a valid assumption as there was no systematic appearance to the missing data. Thus missing at random (MAR) was assumed.Following imputation, the upper and lower bounds along with the mean value of the observed data was compared to the completed dataset for each variable. We observed consistency between the observed and completed datasets for all three metrics, indicating the imputation method is acceptable. In a further test, we checked the conditional distribution assumption of each variable with missing values—each variable containing missing values is normally distributed conditional on all the other variables. To test this, we regressed the observed part of one variable on the corresponding values of the other variables in the completed dataset. The residuals for all the variables that went through this test turned out to be approximately normal, indicating our assumptions had been met.http://who.int/choice/onehealthtool/en/), enabling compatibility between the two tools.The price database is primarily used for two purposes: WHO-CHOICE generalised cost effectiveness analysis (GCEA) and the production of global resource needs assessments, both of which require extensive datasets to be generated and processed for multiple countries. A specific requirement is standardisation across interventions, which is provided through a programme costing calculation Excel workbook. This incorporates the price database, along with the quantity assumptions and scaling factors outlined. Users can select a country and are provided with generic estimates for the country of interest, with the possibility to modify all prices and quantity assumptions. The file differentiates between ongoing costs (expected to occur each year to maintain a current level of service) and set-up costs . This calculation workbook is used in the computation of programme support costs for WHO-CHOICE GCEA analyses. It also maps to the programme costing menus in the OneHealth Tool , both similarities and differences are present (Fig. http://www.who.int/choice and is available as an Annex to this paper. A tool to undertake programme costing, incorporating the quantity assumptions in this paper is also available. Estimates are specific to each country and should be considered as normative, global costs that have not been validated by countries.All data modelled by WHO-CHOICE is available for download from the website This work represents the most comprehensive update of the WHO-CHOICE programme costing prices and quantity assumptions since they were first published in 2003. Between 2000 and 2010, global prices have undergone substantial changes, and continuing to inflate prices from 2000 to the present ignores underlying changes in cost structures that can be complex. Given the substantial contribution programme costing can have on the total resource allocation for implementing health care interventions, ideally data such as these would be continually updated to keep track with these changes in the global economy.Compared to the 2000 estimates, a greater amount of data was able to be sourced from online databases, with econometric or statistical models used to generate missing values. However, for some variables, such as media and construction, freely available global databases do not exist. The development of a global database reporting prices and quantity assumptions collected in costing studies would be a helpful contribution to future updates of this work.Despite major advances in the availability of data since the previous version of this work, there are still some limitations in data availability to respond to the needs of those wishing to develop cost and cost-effectiveness estimates. Firstly, global price databases on prices present missing values for up to 60% of countries, with many of the gaps seen in low income countries, where the greatest need for accurate cost data is present Table . This inWe were unable to use published costing data to further develop quantity assumptions and unit prices due to the general lack of detail provided in such studies. The majority of published costing studies present unit costs, without separating quantity assumptions and unit prices. In order to understand what drives differences in prices across countries we must understand the quantities and prices making up these unit costs. Reporting standards in this area towards greater disaggregation of cost data would drive improvements in programme costing analysis. Similarly, despite common consensus on the increasing marginal costs of implementing interventions in the final phases of scale up , there iFuture costing studies should consider the benefits of integrated approaches to programme delivery . A stron