Neutral lipid storage is enhanced by nitrogen deprivation (ND) in numbers of green microalgal species. However, little is known about the metabolic pathways whose transcription levels are most significantly altered following ND in green microalgae, especially the nonmodel species.Botryosphaerella sudeticus cells in response to ND. Transcriptome of B. sudeticus is de novo assembled based on millions of HiSEQ short sequence reads using CLC Genomics Workbench software. The resulting non-redundant ESTs are annotated based on the best hits generated from the BLASTX homology comparison against the “best” proteins in the model microalgae Chlamydomonas reinhardtii and Chlorella variabilis. By using a pathway-based approach according to KEGG databases, we show that ESTs encoding ribosomal proteins and photosynthetic functions are the most abundantly expressed ESTs in the rapidly growing B. sudeticus cells. We find that ESTs encoding photosynthetic function but not the ribosomal proteins are most drastically downregulated upon ND. Notably, ESTs encoding lipid metabolic pathways are not significantly upregulated. Further analyses indicate that chlorophyll content is markedly decreased by 3-fold and total lipid content is only slightly increased by 50%, consistent with the transcriptional profiling. On the other hand, carbon content and photosynthesis efficiency are only marginally decreased by 7% and 20%, respectively, indicating that photosynthesis is only slightly reduced upon drastic downregulation of photosynthetic ESTs and chlorophyll content upon ND. In addition, TAG content is found to be greatly increased by 50-fold, though total lipid content is only slightly increased by 1.5-fold.To start gaining knowledge on this, we performed transcriptome profiling of the nonmodel green microalga B. sudeticus. Degradation of excess photosynthesis proteins is most likely a mechanism for recycling of nitrogen-rich molecules to synthesize new proteins for preparation of gametogenesis and zygospore formation in adaptation and survival upon ND. Furthermore, our analyses indicate that TAG accumulation is largely attributed to the modification of other pre-existing lipid molecules, rather than de novo synthesis. We propose that this is likely an evolutionarily conserved mechanism in many green microalgae species.Taken together, our results suggest that light-harvesting proteins and chlorophylls are in excess in The online version of this article (doi:10.1186/1471-2164-14-715) contains supplementary material, which is available to authorized users. Economic growth is often accompanied by the increase in energy consumption. Depletion of fossil fuel reserves and rising crude oil prices prompt renewed interest in algae biofuel research. Green microalgae are unicellular photosynthetic organisms capable of converting photon energy into chemical energy and assimilating carbon dioxide to form glucose. It has been proposed that microalgae farming could avoid competing with food crops for arable land and fresh water and the energy yield from algae per acre per year would be much higher than other crops –3. In adC. reinhardtii B. sudetiB. sudeticus.We subsequently investigate if gametogenesis would be induced by ND. We noted that there were ~1% of gametes in log-phase growth culture  cells were stained with a lipophilic dye Nile red and subjected to FACS analysis Figure C. AveragB. sudeticus cultures were maintained by subculturing or 8-fold dilution in fresh BB+N medium prior to exhaustion of nitrate in the medium . We noted that the cell mass continued to increase under ND conditions . To examine the alteration of nitrogen (N) and carbon (C) contents in cells after ND, we performed elemental analysis of N and C in cells. It was clear that N contents in the ND cells was greatly dropped by 2.5-fold compared to that of nitrogen-replete cells   pooled from both growth conditions were subjected to de novo transcriptome assembly using the CLC Genomics Workbench software (see Methods). To this end, 56,000 non-redundant contigs/ scaffolds/ ESTs (ESTs were used hereafter) were generated. Length of ESTs ranged from 200 to 15,487 bps with a median of 369 bps or average of 660 bps  ranged from 0.09 to 17,293 with a median of 2.21  against the “best” proteins in the model green microalgae C. reinhardtii and C. variabilis [B. sudeticus were annotated, of which, 6,559 ESTs had best-hits in C. reinhardtii and 1,066 in C. variabilis [riabilis , 14 withriabilis , 14 . GO terms associated with the highest number of ESTs in three categories were metabolism , membrane , and ATP-binding . The top 10 GO-terms in three categories were shown in Figure Gene Ontology (GO)  and KEGGB. sudeticus (see below).Out of 7,625 ESTs, 1,129 were found to be associated with at least one of the 114 KEGG metabolic pathways . Top 30 Based on KEGG databases, we focused on the coherence analysis of the absolute abundance  and relative abundance  of a subset of the metabolic pathway-associated ESTs. We assumed that co-regulated basal or constitutive transcription of the pathway-associated ESTs would display a high consistency in EST levels in rapidly growing cells. Likewise, co-regulated transcriptional alteration in response to ND would exhibit a high coherence of the pathway-associated EST ratios between levels in ND and nitrogen-replete cells.We used the occurrence density of the pathway-associated ESTs in a range that contained the first 50%, middle 50%, or second 50% of the ESTs based on ranks to approximate the transcriptional coherence of a given metabolic pathways (see Methods). Coherence of the constitutive transcription and ND-induced transcription of the pathway-associated ESTs was determined based on ranks by the absolute abundance and relative abundance or ratio, respectively. A metabolic pathway was designated as a co-transcriptionally regulated (or coherent) pathway if the occurrence density of the pathway-associated ESTs in one of the three half ranges was 2-fold above background and the p-value was less than 0.05 based on ranks by level  or ratio . We also tested the occurrence density in the top 10% of the most abundant ESTs based on ranks by level or in the top (or bottom) 10% of the most upregulated (or downregulated) ESTs based on ranks by ratio  for coherently regulated metabolic pathways (see Methods).We proposed that coherent pathways were potentially co-transcriptionally regulated. Given that the magnitude of alteration in transcription levels following ND was a function of time, magnitude of change in transcription levels would vary in cells sampled at different time point after stress. On the other hand, coherence of transcriptional response for metabolic pathways would be remained throughout the course of response.B. sudeticus was approximated by RPKM (see Methods). We found that 16 out of top 30 most abundant ESTs encoded putative ribosomal proteins, indicating that RP-encoding ESTs are enriched in the topmost expressed ESTs compared to background  in actively growing B. sudeticus cells  are encoded by nuclear genes in green microalgae. On the other hand, chloroplast and mitochondrion or archaea ribosomal proteins  are encoded by both the nuclear and chloroplast or mitochondrion genes, respectively. Transcriptional coordination would be relatively easy to achieve for a group of genes localized in a single genome compared to those in two genomes such as nuclear and chloroplast or mitochondrial genomes. Hence, we hypothesized that the transcription consistency of the eRP-encoding ESTs would be high compared to that of aRP-encoding ESTs. To test this hypothesis, we first determined the relative occurrence density in the 3 half-ranges  of the eRP- and aRP-encoding ESTs  based on the rank by abundance , indicating that protein synthesis is highly active in both cytoplasm and chloroplast/ mitochondrial to support growth of B. sudeticus cells.We noted that the occurrence density of both eRP- and aRP-encoding ESTs was clearly enriched in the top 10% of the most abundant ESTs in actively growing B. sudeticus cells 3 days after ND , suggesting that change in transcription levels of eRP-encoding ESTs is consistent during response to ND  of the pathway-associated ESTs was determined based on ranks by ratio between levels in ND and nitrogen-replete cells Figure A. Five oConsistent with the downregulation of photosynthesis-associated ESTs, chlorophyll contents  in ND cells were also greatly reduced by 3.1-fold compared to that of nitrogen-replete cells   were available in transcriptomic datasets of C. reinhardtii[B. sudeticus and C. reinhardtii was moderate . The correlation between the two independent experiments in C. reinhardtii was relatively high , indicating a good quality of the two datasets  was determined based on rank by level using the common set of 4,148 ESTs in B. sudeticus and C. reinhardtii  in C. reinhardtii. In addition, 2 carbohydrate and 1 amino acid metabolic pathways were coherent and abundant in C. reinhardtii. Coherent pathways tended to be abundant in C. reinhardtii, but not in B. sudeticus.Comparative analysis showed that eRP- and aRP-encoding ESTs were both abundant and coherent in i Figure C. FurtheB. sudeticus but not in C. reinhardtii  with that of C. reinhardtii following ND based on the common set of 4,148 ESTs  of ESTs in s Figure A. On theB. sudeticus and C. reinhardtii, we determined the relative occurrence density in the 3 half-ranges of the pathway-associated ESTs based on rank by ratio  following ND in B. sudeticus and C. reinhardtii. Interestingly, though, the median level of eRP- or aRP-ESTs was significantly altered in C. reinhardtii following sulfur deprivation.In D Figure D. On theB. sudeticus following ND, of which, 3  were also found to showed coherent response in C. reinhardtii in response to ND. While the median level of citrate cycle and ATP biosynthesis was not significantly changed, the median level of photosynthesis was obviously downregulated in B. sudeticus and C. reinhardtii following ND. In fact, it was also greatly downregulated in C. reinhardtii following sulfur deprivation. Consistent with it, aRP- but not eRP-ESTs were also downregulated in C. reinhardtii following sulfur deprivation. These results suggested that transcriptional downregulation of photosynthesis is a common response to nutrient deprivation.We found that there were 7 metabolic pathways exhibited consistent response  in B. sudeticus following ND and porphyrin and chlorophyll metabolism was downregulated  in C. reinhardtii following sulfur deprivation. Folate and its derivatives are cofactors of one-carbon metabolism that is required for the biosynthesis of nucleotides and amino acids and upregulation of folate biosynthesis was in agreement with biosynthesis of novel proteins for gametogenesis upon ND. Chlorophyll is critical to photosynthesis. Hence, downregulation of porphyrin and chlorophyll metabolism was consistent with the repression of photosynthetic transcription.Among the coherent pathways specific to species or stresses Figure E, folateB. sudeticus  of the pathway-associated ESTs. It implies that there is a tight regulation of the abundantly expressed transcripts in microalgal cells. It would save energy for optimal function of complex metabolic pathways in microalgae.Based on the pathway-based approach, we show that the absolute level of ESTs encoding RPs and energy metabolic pathways such as photosynthesis, carbon fixation, and oxidative phosphorylation are highly coherent and abundant in B. sudeticus following ND and C. reinhardtii following ND or sulfur deprivation .Transcriptional response to ND has been thoroughly studied in diatoms –41. In Pafter ND . In addiafter ND . Noticeaafter ND . These rB. sudeticus  sudeticus (UTEX2629) strain was obtained from the Culture Collection of Autotrophic Organisms  with a strain catalog number of CCALA780 and the C. reinhardtii (UTEX89 or CC1009) strain was obtained from Chlamy Center (http://www.chlamy.org). CC1009 is a cell wall proficient and NIT1+ and NIT2+ strain. The cells were cultivated in 1 L of HS medium [-2 sec-1. Culture was supplied with 2% CO2 through bubbling. BB medium without sodium nitrate (or BB-N) was used as nitrogen-depleted medium.The S medium  or Bold’icus UTEX29 strainCell density of cultures was determined using cell dry weight (CDW) in g/L. Approx. 50 ml cell culture were harvested by filtration using the glass fiber filter GF/A  and dried in oven at 80°C overnight. CDW was measured in triplicate using the AG204 balance .The ratio of flagellates in cultures was determined by counting a total of 500 individual cells  in at least five independent fields in triplicate using Zeiss Axiovert 200 M microscope .To assess flocculation, shaken flasks  were brought to standstill for 30 min to score for flocculation or sedimentation of cells at the bottom of flasks.Log-phase culture in BB+N medium at the CDW of ~1.2 g/L is diluted by 8-fold in fresh BB+N and BB-N medium and cultivated for 3 days as nitrogen-replete and nitrogen-depleted cultures, respectively.To determine nitrogen and carbon content of cells, approx. 3mg dry cell powder were subjected to elemental analysis using the the Vario Micro Cube  in the Elemental Analysis Laboratory, Department of Chemistry, National University of Singapore. To determine nitrate in medium, 2 ml culture supernatant was subjected to OD measurement using the Spectroquant Test Kit for Nitrate  according to the manufacturer’s instruction.645+8.05D663[To determine chlorophyll content, cells from 2 ml culture were harvested by centrifugation. The resulting cells were resuspended in 96% ethanol and broken in the glass-bead beater FastPrep homogenizer . After incubation with ethanol for 2 h at 4°C, cell debris was cleared by centrifugation. The resulting supernatant was subjected to OD measurement at the wavelength of 645 nm and 663 nm, with the 96% ethanol as blank. Total chlorophyll content  was estimated using a formula C (mg/L) = 20.2OD-2 sec-1 to mimic the growth conditions. The quantum yield was based on the formula Y(II) = (Fm’-F)//Fm’ [To determine photosynthesis efficiency, cells were subjected to PAM-fluorescence analysis using the fluorometer Imaging-PAM  according to the manufacturer’s instruction. Briefly, the Fm’ and F were determined at the arctic light of 250 µmol photon m-F)//Fm’ .B. sudeticus cells, 4 μl of 0.25 mg/ml Nile red in acetone was directly added to 1 ml of fresh culture. After incubation at RT in dark for 15 min, fluorescence signals in B. sudeticus cells were examined using the Zeiss Axiovert 200 M  with a Zeiss EC Plane-Neofluar 40x/0.75 objective lens. The images were captured by a CoolSNAP HQ monochrome digital camera  and processed using MetaMorph software .To examine the lipid content in B. sudeticus cells were fixed with 10% formaldehyde (w/v) at RT for 15 min. The cells were washed twice with 1×PBS and stained by Nile red at the final concentration of 1 μg/ml for 15 min in dark. In microscopic analysis, Nile red stained cells were examined using the Zeiss Axiovert 200 M  with A Zeiss filter cube no. 15 . In FACS analysis, Nile red stained cells were subjected to the analysis of BD FACS Calibur flow cytometer .Total lipid was exacted with methanol/ chloroform (2:1 by volume) solution and was quantified via gravimetric analysis after evaporation of solvents. Then it was dissolved in chloroform at a concentration of 0.1 mg/µl. Equal amount of lipid was loaded on silica TLC plate  and developed in hexane/ diethyl ether/ acetic acid (35:15:0.1 by volume). TAG  glyceride) and FFA )  was used as standard. Lipid profile on TLC plate was developed using iodine vapor. TAG and FAA contents were estimated through densitometric analysis of TLC profile. Hydrocarbons were exacted in equal amounts of total lipids with hexane. Hydrocarbons were analyzed using Shimadzu GC/MS-QP2010 Plus system  equipped with a HP-5ms Ultra Inert column .B. sudeticus cells using TRIzol® Plus RNA Purification System  according to manufacturer’s protocol. Appr. 4 µg of the resulting total RNA was used for synthesis of cDNA using the TruSeq RNA Sample Prep Kit (Invitrogen-Life Technologies Co.) according to manufacturer’s instruction including synthesis of first and second strands cDNA, end repair, 3’-end adenylation, adapter ligation, fragment enrichment , and library validation, quantification, and quality assessment with a bioanalyzer . The libraries are sequenced using the Illumina HiSeq 2000 Sequencer .Total RNA was extracted from Over 2 Giga-base clean paired-end reads (90 bps in length/ read) from each library were generated using HiSeq2000 technology. A total of 56 million reads from both growth conditions were pooled and subjected to de novo assembly using the CLC Genomics Workbench software . As a result, 56 thousand nonredundant contigs/ scaffolds/ ESTs ranging from 200 to 15,500 bps in length were obtained. More than 90% of total reads from each growth condition were mapped back to the 56 thousand ESTs, ~99% of which were found in both conditions. Read hits per EST were normalized to RPKM (Reads Per Kilobase per Million mapped reads) for estimation of ESTs transcription levels.C. reinhardtii and C. variabilis (genome.jgi-psf.org/Chlre4 and genome.jgi-psf.org/ChlNC64A_1). A total of 6,559 ESTs showed to have a best hit  in the C. reinhardtii “best” proteins. Among the remaining ESTs, 1,066 showed to have a best hit  in the C. variabilis “best” proteins. To this end, 7,625 ESTs (13.6%) in B. sudeticus were annotated based on the best hits in C. reinhardtii and C. variabilis  metabolic pathways were found to have 10 or more pathway-associated ESTs by using the set of 7,625  ESTs  ESTs based on rank by level (or ratio) .Coherence of the constitutive transcription levels (or ND-induced transcription ratios) of various metabolic pathway-associated ESTs is defined as those whose relative occurrence density in one of the 3 half-ranges based on rank by level (or ratio) is 2-fold above background and p-value < 0.05. We assessed whether the coherent pathways were the most abundant (or most differently transcribed) ones in The distribution of pathway-associated ESTs based on ranks  in heat-map display is binned by an average sliding window of 11 consecutive ranks. The entire distribution of the ESTs in each pathway is normalized to 1 or 100%.B. sudeticus, 6,559 have a best hit in the C. reinhardtii “best” proteins. Based on the transcriptome analysis by Miller et al. [Of 7,625 annotated ESTs in r et al. , 4,148 oBinomial test was used to determine the statistical significance of enrichment for a number of pathway associated ESTs  that are present within a range of ranked ESTs . Average occurrence (or background level) of a pathway associated ESTs is the ratio between numbers of the pathway associated ESTs and all ESTs tested.T-test was used to determine the statistical significance of level changes of pathway associated ESTs compared to all ESTs tested (or background). P-values are subjected to Bonferroni correction according to numbers of individual pathways tested against background.http://www.ncbi.nlm.nih.gov/sra) with the SRA Study accession number, SRA026047.The raw HiSEQ2000 pair-end sequencing data used in this study are available at the NCBI’s Sequence Read Achieve Additional file 2: Table S1: List of 7,625 annotated ESTs in B. sudeticus. (XLS 2 MB)Additional file 3: Table S2: List of EST-associated GO terms. (XLS 414 KB)Additional file 4: Table S3: List of EST-associated KEGG metabolic pathways. (XLS 44 KB)Additional file 5: Table S4: List of 66 metabolic pathways and occurrence densities based on 7,625 ESTs. (XLS 38 KB)Additional file 6: Figure S1: Upregulated (>3-fold) pathway-associated ESTs. (PDF 96 KB)Additional file 7: Table S5: List of 4,148 ESTs in B. sudeticus and C. reinhardtii. (XLS 1 MB)Additional file 8: Table S6: List of 53 metabolic pathways and occurrence densities based on 4,148 ESTs. (XLS 54 KB)Additional file 9: Table S7: Occurrence of genes based on ranks in various pathways. (XLSX 2 MB)


Interspecies transmission of equine influenza A(H3N8) virus has resulted in establishment of a canine influenza virus. To determine if something similar could happen with cats, we experimentally infected 14 cats with the equine influenza A(H3N8) virus. All showed clinical signs, shed virus, and transmitted the virus to a contact cohort. Experiments were approved by the Institutional Animal Care and Use Committee and monitored by veterinarians. 50 [median tissue culture infective dose]) in 1.0 mL of phosphate-buffered saline (0.5 mL in each nostril). One day after inoculation, 5 specific pathogen–free cats (contact cohort) were introduced into the same cages. Three noninfected cats (control cohort) were housed in a different room. Clinical monitoring began 1 day before virus inoculation and continued daily for the next 14 days. Nasal swab samples were collected daily for virus titration in MDCK cells. Serum was collected on postinoculation days 5, 7, 9, 12, and 14 and titrated by hemagglutination-inhibition assay with a 1% horse erythrocyte suspension. On postinoculation day 5, a total of 2 cats from the inoculated group were euthanized by intravenous pentobarbital, and on postinfection day 7, another 2 cats from the inoculated group plus 2 cats from the contact cohort were euthanized. Necropsies were performed, and trachea and lung sections were stained with hematoxylin and eosin and by an immunocytochemistry technique that involved a murine monoclonal antibody specific to EIV hemagglutinin.The virus used was A/equine/Heilongjiang/SS1/2013, which had been isolated from a mule in northern China . For virus inoculation, 6 cats were anesthetized with xylazine hydrochloride , after which they were inoculated with virus  and in contact cohort cats during days 4–9 (peaking at day 5); however, average clinical scores were lower for cats in the contact cohort than in the inoculated cohort . Virus sProductive viral infection was evidenced by histopathologic and immunocytochemical examinations. Characteristic lymphocytic infiltration was observed in samples from cats in the inoculated and contact cohorts; intensity was less for cats in the contact cohort . LikewisThat cats are susceptible to EIV by direct inoculation is not surprising because infection of cats with various influenza A viruses has been reported. Feline respiratory tract epithelial cells contain sialic acid α-2,3-galactose β-1,3-N-acetyl galactosamine  receptors for avian and equine influenza viruses and SA α2,6 gal receptors for mammalian influenza virus . Experiments to elucidate the transmission mechanism are being conducted.Because we had used a contemporary strain of EIV, to rule out the possibility that the ability to cause clinical infection is unique to this strain, we repeated the experiment with the prototype EIV, A/equine/Miami/63 (H3N8). Although 6 of 6 infected cats showed no overt clinical signs, and virus shedding was not detectable (<1:10), susceptibility was evidenced by seroconversion for 2 of the 6 inoculated cats, although at a low hemagglutination-inhibition titer (1:40 on postinoculation day 14), thereby ruling out the possibility that A/equine/Heilongjiang/SS1/2013 is an aberrant virus. None of the 3 cats in the contact cohort showed clinical signs, shed virus, or had detectable hemagglutination-inhibition titers. This strain-dependent variation in virulence is not unusual for influenza virus. Interspecies transmission of EIV to dogs and establishment of a new lineage of equine influenza virus in dogs were probably a function of that particular EIV strain, as evidenced by finding that so far only 1 EIV-originated canine influenza virus lineage is circulating. However, transmission of EIV to dogs has occasionally occurred, including during the epizootic of EIV in Australia (


We report here the full-length genome sequence of a novel chimpanzee polyomavirus. Viral sequences were recovered from colon, bladder, and ureter tissue from a western common chimpanzee. The virus is genetically closely related to the human BK polyomavirus. Polyomaviruses (PyV) are small DNA viruses that infect a wide range of mammals, birds, and fish. Since the discovery of the macaque polyomavirus SV40 in 1960, PyVs have been characterized from a range of nonhuman primates, including New World monkeys, Old World monkeys, and apes.Pan troglodytes verus). In tissue samples from this animal, Ch-Regina, we previously detected a PyV (Pan troglodytes schweinfurthii (P. troglodytes verus polyomavirus 8 (PtrovPyV8), has the highest identity (88%) to the recently described Pan troglodytes troglodytes polyomavirus 1 (PtrotPyV1)  , and it The human BK virus persists in the renourinary tract, and BK virus (BKV) has been detected in epithelial tissues of the ureter and bladder . A similKT884050.The complete genome of PtrovPyV8 has been deposited in GenBank under the accession number


Echinacea species have been used traditionally in North America for the control of symptoms of colds, influenza, and other diseases, and some of them have become very popular as “herbal medicines”. Recent studies have revealed that preparations derived from certain species and plant parts, but not all of them, possess potent antiviral activities, at non-cytotoxic concentrations, particularly against membrane-containing viruses. Thus all strains of human and avian influenza viruses tested (including a Tamiflu-resistant strain), as well as herpes simplex virus, respiratory syncytial virus, and rhinoviruses, were very sensitive to a standardized Echinacea purpurea preparation. In mechanistic studies the influenza virus-specific hemagglutinin and neuraminidase were inhibited. In addition some extracts displayed anti-inflammatory activity in virus-infected cells, and numerous other effects on the expression of cellular genes. Multiple components, either discrete compounds or mixtures, appeared to be responsible for the various antiviral activities.Extracts of  In addiAnother problem with the specific target approach, especially in the case of compounds directed at specific viral genes or their products, is the inevitable emergence of virus resistant mutants and their subsequent spread through the community and environment. The conventional answer to this problem has been the suggestion that two or more antiviral drugs, with distinct molecular targets, be used in combination, notwithstanding the likely increase in undesirable side-effects. A logical alternative approach is the use of a non-toxic agent that has the capacity to inhibit many different respiratory viruses simultaneously, and recent evidence indicates that certain herbal extracts might fulfill this requirement ,7,8.2.“Colds” and “flu” are terms that have been coined to describe a combination of common symptoms, supposedly brought about by the actions of specific viral infections of the upper respiratory tract. These symptoms may include such familiar discomforts as sneezing, stuffy nose, irritation of mucous membranes, excess mucus production, sinusitis, cough, sore throat, malaise and fever, as well as exacerbation of asthma and COPD (chronic obstructive pulmonary disease). In some cases symptoms may spread to include the lower respiratory tract and lungs, and result in bronchitis, bronchiolitis, or pneumonia ,8,9. HowIn respiratory infections, whether they start in the nasal passages or oropharynx, or other parts of the airway, the invading virus initially encounters epithelial tissues, composed largely of epithelial cells and occasional dendritic cells and macrophages, which accordingly respond by means of the various antimicrobial strategies that make up the innate immune system, including defense peptides  and the secretion of various pro-inflammatory cytokines and other mediators of inflammation ,13. OtheSince most of the symptoms reflect this common non-specific host response to infecting agents, rather than to the direct cytolytic or cytopathic effects of a specific virus ,7,8, theThe limitations of conventional antiviral therapy and prevention were illustrated in 2002 with the sudden appearance of the SARS (severe acute respiratory syndrome) pandemic. The novel coronavirus responsible for the disease (SARS-CoV) was quickly isolated and its genome sequenced ; howeverEchinacea extracts have become very popular, although not all of them are necessarily beneficial, as will be discussed.Several herbal extracts have been shown recently to possess a combination of bioactivities that could be useful in the control of colds, flu, and bronchitis ,8, and, 3.Echinacea are usually made in the form of extracts, tinctures, teas, sprays etc. derived from various parts of one or more of three species of Echinacea: E. purpurea, E. angustifolia, and E. pallida  is their inadequate characterization and standardization. Consequently different commercial sources, derived from different species and plant parts, and with resulting distinctive chemical compositions, may show different combinations of bio-activities, or in some cases relatively little bioactivity [However, a problem with commercial activity . The resEchinacea [E. purpurea could partially protect cultured cells from infection by influenza A virus, herpes simplex virus type 1, or vesicular stomatitis, viruses. This suggested an intracellular inhibition, although the possibility of a virucidal activity was not reported.Early reports of antiviral activity of chinacea  indicateEchinacea species as a potential source of antiviral activities. Cheminat et al. [E. pallida plants, and examined two of them, cichoric acid and echinacoside, as well as caffeic acid, a constituent of E. purpurea, for activity against the replication of vesicular stomatitis virus (a membrane containing RNA virus) in a mouse cell line. Activity was relatively weak however, except at high and cytotoxic concentrations, and the possibility of virucidal activity was not tested.Later studies supported the concept of t et al.  isolatedet al. [E. purpurea and the ethanol inflorescence extract of E. sanguinea were more substantial. Pure cichoric acid was also moderately active, and could therefore contribute to the activity of certain extracts.Binns et al.  examinedet al.  in Vimalanathan E. purpurea, E. angustifolia and E. pallida (23). The aqueous fraction of E. purpurea roots, which was almost devoid of caffeic acid derivatives and alkylamides, showed impressive activity against HSV and influenza virus  in the ethyl acetate fraction, but no activity in the aqueous extracts. In contrast, E. pallida root extracts were devoid of antiviral activity.Similar studies were carried out to compare root extracts from za virus . The E. E. pallida aerial parts showed significant virucidal activity against HSV-1 and HSV-2 [E. angustifolia aerial parts was as active against HSV as a corresponding E. purpurea extract (unpublished observations), thus indicating that there may be similar antiviral compounds in aerial part extracts of all three common Echinacea species . It is also clear that several different compounds contribute to the overall antiviral activity of a given extract, although many other extracts are devoid of activity.However, in a more recent study, a series of aqueous and ethanol extracts of nd HSV-2 , and som4.Echinacea preparation, such as tinctures, sprays, tablets, teas, etc. could be beneficial in the treatment of colds and flu, although not all preparations are likely to be effective. In fact a recent study on 10 commercial preparations highlighted the variability of antiviral activity between different preparations, although lot-to-lot variation was less evident [The presence of multiple antiviral activities among different extracts and fractions suggests that many kinds of  evident . In gene®  showed that this preparation was very active as a virucidal agent against viruses with membranes, as indicated in ® recommended for oral consumption . Under these conditions more than 105 infectious viruses could be killed within 5 min.Recent detailed studies with the standardized preparation Echinaforce® was found to be less effective against intracellular virus [®, but virus particles shed into the extracellular fluids would be vulnerable [® would be manifest during initial contact with the virus, i.e. at the inception of infection, and also during transmission of virus from infected cells.In contrast Echinaforcear virus . Consequlnerable ,26. Ther® did not result in the emergence of resistant strains, whereas passage of the virus through successive cultures in the presence of Tamiflu rapidly generated Tamiflu-resistance. Furthermore Tamiflu-resistant virus remained fully susceptible to Echinaforce® [® in the population would be less likely to generate resistant strains of virus than Tamiflu or other anti-influenza compounds currently in the market. Recent studies have illustrated the relative ease with which resistant strains of influenza virus can arise [® could also be useful as an accessory treatment for individuals undergoing anti-influenza therapy with agents such as Tamiflu.Additional experiments showed that continuous passage of influenza A virus in cell cultures in the presence of Echinaforcenaforce® . Therefoan arise ,27. Furtin vitro (unpublished results), suggesting that the active compounds could block influenza virus entry and spread by acting on at least two virion targets. However, the susceptibility of other viruses, which do not rely on HA or NA functions, to Echinaforce® indicates that additional molecular targets must be accessible.It was shown by hemagglutination assays that this extract (EF) inhibited the receptor binding activity of influenza A viruses, over a range of EF concentrations including that recommended for oral consumption, suggesting that EF interfered with viral entry into the cells, thus effectively rendering the virus non-infectious . EF also5.® reversed this secretion in both systems [Echinacea treatment. This result was supported by histochemical examination of cultured airway tissues, which revealed the conspicuous presence of muco-polysaccharide-filled goblet cells resulting from rhinovirus infection, whereas EF treated and infected tissues appeared normal [Most sufferers of colds and other respiratory problems would agree that secretion of excessive mucus is one of the more annoying symptoms, and accordingly many pharmaceuticals have been designed to relieve this feature of a cold or flu, usually with the accompaniment of undesirable side effects. Rhinoviruses induced the secretion of excess MUC5A, the dominant respiratory mucin, in bronchial epithelial cells in culture, and in cultured airway tissues, and Echinaforce systems , suggestd normal .6.et al. focused on the application of E. purpurea extracts (including Echinaforce®) to epithelial cells and tissues infected by viruses [Echinacea preparations were able to completely or partly reverse this stimulation [®, it was shown that EF could be added before or after virus infection, with similar success, and also the results were not affected by virus dose or the time of exposure to EF [A series of studies by Sharma  viruses ,29-31. Imulation ,29-31. IEchinacea, but could nevertheless stimulate cytokine secretion, were still susceptible to cytokine reversal.Other viruses, including HSV-1, influenza A virus, adenovirus type 3 and 11, and respiratory syncytial virus, stimulated the secretion of pro-inflammatory cytokines, and in each case the stimulation was reversed by EF  by human bronchial epithelial cells infected with rhinovirus type 14. All of the E. purpurea fractions, comprising aqueous or ethanol extracts of roots, leaves and stems, but to a lesser degree flowers, strongly inhibited the secretion of both cytokines [In an attempt to correlate immune modulatory effects with specific classes of ytokines . These r7.Echinacea preparations on cellular gene expression, mostly in uninfected cultured cells relevant to the immune system, although it is not feasible to compare the studies because of the different cellular systems and because the Echinacea preparations were different.Several studies have reported the effects of et al. [Echinacea product, and Brovelli et al. [Echinacea preparation (stage of development and plant part used), presumably a reflection of their different chemical compositions. Altamirano-Dimas et al. [E. purpurea preparations, a polysaccharide rich aqueous extract and an alkylamide-rich ethanol extract, with or without infection by rhinovirus type 14. Both extracts influenced the expression of many genes, including cytokine genes, although the pattern of expression was different for the two extracts. In addition the virus induced numerous changes, mostly increases in expression, and the extracts tended to decrease  these expression levels. Further analysis of the effects revealed that some of the changes in cytokine expression were interconnected through a specific transcription factor, C/EBPb (CAAT/enhancer-binding protein b). Since Sharma et al. [E. purpurea extract in this same cell-virus system, it is tempting to conclude that many gene expression effects of Echinacea extracts could be due to changes in expression or activation status of multiple transcription factors. This in turn could be brought about by interaction with surface receptors or intracellular modulators.Randolph et al.  describei et al.  found ths et al.  analyzeda et al.  had showet al. [Echinacea extract and target cells, infected or otherwise, are complex and may involve different levels in the signaling pathway network. Nevertheless the important conclusion of these analyses is that rhinovirus induction of numerous cytokines and their secretion can be modulated by an appropriate Echinacea purpurea extract.However in an extension of these analyses Altamirano-Dimas et al. , using cet al. [E. purpurea, on gene expression of immune-related molecules in human dendritic cells, which are part of the adaptive immune response, in contrast to the studies described above, which focused on the innate immune response. Many dendritic cell genes were affected, either up regulated or down-regulated. These studies did not include infected cells, but clearly showed the multiple gene effects of this Echinacea preparation.Wang et al.  describeet al. [E. purpurea, an aqueous extract of roots and an ethanol extract of aerial parts, on selected immune-related proteins in murine dendritic cells, and found a variety of significant responses, reinforcing the concept of multiple consequences of Echinacea exposure of immunologically important cells.Benson et al.  studied 8.Echinacea extracts contain multiple bio-activities which collectively inactivate and/or inhibit many viruses at different levels. Influenza viruses present at least two molecular targets, the hemagglutinin and neuraminidase proteins; but other viruses evidently contain additional virion targets. There are also intracellular mechanisms at play, which include a widespread reversal of virus-induced pro-inflammatory cytokine secretion, mediated through one or more signaling pathways. All of these events occur at non-cytotoxic concentrations.The results reviewed above indicate that certain Echinacea extracts, i.e. caffeic acid derivatives, alkylamides and certain types of polysaccharides, prompted us to search for evidence of alternative bioactive compounds. We found recently that the antiviral activity in ethanol extracts of E. purpurea aerial parts was able to bind to the polymeric matrix polyvinyl-polypyrrolidone (PVPP) and could subsequently be eluted and recovered . Recent studies on PVPP have shown its effectiveness in selectively removing tannins and other polyphenols from plant extracts, by a variety of chemical interactions [E. purpurea aerial parts could be, or could include, polyphenols. Anti-inflammatory activities could be due partly to alkylamides or other constituents.The lack of correlation between antiviral activities and the presence of the known marker compounds for ractions . Thus thEchinacea extracts on gene expression, additional consequences can be anticipated, and some of these could also supplement the beneficial effects of Echinacea in counteracting virus infection. If the active compounds can be isolated and characterized, then further analyses could lead to improvements in the efficacy of the extracts, and in addition provide further evidence to substantiate some of the medical claims made for Echinacea.In view of the numerous effects of 9.Echinacea extracts have shown that some of them, but not all, possess multiple beneficial actions in the treatment of viral respiratory infections: (1) a direct virucidal activity against several respiratory viruses; (2) reversal of the pro-inflammatory response of epithelial cells and tissues to different viruses; (3) reduction in the excessive secretion of mucin by airway cells and tissues; (4) lack of cytotoxic effects or disruption of tissue integrity by Echinacea in airway cell cultures or tissues, at practical antiviral concentrations; (5) additional potentially positive effects on cellular gene expression. A combination of these beneficial activities could reduce the amount of prevailing viable virus, and their transmission, and also lead to amelioration of the virus-induced symptoms.Studies on


Arboviruses: Molecular Biology, Evolution and Control. Nikos Vasilakis and Duane J. Gubler, eds., 2016. 398 pp. Poole, UK: Caister Academic Press. US$319, ISBN 978-1-910190-21-0Flaviviridae), which occurs in every, or almost every, country with a tropical or subtropical environment. According to one recent estimate, dengue virus is responsible for 390 million infections annually, of which 96 million are accompanied by clinical manifestations.Flaviviridae), chikungunya and Venezuelan equine encephalitis viruses (Togaviridae), and Crimean-Congo hemorrhagic fever, Oropouche, and severe febrile thrombocytopenia viruses (Bunyaviridae). Arboviruses of major veterinary significance include Rift Valley fever virus (Bunyaviridae) and bluetongue virus (Reoviridae). Most recognized arboviruses are maintained in natural transmission cycles between wild vertebrate animals and hematophagous arthropods , and cause disease after spillover transmission to humans or domestic animals that are dead-end hosts. However, some arboviruses—most notably dengue, yellow fever, chikungunya, and Zika viruses—have lost the requirement for enzootic amplification, and are maintained in human–mosquito transmission cycles. The recent emergence of Zika virus and chikungunya virus in the Western Hemisphere and the explosive disease outbreaks accompanied by these introductions provide a stark reminder of how easily arboviruses can spread across the globe and cause devastating disease in human populations. Despite the enormous burden that arboviruses impose on human and animal health, very few books devoted specifically to arboviruses have been published in recent years.Arthropod-borne viruses (arboviruses) have a devastating impact on human health. The arbovirus of greatest medical significance is dengue virus . The current status of arboviral disease and reasons for dramatic emergence of epidemic arboviral diseases  are also discussed.Togaviridae), flaviviruses (Flaviviridae), rhabdoviruses (Rhabdoviridae), bunyaviruses (Bunyaviridae), reoviruses (Reoviridae), and orthomyxoviruses (Orthomyxoviridae).Section I (chapters 2–8) covers the molecular biology of arboviruses. Topics included in this section are taxonomy, genomic organization, replication, virus–vector and virus–host interactions, and innate immune evasion. Chapter 2 contains two appended tables; one that lists every traditional arbovirus currently recognized by the International Committee on Taxonomy of Viruses, and another comprised of viruses that infect hematophagous arthropods, but appear to have arthropod-specific host ranges. Many viruses listed in the second table were discovered within the last few years and thus, are not covered in many other virology books. The impact of next-generation sequencing technologies and other recently developed molecular approaches on virus discovery and classification is also covered. This section also compares the genomic organizations, translation products, and replication strategies of the six virus families that contain most of the arboviruses known to infect humans and livestock: alphaviruses (Section II (chapters 9–14) covers arboviral diversity and evolution. This section provides an excellent review of ecological and epidemiological factors that influence arbovirus genetic diversity, evolution, and emergence. The importance of host and vector genetics and virus–host interactions is also discussed. One chapter is devoted to the role of vertical transmission in the adaptation and evolution of arboviruses. The major modes of vertical transmission are explained, and the contributors include a table listing most of the bunyaviruses, flaviviruses, reoviruses, alphaviruses, rhabdoviruses, and asfaviruses known or suspected to be vertically transmitted. There is also a chapter on arbovirus genomics and metagenomics. Protocols for sample preparation, whole genome sequencing, and data analysis are provided.Section III (chapters 15–21) covers arbovirus diagnosis and control. The opening chapter describes serologic and nucleic acid–based detection assays as well as virus isolation methods available for arbovirus diagnosis. Nucleic acid–based diagnostics is the fastest growing field in arboviral diagnosis, and assays covered in this chapter include the widely used reverse transcription polymerase chain reaction as well as less common methods such as fluorogenic assays, reverse transcription loop-mediated isothermal amplification, and nucleic acid sequence–based amplification. The proceeding chapters describe conventional methods available for arbovirus control , and more recent approaches that hold promise for future control efforts . The chapter on vaccination summarizes the current status of dengue virus vaccine development, which has long been complicated by the potential for disease enhancement to occur after exposure to a heterologous dengue virus serotype and the unavailability of a suitable animal model. The 17D yellow fever virus vaccine, which is potentially the safest vaccine ever developed, is also discussed along with the current vaccine status of many other arboviruses of medical and veterinary significance. Section IV consists of a single chapter “Arbovirology: Back to the Future” written by Robert B. Tesh and Charles H. Calisher, two of the most recognized names in the arbovirus community. These two individuals provide the reader with a historical perspective of arbovirology and conclude with a discussion on future trends.Arboviruses: Molecular Biology, Evolution and Control is an outstanding book. An impressive team of contributors was involved in its conception, and each contributor provides an excellent review of their particular research niche. This is arguably the first comprehensive book devoted specifically to arboviruses to be published in the last few decades. The book is highly recommended for every arbovirologist whether it be a first-year graduate student or an established researcher.Overall,


Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both non-clinical and clinical samples using two different microarray data analysis methods. Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae and Togaviridae virus families, but some are from DNA virus families such as Poxviridae. Their survival often depends on a vertebrate or arthropod host  Emerging viruses may be defined as viruses that are newly discovered or have the potential to increase in incidence or geographical range. Some important emerging viruses cause severe acute syndromes such as viral haemorrhagic fevers or encephalitides. They are endemic to tropical and sub-tropical regions. The majority are RNA viruses, from the Due to increased global travel, lifestyle changes and climate change, the risk of importing rare, exotic and emerging diseases to Europe has increased Several metagenomic approaches, such as microarrays The Lawrence Livermore Microbial Detection Array (LLMDA) is a high density oligonucleotide microarray that contains probes for all sequenced viruses and bacteria Exemption for review by the ethical committee system and informed consent was given by the Committee on Biomedical Research Ethics-Capital region in accordance with Danish law on assay development projects.All authors comply with the data availability policy.7 copies/ml and the DENV13-01 sample contained Dengue virus (DENV) type 1 at a concentration of 1.0×106 copies/ml. The WNV10-01 and WNV13-10 samples contained a mixture of flaviviruses ). The WNV10-07 and WNV13-11 samples contained a mixture of DENV type 3, tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV), each at a concentration of 1.0×106 copies/ml.Within the European Network for Diagnostics of Imported Viral Diseases (ENIVD) we gathered a wide range of emerging viruses, as inactivated culture supernatants or as purified viral DNA or RNA . Viruses6 IU/ml) determined by standardisation against the WHO control. As negative controls we used virus-negative clinical samples .We used clinical samples received for routine diagnostic analysis at Statens Serum Institut (SSI), Copenhagen, Denmark ), from the CCH Fever Project bio-bank at the Swedish Institute for Communicable Disease Control (Sweden), and from the Department of Microbiology, Aristotle University of Thessaloniki (Greece). The samples were : i) One As previously described 6) (Eurofins MWG Operon). Briefly, 11–12 µl viral RNA was mixed with 1 µl 10 mM dNTP mix and 1 µl 250 ng/µl P-N6, incubated at 85°C for 5 min, and cooled on ice. Next, 4 µl 5x first strand buffer, 1 µl 0.1 M DTT, 1 µl RNaseOUT (40 U/µl)  and 1 µl Superscript III RT enzyme (200 U/µl) was added, and the sample mixed and incubated at 25°C for 10 min, 42°C for 60 min and 95°C for 5 min. ii) The RT-reaction included in the WTA kit (Qiagen), which uses T-Script reverse transcriptase combined with random and oligo-dT primers. RT was performed according to the manufacturer’s instructions. iii) The VILO method, which uses a cDNA Synthesis kit (Invitrogen) containing Superscript III reverse transcriptase combined with random primers. The method was performed as previously described Reverse transcription (RT) on purified viral RNA was performed with three different methods: i) The P-N6/SSIII method, which uses the Superscript III Reverse Transcription kit (Invitrogen), combined with 5′-phosphorylated random hexamers , the DENV13-01 QCMD sample (1.0×106 copies/ml) and the WNV13-01 QCMD sample (1.0×107 copies/ml), under the assumption that no viral NA was lost during purification.The technique used for routine diagnostic virus analysis at SSI is quality-assured real-time PCR . To confirm presence of virus in the samples and quantify the virus before and after WTA, we performed virus-specific real-time PCR. We used in-house assays for DENV, WNV, orthopoxvirus, parapoxvirus, Usutu virus, Hantaan virus, Toscana virus, BK virus (BKV) and rotavirus A; and previously published assays for JC virus (JCV) We analysed samples with the LLMDAv2 microarray, developed at the Lawrence Livermore National Laboratory (LLNL), USA and described elsewhere http://ncbi.nlm.nih.gov/geo/with the accession number GSE55576. All microarray data used in this study are MIAME compliant.Microarray data were submitted to the Gene Expression Omnibus (GEO) database 6) hereafter called P-N6/SSIII. This was done in order to phosphorylate the 5′-end of the cDNA fragments so that new phosphodiester bonds could be formed during the ligation step 6 IU/ml)  6 IU/ml) , on two 6 IU/ml) , and on 6 IU/ml) . For all6, DOBV (Hantavirus) with a fold increase of 4.5×106 and Puumala virus (Hantavirus) with a fold increase of 1.4×104  , purified viral NA or QCMD panel samples . The WT  1.4×104 . When weication) , we obse6 copies/ml  containing mixtures of four and three different flaviviruses, respectively, each at a concentration of 1.0×10opies/ml . All sevopies/ml . This mo6 IU/ml), a RVFV-positive supernatant (3.3×106 copies/ml), a WNV-positive QCMD panel sample (WNV13-01) (1.2×107 copies/ml), a DENV-positive QCMD panel sample (DENV13-01) (1.0×106 copies/ml) and two QCMD panel samples (WNV13-10 and WNV13-11) containing mixtures of JEV, DENV-1, DENV-2, DENV-4 and YFV, DENV-3, TBEV respectively (1.0×106 copies/ml)  . Dilutio6 to 103 copies/ml yielded sufficient viral material for successful identification by the LLMDAv2, while dilutions of 102 copies/ml were not detected by the microarray  than the rest of the flaviviruses analysed. The WNV13-11 sample was documented as containing YFV, DENV-3 and TBEV, each at 1.0×106 copies/ml; however, analysis of the Ct-values of YFV and TBEV before amplification showed a higher value for YFV (Ct = 27) compared to TBEV (Ct = 23)  , which cectively . From thThe LLMDA microarray Mus dunni endogeneous virus (MDEV), murine leukemia virus (MuLV) and mouse mammary tumour virus (MMTV). These additional viruses were not identified by the SSI method because non-human, non-zoonotic pathogens were considered clinically irrelevant and excluded in the SSI data analysis.In all 21 samples analysed, both methods identified the correct virus . In moreOrthopoxviridae family as present. The SSI analysis identified both samples as mixtures of cowpox, monkeypox, vaccinia and variola viruses, while the CliMax analysis identified the correct cowpox or monkeypox virus together with the variola minor virus (th percentile of the negative control intensities) showed that all such probes with alignments to the variola minor virus genome had strong matches in the cowpox and monkeypox genomes; so that the identification of variola minor virus in the CliMax analysis in these samples is most likely due to cross-hybridization of these probes.For four of the samples , the SSI method had difficulties in distinguishing between different genus-members and subtypes. In the Usutu virus sample (Flavivirus), the SSI method identified both Usutu virus and JEV, another Flavivirus species, as being present, while the CliMax analysis correctly identified Usutu virus only. In the PV sample, the CLiMax analysis correctly identified PV-1 and PV-2, while the SSI analysis made an additional false-positive detection of PV subtype 3 . In the or virus , which bFor five samples  the CLiMax analysis identified additional viruses that were not observed using the SSI analysis . To bettWe observed a similar fragmented alignment pattern for RVFV segment S in the Marburg sample, indicating non-specific cross-hybridisation (data not shown). In contrast, we obtained uniform positive probe distributions for mammalian orthoreovirus 1 and 3 (MRV) genomes in the Hantaan virus sample, for CCHFV and Simian virus 5 (SV5) genomes in the RVFV sample, and for BVDV-1 in the Naples virus sample, indicating that these complete viral genomes were truly present (data not shown). CCHFV specific PCR could not confirm the presence of CCHFV in the RVFV sample (data not shown). The other additional findings were all considered clinically irrelevant and therefore not further investigated by PCR.The presence of several viruses in a sample did not interfere with identification, as can be seen by the microarray analysis of the two panels of samples (WNV10-01 and -07) containing mixtures of different flaviviruses . MicroarIn summary, the LLMDAv2 correctly identified single and multiple viruses present in non-clinical samples with a very low level of false positive signals. The CLiMax analysis method identified every virus present in the samples whereas the simpler SSI analysis method only identified clinically relevant human pathogens.We tested the LLMDAv2 microarray on 18 clinical samples previously identified as positive by real-time PCR for emerging viruses. The correct virus was identified in 17 samples using both the SSI  and CLiM4 and 4.9×105 copies/ml before WTA and concentrations between 3.0×105 and 2.8×107 copies/ml after WT amplification (3 copies/ml) determined for dilutions of the WNV-positive QCMD sample (WNV13-01)  . AnalysiNV13-01) , however4 and 1.8×107 copies/ml before WT amplification and between 1.4×105 and 2.5×1012 copies/ml after WTA  . This sample was also near the limit of detection with real-time PCR, with a Ct value of 37 before amplification. In summary, 11 out of 12 clinical samples analysed had viral concentrations above the detection limit of the LLMDAv2.The DENV-positive serum samples were determined to have concentrations between 1.1×10fter WTA . The DENpies/ml) ; and a c3–104 copies/ml for different emerging RNA viruses, which is in the range of clinical relevance, but not as sensitive as specific real-time PCR. However, the use of PCR requires a specific hypothesis as to the causative agent, which is not the case with the LLMDA array. We use a modified random WTA method to amplify the RNA virus and show that least 105 copies/ml of amplified material is needed in order to have a successful identification by the LLMDAv2. This is equivalent to the recently published data that show detection of 105 copies of vaccinia virus DNA without any amplification prior to hybridization to the 4x72K version of the LLMDA The disease symptoms for emerging viruses are often similar to those of other more common viruses, posing a diagnostic challenge to clinicians unfamiliar with the novel organism. In the case of emerging viruses it is crucial for patient treatment and for containment of a potential epidemic to quickly identify the correct virus. We demonstrate the ability of the LLMDAv2 array combined with a modified WTA protocol to correctly identify 29 different emerging viruses in both clinical and non-clinical samples. Previously we have also shown that LLMDAv2 can detect a broad range of common viruses in clinical samples The samples used in this study to measure sensitivity were all dilutions of viral samples or supernatants and do not represent clinical samples containing low viral concentrations. Therefore, further experiments to investigate clinical sensitivity are warranted. Previous reports have shown high clinical sensitivity (86–97%) and specificity (98–99%) of another microarray, the Virochip Overall, the LLMDAv2 demonstrates high specificity and sensitivity with few false positives. The majority of additional hits found by the microarray data analysis are retroviruses normally found in mammalian genomes . They are clinically irrelevant and most probably originate from host or cell culture DNA. The BEV identified in the Ebola virus, cowpox virus and monkeypox virus SN samples is not surprising, since cross-hybridization of endogenous retroviruses in African green monkey-derived Vero E6 cell cultures to the BEV probes has been previously reported The CLiMax software is numerically intensive and requires a large-memory LINUX server harbouring a library of probe-target binding probabilities that are the basis for pathogen identification The analysis developed in-house at SSI processes microarray feature intensities produced by the NimbleScan software within a Microsoft Excel framework Table S1Modified WT amplification of non-clinical samples.(DOCX)Click here for additional data file.


The nature of cost-saving effects of early referral to a nephrologist in patients with chronic kidney disease (CKD) is not fully evaluated. We evaluated the health care costs before and after dialysis according to the referral time.A total of 879 patients who were newly diagnosed as having end-stage renal disease from August 2008 to June 2011 were prospectively enrolled. The early referral (ER) group was defined as patients who were referred to a nephrologist more than a year before dialysis and had visited a nephrology clinic 2 or more times. Patients whose referral time was less than a year were considered the late referral (LR) group. Information about medical costs was acquired from the claim data of the Korea Health Insurance Review and Assessment Service.The total medical costs during the first 12 months after the initiation of dialysis were not different between the 526 ER patients and the 353 LR patients. However, the costs of the ER patients during the first month were significantly lower than those of the LR patients . The total 12-month health care costs before the initiation of dialysis were significantly lower in the ER group . In the multivariate analysis, ER significantly lowered the health care costs during the 12 months before  and the first month  after the initiation of dialysis.The ER of patients with CKD to a nephrologist is associated with decreased medical costs during the pretreatment period of renal replacement therapy and the early period of dialysis initiation. Chronic kidney disease (CKD) is a major public health problem that is continually growing worldwide. The overall prevalence of CKD around the world is 10–16% Along with the increase in ESRD prevalence, the economic costs resulting from the care of these patients have increased. In the United States, the annual medical cost of dialysis patients borne by the Centers for Medicare and Medicaid in 2008 was 26.8 billion USD, which was 5.9% of the total costs of the entire population, and the annual increase rate reached 13.2% The late referral (LR) of patients to a nephrologist in the course of CKD progression has increased the morbidity and mortality This study was nested within an ongoing cohort study  of patients with ESRD in South Korea. The CRC for ESRD is a nationwide multi-center web-based comprehensive prospective cohort of CKD patients on dialysis whose aim is to analyze the treatment effects on survival or quality of life and cost-effectiveness by dialysis modality We selected and studied 1011 adult patients who were newly diagnosed as having ESRD and started dialysis between August 2008 and June 2011 from the new patient registry in the CRC for ESRD cohort. Of the 132 patients who were excluded, the data regarding visits to a nephrologist were missing for 61 patients, and data regarding the type of initial dialysis were unknown for 33 patients. In addition, 38 patients were excluded due to the lack of cost data because of an unmatched identifier. The early referral group (ER) was defined as patients who were referred to a nephrologist more than 1 year before dialysis initiation and who had visited a nephrology clinic 2 or more times; the remaining patients whose referral time was less than a year were considered the late referral group (LR) Information about the participants was primarily collected from the data server of the CRC for ESRD for analysis. All traceable identifiers were removed prior to the analysis to ensure patient confidentiality. The clinical and laboratory data had been stored in the form of web-based medical questionnaires. The questionnaire items were filled in by data coordinators, who were trained in each center to collect patient data by a combination of chart reviews and direct interviews using a standardized form. The questionnaires included data on demographics, previous medical history, laboratory results, dialysis modality and prescription, type of permanent access used for the first dialysis, and medications. The estimated glomerular filtration rate (eGFR) was calculated by CKD-EPI equations Information about the medical costs during the 1 year periods just before and after the initiation of dialysis was acquired from the claim data of the Korea Health Insurance Review and Assessment Service (HIRA). In Korea, government-initiated public health insurance is mandatory, and private health insurance without national health insurance is not allowed for direct medical costs. All populations are obligatorily registered in the national health insurance service, and almost all legitimate medical costs are controlled and reimbursed via the Korea HIRA. We calculated the medical costs by an official request for the claim data of medical costs from the Korea HIRA and data integration by the coded identifiers of patients in the CRC for ESRD. To determine the economic cost of an illness, three types of costs should be considered: direct costs, indirect costs (productivity loss), and intangible costs. In this study, we evaluated direct medical expenditures, including physician visit costs, admission costs, surgery costs, physical therapy costs, and total medicine costs. The following costs were excluded: traffic costs, which are considered a direct non-medical cost; traditional Chinese herbal medicine costs; indirect costs; and intangible costs, which cannot be directly collected through the claim data of the Korea HIRA. All of the costs were calculated in the currency unit of the Korean Won (KRW) .a priori and by clinical significance, and included the type of referral, age, sex, type of dialysis modality, body mass index, causes of underlying kidney disease, hemoglobin level, and serum albumin level. Multivariate model included variables that were proved to be associated  with the total medical costs. SAS/STAT 9.2  was used in the analysis of the medical costs, and IBM SPSS ver. 20.0 was used to compare the demographic and clinical parameters. A P value <0.05 was considered statistically significant.In the descriptive analysis of the demographic and clinical characteristics and laboratory results, continuous variables were expressed as the mean and standard deviation, and categorical variables were described numerically with a percentage. Comparisons between the groups based on referral time were performed using a t-test for continuous variables and Chi-square or Fisher's exact tests for categorical variables when appropriate. We presented the mean with the standard error of the mean for the entire cost. The associations between medical costs and independent parameters were assessed using univariate and multivariate linear regression methods. Variables in the univariate models were selected The patients' clinical and laboratory characteristics are summarized in At the time of the referral to a nephrologist, ages were younger, the blood pressure (BP) and serum creatinine level were lower, and the hemoglobin level and eGFR were higher in the ER group than in the LR group. At the time of dialysis initiation, most findings, including the age, sex, co-morbidity index, serum creatinine, eGFR, hemoglobin levels, calcium and phosphate levels, urate levels, and total cholesterol levels were similar in both groups. However, diastolic BP  and LDL cholesterol levels  were lower in the ER group.The monthly medical costs before and after the initiation of dialysis are summarized in We evaluated the impact of various clinical and laboratory parameters on medical costs by univariate and multivariate analyses. The univariate and multivariate analyses on the costs during the 1 year before dialysis also showed a significant association of the referral time with the medical costs . In the Frequency of medication use, including antihypertensive drug, antiplatelet agents, anticoagulants, vitamin D, and phosphate binders, were not different between the two groups. Erythropoiesis-stimulating agents use was more  in the ER group. In the ER group, frequency of native arteriovenous fistula was higher  and temporary tunneled-catheter use was lower .We evaluated the benefit of ER to a nephrologist with respect to economic costs in a prospective cohort of patients who started dialysis for ESRD. We found that ER to a nephrologist significantly reduced medical costs during the periods of 1 year before and 1 month after the initiation of renal replacement therapy using national health insurance claim data. Patients with advanced CKD are frequently complicated with anemia, electrolyte imbalances, hypertension, mineral bone disorders, and other risk factors of cardiovascular diseases. Therefore, correcting and reversing the causes of renal impairment and managing the comorbidities in CKD are important to improve clinical outcomes and patient survival. Education of CKD patients about dialysis can reduce the incidence of emergent hemodialysis via a central venous catheter Despite the various benefits of early nephrology referral, there are still many patients who are referred to a nephrology clinic too late for impending renal replacement therapy. The problems related to LR are common in many countries, and the proportion of patients who are referred when facing the impending dialysis is up to 31–38% There are several causes of the LR of patients with CKD to a nephrologist despite the clinical benefits of ER. With respect to clinicians or medical facilities, the lack of communication between doctors or hospitals, the absence of definite guidelines for the referral time to a nephrologist, and ambivalent opinions about dialysis in the elderly or high risk patients are factors that affecting LR Despite the unique findings and advantages, this study has several limitations. At first, there can be concerns about the lead time bias that patients in the ER group had better renal function, lower blood pressure, less severe anemia, and lower LDL cholesterol levels at the time of referral, and these reduced comorbidities could influence to the reduced medical costs in the ER group. These facts that patients in the ER group showed better clinical conditions are not surprising because they were referred about 5 years earlier than those in the LR group. At least, almost all parameters including age and co-morbidity index at the time of dialysis were similar between the ER and LR groups, and it is hard to say that patients in the ER group had better renal function or lesser comorbidities at the same time 6 or 12 months before dialysis initiation. If we could adjust parameters at the 12 months before dialysis instead of parameters at the time of dialysis initiation, multivariate regression model would be more accurately fitted to predict medical costs during the 12 months before dialysis initiation. However, due to the limited data collection in this cohort study, we could not gather clinical parameters or laboratory data at the 12 months before dialysis initiation, but only get data at the time of referral and at the time of dialysis initiation. Secondly, we only analyzed and compared the medical costs during the 12 months before and after dialysis initiation, not the whole or average medical costs from referral to dialysis initiation. Considering the 5 year mean duration from referral to dialysis in the ER group, if we could have compared the total 5 year medical costs or yearly medical costs during the 5 years between the two groups, the cost-saving effects of ER would be proved apparently without bias. The reasons why we focused on the medical costs during the 12 months before dialysis initiation were that we had divided the ER and LR groups by the referral time of 12 months and aimed to compare the medical costs around the time just before and after dialysis initiation. Actually, during the 12 months before dialysis initiation, monthly medical costs and the difference of medical costs between the ER and LR groups increased steadily. A considerable part of cost-saving effects of ER derived from the reduced medical costs during the several months of impending dialysis. It is still important that patients in the LR group spend much more medical costs probably due to the increased length of hospital stay and increased use of temporary vascular catheters immediately before dialysis initiation. Lastly, we could not investigate the relationship between medical costs and other clinical and laboratory parameters including eGFR, co-morbidity index, blood pressure, and cholesterol levels in univariate analysis. However, it might not lessen the strength of our results because these parameters were similar between the two groups.The care of patients with CKD requires not only evaluation and treatment of decreased renal function but also a multidisciplinary approach, including proper blood pressure control, appropriate selection of medications and their dosages, the avoidance of nephrotoxic drugs, education about diet, preparation for renal placement therapy, psychological consultations, and economic evaluations. Timely referral is essential to improve patient outcomes and survival, but a considerable proportion of patients are referred late to dialysis for various reasons, including a lack of knowledge, negative preconceptions about dialysis, and economic difficulties. In this study, we demonstrated that the ER of CKD patients could reduce economic costs during the period before and after the initiation of renal replacement therapy. Considering the benefits of ER to a nephrologist in both economic and clinical terms, clinicians should focus on the timely referral of patients with CKD.


In recent years, cold-active esterases have received increased attention due to their attractive properties for some industrial applications such as high catalytic activity at low temperatures.estS, 909 bp) from Serratia sp. was identified, cloned and expressed in Escherichia coli DE3 (BL21). The estS encoded a protein (EstS) of 302 amino acids with a predicted molecular weight of 32.5 kDa. It showed the highest activity at 10 °C and pH 8.5. EstS was cold active and retained ~92 % of its original activity at 0 °C. Thermal inactivation analysis showed that the T1/2 value of EstS was 50 min at 50 °C  after 1 h incubation. EstS is also quite stable in high salt conditions and displayed better catalytic activity in the presence of 4 M NaCl. To improve the thermo-stability of EstS, variants of estS gene were created by error-prone PCR. A mutant 1-D5  that showed higher thermo-stability than its wild type predecessor was selected. 1-D5 showed enhanced T1/2 of 70 min at 50 °C and retained 63.29 % of activity after incubation at 50 °C for 60 min, which were about 22 % higher than the wild type (WT). CD spectrum showed that the secondary structure of WT and 1-D5 are more or less similar, but an increase in β-sheets was recorded, which enhanced the thermostability of mutant protein.An esterase-encoding gene (EstS was a novel cold-active and salt-tolerant esterase and half-life of mutant 1-D5 was enhanced by 1.4 times compared with WT. The features of EstS are interesting and can be exploited for commercial applications. The results have also provided useful information about the structure and function of Est protein. Psychrobacter pacificensis, was salt tolerant, highly active at low temperatures, and its biochemical characteristics made it important for commercial applications  from Serratia liquefaciens ATCC 27592 , esterase [GenBank: WP_044551387.1] from Serratia liquefaciens FK01 (92 %), and esterase [GenBank: WP_037415500.1] from Serratia grimesii (82 %). A multiple sequence alignment of EstS was performed with thermophilic carboxylesterase Est2 [PDB: 1EVQ_A] from Alicyclobacillus acidocaldarius (identity: 41 %), esterase PestE [PDB: 2YH2_A] from Pyrobaculum calidifontis (41 %), carboxylesterase Este1 [PDB: 2C7B_A] from a metagenomic library (40 %), esterase Lpest1 [PDB: 4C88_A] from Lactobacillus plantarum (29 %), and all of them were retrieved using BLASTP in the NCBI and PDB database .The protein WT and mutant fused with GST tag (58 kDa) were efficiently induced and overexpressed as a soluble, catalytically active form in host strain at 15 °C. The purified WT and mutant ~32 kDa) were detected by SDS-PAGE as a single band, which was consistent with the value predicted from the deduced amino-acid sequence . EstS showed the maximum hydrolytic activity towards p-NP acetate (C2), but no activity toward p-NP palmitate , 1 and 5 mM Mn2+ , whereas it was fairly inhibited by 1, 5 mM Zn2+  and Cu2+  and 5 mM PMSF (89 %). The mutant 1-D5 showed a similar behavior in the presence of metal ions and relative activity was slightly increased by Mn2+ and strongly decreased by the presence of Zn2+ and PMSF. However, the activity of EstS was not affected by the presence of EDTA, Ca2+, Ba2+ and Sr2+  and was almost completely inhibited by N-butyl alcohol and acetonitrile. Our data also showed that ethanol (10–30 %) increased the activity of the enzyme WT by more than 10 % . Both the predicted models of the WT and mutant enzyme exhibit the typical α/β hydrolase fold, which was characteristic of lipolytic enzymes . The specificity towards short chain acyl esters indicated that purified protein (EstS) was an esterase. EstS demonstrated the T1/2 of approximately 50 min at 50 °C and retained 41.23 % activity after 1 h incubation. In addition, the activity of EstS WT was increased by low concentration (1 mM) of Mg2+ and Mn2+, partly inhibited by Cu2+ and Zn2+, and completely inhibited by the addition of acetonitrile, n-butanol and SDS. EstS retain its activity and stability between pH 5.5 and pH 9.5 after 24 h at 4 °C. Furthermore, we also report the engineering of EstS by directed evolution. A thermo-stable mutant 1-D5 was selected from the random mutant library constructed by error-prone PCR. 1-D5 showed change in three amino acids . No change in optimum temperature and pH of 1-D5 was observed in comparison to EstS. But the T1/2 at 50 °C of 1-D5 was about 70 min and it retained 63.3 % of activity at 50 °C for 1 h, which were about 22 % higher than WT.In this study, we have identified and characterized an esterase (EstS) from a marine bacterium Alcanivorax dieselolei B-5(T) which showed optimal activity at 20 °C and retained 95 % of its original activity at 0 °C  of Serratia liquefaciens ATCC 27592. The whole genome of S. liquefaciens ATCC 27592 [GenBank: CP006252.1] was sequenced and published in GenBank by Nicholson et al .The kinetic parameters , including three executable programs   repository, [GenBank accession number:KU362566].


In Cox-regression analysis, higher survival rate was related to virus-positive biopsies (p < 0.05). When comparing long-term prognosis among different viral groups, a trend towards better prognosis was observed in the presence of isolated Parvovirus B19 (PVB19) (p = 0.07). In our series, presence of a virus-positive EMB (mainly PVB19) was associated with improvement over time in cardiac function and better long-term prognosis.The presence and type of viral genomes have been suggested as the main etiology for inflammatory dilated cardiomyopathy. Information on the clinical implication of this finding in a large population of children is lacking. We evaluated the prevalence, type, and clinical impact of specific viral genomes in endomyocardial biopsies (EMB) collected between 2001 and 2013 among 63 children admitted to our hospital for acute heart failure (median age 2.8 years). Viral genome was searched by polymerase chain reaction (PCR). Patients underwent a complete two-dimensional echocardiographic examination at hospital admission and at discharge and were followed-up for 10 years. Twenty-seven adverse events (7 deaths and 20 cardiac transplantations) occurred during the follow-up. Viral genome was amplified in 19/63 biopsies (35%); PVB19 was the most commonly isolated virus. Presence of specific viral genome was associated with a significant recovery in ejection fraction, compared to patients without viral evidence ( Acute myocarditis is a serious medical condition associated with high morbidity and mortality in both children and adults. Myocarditis may be initiated by a number of different environmental agents, such as viruses, bacteria, parasitic organisms, and drugs . Dilatedi.e., MRI) [in situ hybridization [Despite the recent promising results obtained from noninvasive diagnosis and prognostication of acute myocarditis  , endomyoe., MRI) ,7. Inflae., MRI) ,8,9,10, e., MRI) , reversee., MRI) , or in sdization . Throughdization . In the dization , both indization ,17,18 andization ,20.The aim of our study was to evaluate the presence of specific viruses in EMB specimens collected between 2001 and 2013 from children who were admitted to our hospital for acute heart failure and subsequently diagnosed with either myocarditis or DCM. In addition, we evaluated differences in clinical and echocardiographic features and long-term clinical outcome according to the presence and different types of viruses amplified in EMBs.Clinical characteristics of the population are showed in Viral genome was amplified in 19/63 cases (30%). In 17, a single virus was identified, while a combined infection was found in two. In particular, isolated PVB19 was detected in 17 EMBs (40.7%), EBV (Epstein–Barr virus) in two (25.6%), CMV  in one (10.2%), HSV (Herpes Simplex) in one (2.5%). ENV (enteroviruses) and ADV (adenovirus) were never detected. In two cases, PVB19 was associated with HSV or EBV. We report the results of the PCR viral amplification in the three classified cardiac diseases . Among t2 with a mean ejection fraction (EF) of 30.8% ± 10.5%.Mean left ventricular end-diastolic volume (EDV) at the time of diagnosis was 118.8 ± 60.5 mL/m2 in children with virus, and 103 ± 38 mL/m2 in children without virus (p < 0.05). Similarly, baseline EF was 36% ± 10% in the children with virus, and 26% ± 0% in children without virus (p < 0.01). At the time of discharge, a larger improvement in EF was found in patients with virus-positive EMB (+18%) as compared to those with virus-negative EMB .As shown in 2 in children with myocarditis, 106.7 ± 23 mL/m2 in children with borderline myocarditis, and 134.9 ± 79 mL/m2 in children with DCM . EF was 30.9% ± 10% in the children with myocarditis, 31.1% ± 14% in those with borderline myocarditis, and 30.7% ± 10% in patients with DCM (p = Not Significant).As shown in p for both vs. DCM <0.01).At the time of discharge, a significant increase in left ventricular EF could be observed in all study sample groups, with a markedly higher improvement in patients with myocarditis (+22%) and borderline myocarditis (+18%) as compared to +12% in those with DCM  were further sub-divided according to the absence or presence of viral-genome-positive PCR in order to define four subgroups: DCM-negative PCR (n = 33), DCM-positive PCR (n = 7), myocarditis (MYO)-negative PCR (n = 11), and MYO-positive PCR (n = 12). When comparing echocardiographic data of these groups, only a minor effect of positive PCR on left ventricular (LV) diastolic volumes could be observed , but a significant difference was found in EF recovery. In details, from admission to discharge, while in the DCM group the presence of positive PCR was associated with a lower increase of EF (+6.5%) compared to DCM with negative PCR ), in the myocarditis group, the presence of positive viruses was related to a significant recovery in EF (+25%) as compared to patients with myocarditis and negative viral presence .In p < 0.001). When dichotomizing our population on the basis of viral presence, virus-positive PCR was associated with a significantly higher rate of freedom from events as compared to virus-negative PCR patients .During clinical follow-up , 27 events occurred, including 7 cardiac deaths and 20 cardiac transplants. The incidence of adverse cardiac events was significantly lower in the group of patients with either clear-cut or borderline myocarditis (26%) as compared to the group without myocarditis , and in the DCM group .When further analyzing the population as four separate groups on the basis of diagnosis and detection of viral genome as previously described, virus presence was associated with a markedly better outcome both in patients with myocarditis (8.3% p < 0.05).In Cox regression analysis, after correction for differences among groups in age, gender, and EF improvement over time, both the presence of myocarditis (HR (Hazard Ratio): 0.23; 95% CI: 0.12–0.42) and of a virus-positive EMB  were independent predictors of a better outcome , possibly also due to the low incidence of events in the virus-positive EMB group.There were no significant differences in event rate when separating patients with virus-positive EMB according to differences in virus type . We need to point out, however, that the main limitation of our study is that other viruses (like HSV6) or bacteria  known to be involved in myocarditis were not tested and could theoretically be involved in causing worse prognosis.In our study sample of 63 young patients, a negative outcome was observed in 43% of the population (including 7 cardiovascular related deaths and 20 children requiring heart transplant). As expected, a worse outcome was observed in children with DCM; however, in regression analysis we found that the presence or absence of a virus-positive EMB was also an independent predictor of events. Presence of specific viruses assessed by PCR was detected in approximately 30% of children. Viral presence was associated with a significant improvement during the hospitalization time in cardiac function and in a significantly higher survival rate at follow-up in the whole study sample, both in myocarditis and in DCM. It should be noted that while in the absence of virus on EMB, incidence of events was 57%, in the presence of virus-positive EMB, only two events were recorded  is characterized by a “transient” cardiac dysfunction, possibly secondary to the effect of the aforementioned endothelial dysfunction caused acutely by the virus. This dysfunction is often reversible, and the presence of viral genome appears to be associated with a more favorable long-term prognosis.Our study shows that over one third of our patients experienced a negative clinical outcome at follow-up (death or transplant), and young patients with myocarditis, either overt or borderline, tend to recover more often than those with idiopathic DCM.Considering the limitation underlined above for our study regarding the specific group of viruses analysed, viral etiology was not common in our clinical setting, as it could be identified in only approximately 30% of the study sample. However, viral presence was associated with a significant early improvement in cardiac function and better long-term prognosis, with a detectable independent effect, regardless of whether the viral genome was observed in children with a diagnosis of myocarditis or DCM.In conclusion, our findings suggest that performing EMB with PCR analysis in paediatric patients presenting with acute heart failure—not sustained by either structural cardiac anomalies, neuromuscular diseases, or systemic metabolic syndromes—provides useful prognostic information. The presence of specific viruses was indeed detected in only 30% of patients, PVB19 being the most commonly isolated virus. Better prognosis was associated with the presence of viral etiology, although the main limitation of our study is that important viruses or bacteria were not tested. These could theoretically be involved in causing a worse prognosis.Between January 2001 and February 2013, 63 patients, from newborn to teenagers, were referred to our hospital for acute heart failure . InclusiAfter hospital discharge, patients were followed clinically for up to 10 years  at 6-month intervals, with physical examination and echocardiography if clinically necessary. Cardiac related death or transplant were considered as primary endpoints of the study.2, including up to 4 monocytes/mm2 with the presence of CD3-positive T lymphocytes ≥7 cells/mm2. We recognized three types of cardiac histological patterns: acute lymphocytic myocarditis, borderline myocarditis, or no myocarditis.EMBs were obtained from the right side of the septum in each patient by means of a flexible 6 French bioptome using transjugular or femoral vein approach. Seven or eight EMB specimens were obtained from each child, frozen in liquid nitrogen and stored at −80 °C for subsequent extraction of RNA and DNA for PCR analysis. According to the “Dallas criteria” , myocardWe searched for the following DNA viruses: ADV, CMV, HSV1/HSV2, EBV, and PVB19, using pre-synthesized primers, based on published sequences or purchased from Amplimedical SpA, Bioline Division . RNA of enteroviruses (ENV)  were also searched.t-test for continuous variables and by Chi-square test . Analysis of variance (ANOVA) and analysis of covariance (ANCOVA) were performed to analyze differences among two or more groups, when correcting for relevant confounders. Paired t-test for continuous variables and Chi-square with McNemar’s test for categorical variables was used to compare repeated measures performed at admission and discharge. The Kaplan–Mayer curves with product limit estimator was used to calculate freedom from mortality and/or heart transplantation between different groups. Multivariate Cox regression analysis was used to compute hazard ratios in order to identify predictors of clinical outcome, independently of significant clinical and echocardiographic confounders. A p value below 0.05 was considered statistically significant.Results were expressed as mean ± SD. All variables were tested for normality using the Kolmogorov–Smirnov test. Data among groups were compared by Student