Pseudogymnoascus destructans, has spread quickly in North America and has become one of the most severe wildlife epidemics of our time. While P. destructans is spreading rapidly in North America, nothing is known about the sexual capacity of this fungus. To gain insight into the genes involved in sexual reproduction, we characterized the mating-type locus (MAT) of two Pseudogymnoascus spp. that are closely related to P. destructans and homothallic (self-fertile). As with other homothallic Ascomycota, the MAT locus of these two species encodes a conserved α-box protein (MAT1-1-1) as well as two high-mobility group (HMG) box proteins (MAT1-1-3 and MAT1-2-1). Comparisons with the MAT locus of the North American isolate of P. destructans (the ex-type isolate) revealed that this isolate of P. destructans was missing a clear homolog of the conserved HMG box protein (MAT1-2-1). These data prompted the discovery and molecular characterization of a heterothallic mating system in isolates of P. destructans from the Czech Republic. Both mating types of P. destructans were found to coexist within hibernacula, suggesting the presence of mating populations in Europe. Although populations of P. destructans in North America are thought to be clonal and of one mating type, the potential for sexual recombination indicates that continued vigilance is needed regarding introductions of additional isolates of this pathogen.White-nose syndrome (WNS) of bats has devastated bat populations in eastern North America since its discovery in 2006. WNS, caused by the fungus  The causative agent of WNS is a psychrophilic fungus named Pseudogymnoascus destructans (=Geomyces destructans)  of hibernating bats has spread to more than 25 states and 5 Canadian provinces, killing more than 5.5 million bats (ructans)  that hase.g., humans), mating in fungi can involve multiple mating types, but there are no male and female genders and, thus, no sex chromosomes . Fungal mating types are determined by a single genetic locus termed the mating-type locus (MAT locus), which consists of highly divergent nonhomologous genes that are termed idiomorphs  transcription factor   .Cryptococcus neoformans  (S. sclerotiorum (white mold)  movement of P. destructans.Although the infectious particles of many fungal pathogens are asexual spores, sexual spores can be infectious, as in the human pathogenic fungus Pseudogymnoascus (P. destructans as exemplars for understanding the mating-type locus in Pseudogymnoascus. We cloned and sequenced the mating-type (MAT) locus from these two homothallic species and discovered that these species share a nearly identical gene structure at the MAT locus (see Results section). Comparison of the homothallic Pseudogymnoascus MAT locus to the P. destructans genome reference strain suggested that the bat pathogen was likely heterothallic. We screened isolates of P. destructans from central Europe and discovered the opposite mating type (MAT1-2). Differential expression of P. destructans genes involved in mating was also examined in cultures of each mating type individually as well as in mixed culture.Based on a recent phylogenetic study of the genus mnoascus , we seleP. destructans were isolated from muzzles and wings of bats with suspected WNS (Myotis myotis and Plecotus auritus) using sterile cotton or plastic swabs and cultured on yeast extract glucose chloramphenicol agar or Sabouraud dextrose agar at 10°. For routine laboratory experiments, isolates were maintained on a combination of glucose minimal medium (GMM) .Fungal strains used in this study are listed in um (GMM)  and Chamum (GMM) . All isoum (GMM) . BLAST sg at 4°. DNA was precipitated by adding 1.0 ml of 95% ethanol and centrifuged for 10 min . The DNA pellet was washed with 70% ethanol and subsequently resuspended in 10 mM Tris-HCl (pH 8.0) containing 20 units of RNAseA (5′).Fungal cultures were grown in liquid stationary culture for 3 wk in Champe’s medium , myceliuP. destructans MAT1-1-1 (α-box) were used to amplify a PCR fragment of the MAT1-1-1 gene from the homothallic Pseudogymnoascus species (WSF 3629 and 23342-1-I1); ∼900-bp fragment was obtained for isolate WSF 3629 and ∼400-bp fragment was obtained for 23342-1-I1. The PCR fragments were subsequently cloned using pCR-Blunt II-TOPO (Life Technologies) and sequenced. Sequencing of the region flanking the MAT1-1-1 gene was achieved by using a modified version of thermal asymmetrical interlaced PCR (TAIL-PCR) called fusion primer and nested integrated PCR (FPNI-PCR)  and subsequently transferred to the surface of solid GMM medium agar plates: one plate for each mating type as well as one that was a 1:1 mixture of mycelium from MAT1-1 and MAT1-2 strains. The plates were wrapped in Parafilm-M (Bemis) and aluminum foil and incubated at 15° for an additional 14 d. Mycelium was then scrapped off the surface of the plates using a sterile glass slide, immediately frozen in liquid nitrogen, and lyophilized overnight. Total RNA was extracted from the lyophilized tissue using Isol-RNA Lysis Reagent (5 Prime) following manufacturer’s recommendations, treated with DNase I (NEB) according to the manufacturer’s protocol, and subsequently used to make cDNA using the iScript cDNA Synthesis Kit (Biorad). Genes involved in sexual reproduction in other filamentous fungi were identified through BLASTp searching of the P. destructans reference genome and primers were designed for the mating-type genes , the pheromone pathway , the G-protein signaling pathway , and the velvet complex ; actin (act1–GMDG_01001.1) was used as a loading control. Between 32 and 42 amplification cycles were used to detect transcription of genes putatively involved in sexual reproduction.Conidia were harvested in sterile water supplemented with 0.01% Tween-80 from 8-week-old cultures of MAT locus of P. destructans was identified by a BLASTp (P. destructans draft genome assembly with the MAT α-box (MAT1-1-1) protein sequence from Aspergillus nidulans AN2755 (apn2 and sla2 flank the MAT locus (apn2 and GMDG_01210.1 as sla2. Using the Conserved Domain Database (CDD) search (MAT α-box domain (pfam04769). Interestingly, GMDG_01208.1 is also located in the MAT locus and has a predicted HMG-box domain (cd01389). A BLASTp search using GMDG_01208.1 of the nonredundant protein database (nr) at NCBI revealed the top hits to be MAT1-2-1 proteins . Therefore, we initially thought that P. destructans could be homothallic because the MAT locus harbored both MAT1-1-1 (α-box) and MAT1-2-1 (HMG-box) genes. However, because we have never observed fruiting bodies from P. destructans 20631-21 in culture and the MAT locus of some fungi contains two HMG-box domain genes, we could not rule out that P. destructans 20631-21 was a MAT1-1 genotype.The AT locus ; thus, wPseudogymnoascus are known to be homothallic, and thus produce sexual fruiting bodies in culture  and Pseudogymnoascus sp. 23342-1-I1 (clade D)  . Pseudognothecia , which anothecia , asci . Subsequent rounds of fusion primer and nested integrated PCR (FPNI-PCR)  (ab-initio gene prediction programs AUGUSTUS 2.7 (www.softberry.com), we deduced that the homothallic MAT locus from both WSF 3629 and 23342-1-I1 contain a nearly identical gene structure consisting of five predicted open reading frames (ORFs). A combination of BLAST (MAT α-box protein (MAT1-1-1) and two high-mobility group (HMG) domain-containing proteins (MAT1-2-1 and MAT1-1-3) (MAT1-1-6 and MAT1-2-5); however, BLAST search did not reveal any significant homology with other known proteins, suggesting that these predicted ORFs are either novel MAT genes unique to the Pseudeurotiaceae or perhaps pseudogenes. Pairwise comparison of the MAT locus from the homothallic Pseudogymnoascus species to the P. destructans genome sequenced reference strain (20631-21) indicated that the genome reference strain was missing the MAT1-2-1 HMG box domain containing gene, as well as the hypothetical MAT1-2-5 gene, indicating that it was a MAT1-1 (α-box) mating type.After confirmation of homothallism in two ctively) . Using tAT1-1-3) . This anP. destructans genome reference strain (20631-21) is a North American isolate that has been hypothesized to be spreading clonally . We screened 23 isolates of P. destructans for the presence of the MAT1-1-1 gene via Southern blotting and found that five of the isolates  were missing MAT1-1-1 , suggesting that this isolate is the opposite mating type , which is involved in production of the α-mating pheromone. The α-pheromone is recognized by the G-protein-coupled receptors (PRE1 and/or PRE2), which our data suggest are, in turn, under the control of the MAT1-2 locus in P. destructans  as well as in the velvet complex  were not drastically altered in either of the mating types or in mixed culture  and two conserved HMG box domain proteins (MAT1-1-3 and MAT1-2-1). This is consistent with the MAT locus of other well-studied homothallic Ascomycota species such as Sordaria macrospora  revealed that it was missing the MAT1-2-1 HMG box protein, suggesting it was heterothallic. Interestingly, there are two more predicted ORFs in the homothallic MAT locus, MAT1-1-6 and MAT1-2-5, which appear to have no known functional domains or homology to other known proteins and thus may represent novel MAT genes in the Pseudeurotiaceae.To gain insight into the molecular components of sexual reproduction in the Pseudeurotiaceae, we selected two homothallic (self-fertile) isolates from a recent study characterizing species related to tructans . CloningP. destructans coexisting in European hibernacula, indicating that in central Europe there is the potential for mating populations. Although these data suggest that, in our limited sampling, the MAT1-1 mating type is found more frequently on Myotis myotis (18 out of 23), more sampling of European fungal isolates is necessary to understand the prevalence of mating types in P. destructans. Preliminary experiments inducing sexual reproduction in the laboratory have not yielded results to date; this is not surprising because P. destructans is slow-growing and sexual reproduction may not occur for long time periods, as exemplified by other members of the genus  was only expressed in the MAT1-1 background. Moreover, both of the G-coupled-protein receptors (PRE1 and PRE2) hypothesized to recognize the α-pheromone are only expressed in the MAT1-2 background. Although we did not detect differences in expression of genes involved in sexual development in other fungi, which included the signal transduction cascade     and the P. destructans in North America, this important discovery of heterothallic mating types highlights the need for continued vigilance in preventing additional introductions of this pathogen in North America. Further work is needed to find and characterize the cryptic sexual cycle of P. destructans, although determination of the mating types of isolates will be crucial to successfully characterizing sexual reproduction in this fungal pathogen under laboratory conditions. Sexual recombination may allow P. destructans to quickly adapt to its environment and hosts, despite its slow growth. Pertinent to pathogenicity of P. destructans, mating types in other fungi have been correlated to virulence (Given the apparent clonality of


Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.DOI:http://dx.doi.org/10.7554/eLife.03679.001 Viruses can infect, take control of and replicate themselves inside the living cells of other organisms. Some viral diseases can be treated with antiviral drugs, which stop viral infections either by making it more difficult for viruses to enter cells or by preventing the virus replicating once inside. As antiviral drugs are currently only available to treat a handful of viral infections, efforts are underway to develop and test experimental antiviral drugs.One such experimental drug is called favipiravir, which is proving to be effective against several viruses that store their genetic information in the form of RNA molecules. These viruses include those that cause diseases such as influenza, gastroenteritis, and Ebola. Along with ongoing work determining how safe and effective favipiravir is for treating viral infections, researchers are also attempting to better understand how favipiravir works.Whenever a strand of RNA is copied to allow a new virus to form, there is a risk that mistakes—or mutations—that could harm the virus are introduced into the genetic code. Previous experiments performed on cells grown in the laboratory suggested that favipiravir works against RNA viruses by increasing how often these mutations occur. RNA viruses naturally experience a large number of mutations and the ability to make mutations is in fact a benefit for viruses as it allows them to evolve rapidly and to escape immune responses. However, there is a limit to how many mutations can be tolerated in the viral genome before it can no longer replicate. Therefore, a slight increase in how often mutations occur—as thought to be caused by favipiravir—is able to stop the RNA virus replicating and halt the infection. However, favipiravir's mode of action had yet to be confirmed in living animals.Using mice, Arias et al. tested favipiravir's ability to treat a persistent infection by norovirus—the most common cause of viral gastroenteritis in humans and also responsible for life-threatening chronic diarrhoea in immunodeficient patients. Treatment increased the number of mutations that occurred when the viral RNA replicated and could reduce the amount of virus in the mice to undetectable levels. In addition, favipiravir did not show toxicity in mice after 8 weeks of treatment. This suggests that favipiravir has the potential to be used safely and effectively to treat norovirus and other RNA viruses, although further studies are required before it can be developed into a clinical treatment.DOI:http://dx.doi.org/10.7554/eLife.03679.002 Due to elevated error frequencies during the replication of their genetic material, RNA virus populations exist as complex distributions of mutant genomes also known as quasispecies . GeneticSeveral nucleoside analogues  and non-nucleoside compounds (amiloride) display antiviral activities in cell culture against a wide range of RNA viruses and appear to act via increased mutagenesis . IncreasDespite all the evidence in cell culture, data confirming lethal mutagenesis as a plausible therapeutic approach in vivo remains limited. Although some of the antiviral compounds with mutagenic activity in cell culture are also reported to have antiviral activity in vivo, controversy exists regarding the therapeutic mechanism. To date, the most successful antiviral compound with mutagenic activity in cell culture is purine analogue ribavirin . RibavirSeveral novel compounds eliciting antiviral mutagenesis in cell culture were recently identified . Among tWith the aim of demonstrating lethal mutagenesis as a conceivable approach to treat viral infections in vivo, we investigated whether ribavirin and favipiravir elicit antiviral activities in a mouse model of persistent norovirus infection. Favipiravir caused significant decreases in virus titres and viral RNA levels and led to the clearance of infectious virus in the faeces of seven out of nine animals . Blind passaging of faecal and tissue samples in cell culture confirmed that favipiravir treatment led to viral extinction in some animals. In contrast, ribavirin showed limited efficacy in vivo which correlated with lower antiviral activity in cell culture when compared to favipiravir. Favipiravir antiviral activity in vivo was associated with a significant increase in viral mutation frequencies. Viral RNA isolated from faeces of treated animals showed decreased specific infectivity, and infectious virus from faeces showed decreased fitness in tissue culture. These data suggest that favipiravir causes increased mutagenesis leading to decreased infectivity and fitness of viral genomes, both features of viral populations approaching extinction during mutagenesis. These results constitute a proof of concept for lethal mutagenesis in vivo and support antiviral therapies based on mutagenic compounds at the clinical level. The data also support the use of favipiravir in the treatment of norovirus infections for which there are not yet licenced antivirals or vaccines available and highlight a need for further studies on favipiravir and other improved derivatives as possible broad-range antiviral strategies.−/− mice  strains, MNV-1 and MNV-3. MNV-1 was the first MNV strain isolated as the causative agent of a fatal infection in Stat1−/− mice . Later s−/− mice . MNV is −/− mice . MNV-1 e−/− mice . Previou−/− mice . However10 decrease for ribavirin and a 4-log10 for favipiravir (The treatment of RAW264.7 cells infected with MNV (MOI of 0.01 TCID50/cell) with ribavirin or favipiravir resulted in significant reductions in virus yields, reaching up to a 3-logipiravir . A decreipiravir .10.7554/To further investigate the mechanism of inhibition, we determined the effect of ribavirin and favipiravir upon MNV replication in single-cycle replication kinetics carried out by infecting RAW264.7 cells at high MOI. Favipiravir inhibition occurred in a gradual manner, with the greatest reduction in virus titre being observed at later time points . This reAlthough ribavirin and favipiravir cause increased mutagenesis in several RNA viruses , the mecA signature feature for error catastrophe in virus populations subjected to mutagenesis is a decrease in virus specific infectivity . To inveTo further investigate the effect of mutagenesis on virus fitness, RAW264.7 cells were infected with virus obtained after 5 passages in the presence of ribavirin or favipiravir  at the sThe efficacy of ribavirin and favipiravir as antiviral compounds in vivo was next investigated in C57BL/6 mice persistently infected with MNV-3. Animals were infected 4 weeks before the beginning of the treatment to allow the establishment of a persistent infection. In a preliminary experiment , we founAfter 53 days of treatment with favipiravir, infectious virus titres were undetectable in the faeces of 7 out of 9 animals, while only 2 out of 10 animals had undetectable titres in the control group, suggesting that the infections were cleared as a result of favipiravir treatment . SimilarTo investigate whether favipiravir treatment in vivo resulted in the selection of adapted MNV-3 variants, we carried out infections in RAW264.7 cells with virus samples recovered from mice. We did not identify differences in the sensitivity to favipiravir between samples isolated from treated and untreated animals, suggesting no adaptation to the treatment .Quantification of MNV RNA in faecal samples also showed lower viral levels in treated mice than in control animals for the duration of the study . The samTo clarify if favipiravir antiviral activity observed in vivo was associated with mutagenesis, we examined the mutation frequency of viral populations shed in the faeces of five different animals in each group. The mutation frequencies found in virus samples from favipiravir-treated animals were greater than in placebo-treated mice with an average of a 2.9-fold increase relative to control animals being observed . We alsoTo investigate whether favipiravir-induced mutagenesis in vivo resulted in decreased infectivity of viral populations, we firstly isolated and quantified viral RNA from faeces at various times during the treatment of animals with favipiravir and used an equivalent genome copy number to transfect BHK-21 cells . BHK-21 To compare whether mutagenesis in treated animals resulted in lower fitness of virus samples recovered, we determined the relative replication rates of virus populations recovered from mice after 18 days of treatment. To this aim, we carried out MOI controlled infections (0.01 TCID50/cell) in RAW264.7 cells using virus previously isolated from faecal samples and propagated once in cell culture. Virus yields obtained using virus from favipiravir-treated animal samples were lower on average than those isolated from placebo-treated animal samples, suggesting that mutagenesis induced by favipiravir results in a viral fitness cost in vivo . Thus, fLethal mutagenesis has been the subject of numerous studies in cell culture in the last few years as an alternative approach to classical antiviral therapies . Due to Favipiravir was initially identified as an antiviral compound for the treatment of influenza virus infection  and currIn addition to its activity in vivo against influenza virus and data shown here on MNV, favipiravir is efficient in the control and clearance of a broad number of RNA viruses including picornavirus, parmyxovirus, bunyavirus, arenavirus, and togavirus . ImportaThese results are also relevant for the development of antiviral strategies to control human norovirus (HuNoV) infections for which there are currently no licenced vaccines or antiviral therapies. Due to the absence of cell culture systems to recover and propagate infectious HuNoVs, MNV has been suggested as a potential surrogate system . HuNoVs Recent evidence suggests that the combination of a mutagenic compound with a classical antiviral molecule can be more efficient in the extinction of a virus than the use of the compound alone or the combination of classical inhibitors only . InhibitStudies with mice were performed in the Department of Pathology BSU Unit (PCD 80/2802) after ethical review by the University of Cambridge Review Panel and subsequent approval by the UK Home Office (PPL70/7689). All animal procedures and care conformed strictly to the UK Home Office Guidelines under The Animals (Scientific Procedures) Act 1986.2. MNV-1 and MNV-3 strains used in this study were obtained after reverse genetics recovery of infectious virus as previously described (Procedures for the cultivation of cells and MNV infections have been previously described . Murine escribed . Recover6 cells in 35 mm diameter dish). Supernatant was then removed and replaced by 1 ml of complete DMEM containing either 200, 400, or 800 μM ribavirin or favipiravir and the cells were incubated for two additional hours at 37°C and 10% CO2. Cells were inoculated with 200 μl of virus at the multiplicity of infection (MOI) indicated and incubated for 1 hr at 37°C and 10% CO2. Supernatants were removed, cells washed to eliminate unattached virus, and 2 ml of fresh media containing ribavirin or favipiravir were added to each well. Cell cultures were collected at 24 hr post-infection and virus released through two consecutive freeze–thaw cycles.RAW264.7 cells were grown until they formed confluent monolayers  were applied to a new monolayer of cells.Viral RNA was extracted from 100 μl of viral samples, either supernatant from lysed infected cultures or PBS-resuspended faeces from animals, using EconoSpin columns , following protocols provided by the manufacturer. Viral RNA was quantified using a two-step qPCR approach following protocols described previously .Escherichia coli colonies were identified by PCR screening with primers flanking the vector-cloning site and GoTaq polymerase . The resultant PCR products corresponding to individual MNV cDNA clones were sequenced and the mutation frequency in each population calculated.For the calculation of mutation frequency in any virus population, we have followed protocols previously described . Briefly4 TCID50 units of MNV-3 as previously described  or presence of 200 μM favipiravir.To determine whether virus replicating in vivo has acquired resistance to favipiravir, virus samples obtained from animal faeces at treatment day 53 were blind passaged in RAW264.7 cells as mentioned above. The amplified virus samples were applied to 10 review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “A mutagenic nucleoside can drive virus extinction during replication in vivo” for consideration at The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Wes Sundquist (Reviewing editor) and Raul Andino, one of the other two reviewers.The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.This manuscript documents the increased efficiency of favipiravir over ribavirin in inducing lethal mutagenesis in RNA viral infection. The loss of infectivity and the increased genetic burden during passage in tissue culture and in mice is chronicled for murine norovirus. The persistent norovirus infection developed by this laboratory is an excellent system to study clearance of chronic viral infections.The work will be an important contribution to the field once several issues have been addressed:1) The authors have overstated their case and should “tone down” their rhetoric in several places. These include:a) The claim that this is “the first proof of principle for lethal mutagenesis as a feasible therapeutic approach”. “Feasible” is a very subjective term. Many papers have shown inhibition of virus growth and pathogenesis in mouse models in the presence of ribavirin, and many papers have shown increased mutagenesis in viruses grown in cell culture. It is true that there are very few studies that have both shown increased mutation accumulation as well as treatment effectiveness in the same paper. Other examples of the in vivo effects of ribavirin on a reduction in viral titers and either an increase or an altered mutation spectrum in mice includes:b) The exaggerated claim in the title that the compound “can drive viral extinction during replication in vivo”. c) Similarly, in 2) The issue of “viral extinction” is an important one. For a virus to be driven to extinction, it should not recrudesce in the absence of drug. Have the authors done this experiment? 3) The enhanced efficacy of ribavirin at early time points could either be “due to a different mechanism of action or an additional antiviral activity” or the different mutational spectrum could render it less possible for adaptive mutations to accumulate. This is the major argument against the concept of lethal mutagenesis, promoted by several laboratories including that of James Bull.4) That the mutation rate increases in correlation with compound treatment does not mean that it was causal. Of course, it is likely that it was, but the authors should tone down their statements of causality. However, as the authors state on in the Introduction: “some studies have not found increased mutagenesis as a factor contributing to the associated antiviral activity”. Please parse out what is different, then, about the logic of the present study.5) 6) The favipiravir mechanism of action was suggested to direct inhibition of the RNA-dependent RNA polymerase, and the paper would be strengthened by establishing whether or not the antiviral effect is not in part a direct inhibition of RNA synthesis. Directly inhibition of RdRp could be tested by doing kinetics experiments in intact cells using RT-PCR because mutagenic effects should only be apparent after accumulation of mutations that manifest after few rounds of replication (particularly at high MOI), whereas direct effects on the RdRp may be clear on one step-replication curve. An alternative is to perform biochemical analyses of the effects of favipiravir on the purified polymerase.7) Another important issue is whether or not the virus can develop resistance to favipiravir, and could also demonstrate that the central tenet of the study is correct. Have the authors done such experiments?8) 9) 10) 11) 12) “A signature feature for error catastrophe in a virus population subjected to mutagenesis is a decrease in virus infectivity.” The authors probably mean a decrease in specific infectivity since all antivirals would be expected to decrease virus infectivity.13) The authors state that the vRNA levels from the colon and caecum are lower or undetectable in the treated mice, but this is only true for the caecum, not the colon. 1) The authors have overstated their case and should “tone down” their rhetoric in several places. These include:a) The claim that this is “the first proof of principle for lethal mutagenesis as a feasible therapeutic approach”. “Feasible” is a very subjective term. Many papers have shown inhibition of virus growth and pathogenesis in mouse models in the presence of ribavirin, and many papers have shown increased mutagenesis in viruses grown in cell culture. It is true that there are very few studies that have both shown increased mutation accumulation as well as treatment effectiveness in the same paper. Other examples of the in vivo effects of ribavirin on a reduction in viral titers and either an increase or an altered mutation spectrum in mice includes:, J. Virol. 87:10997., cited in paper. This paper shows a change in mutation profile in the presence of ribavirin in humans concomitant with a decrease in viral load.In hindsight, we agree with the reviewers in this point. As they state, others have provided evidence to suggest that ribavirin could be causing lethal mutagenesis in vivo. We have edited the manuscript to tone down the rhetoric and make the text more accurate. We have also modified the title of the paper as suggested below to: ‘Favipiravir elicits antiviral mutagenesis during virus replication in vivo’We have also modified the text to highlight the advances made in the present manuscript in the field of lethal mutagenesis. Specifically, our data support that lethal mutagenesis occurs in vivo since increased mutagenesis is associated with decreased specific infectivity and fitness of the virus population. We have now modified the text accordingly.b) The exaggerated claim in the title that the compound “can drive viral extinction during replication in vivo”.We agree with the reviewers that our title might be exceeding the evidence obtained. Thus, we have changed the title to more clearly reflect our data.shows reduction of virus below the detection limit, but virus also 'extinguishes' in this experiment in the untreated mice, just later. Therefore, the mice were not actually 'chronically infected'.MNV establishes a persistent infection that can be typically tracked by PCR for at least 8 months . Virus rebound was observed in all untreated animals while 3 out of 9 favipiravir-treated animals remained negative (included as a supplementary figure). We have modified the text now to include this information.We have also modified c) Similarly, in, the authors show the disappearance of viral RNA from the faeces of 7/9 favipiravir-treated animals while this occurred in only 2/10 control animals. This is a trend but probably not significant and not sufficient to make it into the title. It is not clear in this case, also, that the mice in this experiment were 'chronically infected' because the amount of RNA was certainly trending downward even in the control.We have assumed that the reviewers here refer to 2) The issue of “viral extinction” is an important one. For a virus to be driven to extinction, it should not recrudesce in the absence of drug. Have the authors done this experiment? We have performed additional experiments and confirmed that extinction occurs in cell culture for MNV-3 treated with 400μM favipiravir . Virus pWe are unable to determine whether a rebound in viral infection occurs in the absence of favipiravir in vivo, as we sacrificed the animals at day 53 (end-point). To determine whether virus infection has been cleared in favipiravir-treated animals, we have blind passaged samples isolated from faeces and tissues collected post-mortem (day 53) in RAW264.7 cells. No virus rebound was observed in any faecal or tissue sample from 3 treated animals  after 3 blind passages cell culture, which suggests that virus extinction has occurred in these animals. This new data are included as We have now included the data of blind passages of animal samples in the manuscript.3) The enhanced efficacy of ribavirin at early time points could either be “due to a different mechanism of action or an additional antiviral activity” or the different mutational spectrum could render it less possible for adaptive mutations to accumulate. This is the major argument against the concept of lethal mutagenesis, promoted by several laboratories including that of James Bull.We have now modified the text to include this possible alternative activity.4) That the mutation rate increases in correlation with compound treatment does not mean that it was causal. Of course, it is likely that it was, but the authors should tone down their statements of causality. However, as the authors state on in the Introduction: “some studies have not found increased mutagenesis as a factor contributing to the associated antiviral activity”. Please parse out what is different, then, about the logic of the present study.In this paper we have demonstrated that increased mutagenesis in vivo is associated with decreased specific infectivity of viral RNA isolated , and als5). “Semi-quantitative PCR” does not contribute to the conclusions of the study and these panels should be deleted.We agree with the reviewer and we have now transferred these data to a supplementary figure . We cons6) The favipiravir mechanism of action was suggested to direct inhibition of the RNA-dependent RNA polymerase, and the paper would be strengthened by establishing whether or not the antiviral effect is not in part a direct inhibition of RNA synthesis. Directly inhibition of RdRp could be tested by doing kinetics experiments in intact cells using RT-PCR because mutagenic effects should only be apparent after accumulation of mutations that manifest after few rounds of replication (particularly at high MOI), whereas direct effects on the RdRp may be clear on one step-replication curve. An alternative is to perform biochemical analyses of the effects of favipiravir on the purified polymerase.Our time-course data of high MOI-infected cells shows similar infectious virus production kinetics during the exponential phase of replication in the absence and presence of favipiravir, suggesting no effect of favipiravir on RNA replication. We have further investigated the effect of favipiravir and ribavirin on MNV RNA levels. We have obtained similar results to those obtained in kinetics of infectious virus, which further supports that favipiravir does not affect the exponential phase of MNV RNA replication (data not shown). We have now modified the text to include this information.7) Another important issue is whether or not the virus can develop resistance to favipiravir, and could also demonstrate that the central tenet of the study is correct. Have the authors done such experiments?We have not directly investigated whether MNV can adapt to replicate in the presence of favipiravir in tissue culture. The sequence analysis of MNV-1 and MNV-3 subjected to 5 passages in the presence of 200μM favipiravir did not identify any adaptive mutation in the virus polymerase.Likewise, no signature changes have been found in the polymerase genes of populations isolated from treated animals (data not shown). To determine whether treatment with favipiravir selects for adaptive mutations, we have carried out an additional experiment to show if virus isolated from treated animals at treatment day 53 are less sensitive to favipiravir. However, the data obtained suggests that virus isolated from treated animals remains sensitive to favipiravir, with no difference observed between treated and untreated animal virus samples. This information is now explained in the text, and included as a new supplementary figure.8)does not seem to provide any new information. Please clarify or delete.9). The authors should provide the concentration of compound on the X axis of the graphs or provide a legend to make it clear. The figure should indicate the compound, concentration, and virus strain directly in the image.We have modified the figure as suggested by the referees.10)should be removed or clarified. These figures show that ribavirin inhibited norovirus replication at early time points, but that favipiravir gradually inhibited replication. The authors argue that this difference indicates that the two compounds act via different mechanism of actions. However, it is alternatively possible that the delayed inhibition shown by favipiravir compared to ribavirin reflects the fact that it takes several more steps to convert favipiravir to the active form compared to ribavirin. For example, favipiravir is predicted to be ribosylated and polyphosphorylated :981-6) whereas ribavirin only needs to be converted to the triphosphorylated form. Additional experiments would need to be performed to draw conclusions from this data.As mentioned above, we have carried out experiments with extended pre-incubation times (∼15 hours). Longer pre-incubation times with favipiravir do not substantially alter the observation made previously, suggesting that favipiravir do not inhibit MNV RNA synthesis. Further experiments to determine the kinetics of viral RNA synthesis in the presence of these compounds have correlated well with infectious virus kinetics, with no difference observed between untreated and favipiravir-treated cells at the exponential phase of replication. These new results have been included in the text.11). A) What is the total number of mutations identified for each treatment group? B) Please supply the individual mutation numbers in a table format with raw numbers so that the reader can determine the actual numbers that the % represents.We have modified 12) “A signature feature for error catastrophe in a virus population subjected to mutagenesis is a decrease in virus infectivity.” The authors probably mean a decrease in specific infectivity since all antivirals would be expected to decrease virus infectivity.We have amended this sentence in the text.13) The authors state that the vRNA levels from the colon and caecum are lower or undetectable in the treated mice, but this is only true for the caecum, not the colon.We have reworded this part to make it clearer now. RNA levels are significantly lower in caecum. In colon are also lower but not significant.


In a recent mini-review published in Frontiers in Medicine, Ben Berkhout pointed out that the interactions between co-localizing viruses and commensal bacteria in the gastric tract are important for the survival and transmission of a number of virus families , the archetypal milk borne virus. As mentioned in the review article, MMTV is a member of the Retroviridae that notably also includes the human disease associated members human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV), both of which can also be transmitted to infants by breast feeding. Transmission can occur either by infection with free virions or by delivery of infected cells/cell associated virus where the virus is then transferred through virological synapses that are formed between an infected source cell and a susceptible target cell  in the plasma arising from the outer membrane of “bad” gram negative bacteria. Infants exposed to HIV-1 but not infected by the virus were compared to those that became infected and it was found that higher pre-infection plasma LPS levels are a good predictor of milk born HIV-1 infection (Kourtis et al., It has long been known that expression of the MMTV envelope is also regulated by LPS, which could be of relevance for later cell-cell transmission of the virus (Sharma et al., MMTV is well-known as a retrovirus that has evolved to coerce, manipulate or even collude with both the host immune system and its interaction with commensal bacteria in the gut. Over 20 years ago, it was discovered that MMTV encodes a superantigen that is functionally, though not structurally, similar to that found in certain bacterial strains. The superantigen is expressed in infected B cells and presented to whole classes of T-cells which release cytokines that cause the amplification of the infected B cells thereby increasing the pool of MMTV infected cells. Thus, this is an example of a virus independently evolving yet another bacterial like mechanism to facilitate its ability to infect its host via the gut Ross, . Howeverde novo infections with other retroviruses or even with other infectious agents.As discussed by Young and colleagues, antibodies have established roles in controlling intestinal bacteria and neutralizing products that they produce such as LPS. Using immunodeficient mice, this group showed that infectious virus can arise from usually silent endogenous viruses by exposure to commensal bacteria—presumably expression of these endogenous viruses is normally held in check by the immune response (Young et al., The mini-review by Dr. Berkhout states that there is no human counterpart to MMTV—but this is currently a highly controversial statement. Indeed a number of independent groups have documented that there may very well be a human counterpart to MMTV (for recent reviews see Salmons and Gunzburg, In summary, the mini-review by Dr. Berkhout is intriguing and scratches the surface of this interesting and somewhat neglected topic. Another recent but short review by Wilks and Golovkina proposes that certain commensal microbiota can either provide protection against virus infection or can promote infection. They further subdivide promotion or facilitation of infection by commensal microbiota into direct and indirect effects (Wilks and Golovkina, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this short review, we present our current understanding of the role of viruses on honey bee health and address some overarching questions in honey bee virology.Apis mellifera) are the primary pollinators of numerous food crops, including fruits, nuts, vegetables, and oilseeds [High annual losses of honey bees, as well as range reductions and local extinctions of wild and native pollinator species, are concerning because bees are important plant pollinators . Approxioilseeds . Annualloilseeds . The laroilseeds . This sioilseeds . Since 2oilseeds ,7. A smaoilseeds ,8. Compaoilseeds , Acute boilseeds , and Defoilseeds ,12, thouoilseeds ). High aoilseeds . Despiteoilseeds . Globalloilseeds .High annual honey bee colony losses in regions that are vital to global food production are unsustainable; therefore, in order to reduce bee colony losses, it is important to identify the factors most responsible. Multiple factors, including pathogens, agrochemical exposure, lack of quality forage, and reduced habitat, negatively impact bee health . Single-stranded positive sense RNA viruses make up the largest group of honey bee-infecting pathogens. These viruses include the following: the Dicistroviruses, Israeli acute paralysis virus, Kashmir bee virus, Acute bee paralysis virus, and Black queen cell virus; the Iflaviruses, Deformed wing virus, Kakugo virus, Varroa destructor virus-1, Sacbrood virus, and Slow bee paralysis virus; and taxonomically unclassified viruses, Chronic bee paralysis virus, and the recently discovered Lake Sinai viruses, which are a phylogenetically unique, globally distributed group of viruses, including LSV1-7 and other variants . RNAHoney bee viruses are transmitted both horizontally and vertically  . Virus tVarroa destructor is a major vector of honey bee viruses, including DWV, VDV-1, and IAPV. Varroa destructor mites were first identified in Asia as a parasite of A. cerana (Eastern honey bee) and later introduced into Europe (1960–70s) and North America (1980s) . Varroany death . The synor mites . Likewisor mites . In addior mites . The intor mites ,29, wheror mites ,30. Althor mites ,31; therApis hymenopteran species obtained near honey bee colonies and implicated inter-species transmission by phylogenetic analyses of viral sequences, which did not cluster by host species [Bombus impatiens) via shared food sources [While bee-infecting viruses are primarily studied in honey bees, and thus upon discovery are called “honey bee viruses,” many of these viruses infect other bee species ,32,33. I species . Further sources . Similar sources ,35. Bumb sources . TherefoBees exhibit pathogen defense mechanisms at both the colony and individual levels. Colony level or social immunity includes behaviors such as grooming and removal of infected and/or mite-parasitized larvae . IndividDrosophila melanogaster, because of the availability of mutant fly lines, and Aedes aegypti and Anopheles gambi mosquitos [Drosophila model [RNA interference (RNAi) plays an important role in insect antiviral defense . RNAi isosquitos . Numerouosquitos ,41 and aosquitos ). The moosquitos  (revieweosquitos ). Furtheosquitos . Colony osquitos . The useosquitos . Unlike la model , treatmela model ,48, suggla model ,45).Drosophila C virus [In order to better characterize honey bee antiviral defense mechanisms, several studies examined transcriptional responses to virus infection ,49–51. T C virus . In addiHoney bee virology is a rapidly growing field currently in its infancy. Researchers are employing advanced colony level tools  and molecular level techniques, including high-throughput sequencing, bisulfite sequencing, and qPCR, to investigate the impact of viruses on bee health at the colony, individual, and cellular levels. Cultured cells, both primary cells and immortalized cell lines, provide the opportunity to study host—pathogen and pathogen—pathogen dynamics while minimizing confounding factors  inherent to performing in vivo studies that utilize honey bees obtained from managed colonies in natural settings. Recently, an immortalized honey bee cell line (AmE-711) was utilized for the first time to investigate host—virus and virus—virus interactions in mixed infections . Both inIn conclusion, this is an exciting time in honey bee virology. Important research topics include (1) understanding honey bee antiviral responses, including those triggered by dsRNA and siRNA, at the cellular and molecular levels, and identifying viral counter measures; (2) identifying the most pathogenic virus strains and determining what factors govern their virulence and transmission; (3) investigating the role of the bee microbiome on virus infection and bee health; and (4) determining how synergistic variables, including agrochemical exposure and nutritional stress, impact viral pathogenesis. Further investigation of these and other topics will advance our understanding of bee biology, host—pathogen interactions, colony health, and may lead to the development of strategies that limit colony losses.


Gallstone disease (GSD) is a common gastrointestinal disorder throughout the world. The authors explored the incidence of GSD in Taiwan and its condition-associated predictive factors.The initial study cohort comprised 2386 healthy adult participants, who were voluntarily admitted to a teaching hospital for a physical check-up in 2002 in Taipei, Taiwan. After excluding 126 patients who exhibited prevalent GSD, 2260 non-GSD participants received annual follow-up screenings for GSD until 31 December, 2007. Of those, 1296 (57.3%) patients were re-examined to collect blood samples and conduct ultrasound sonography.2 versus < 24 kg/m2, RR = 1.64 [95% CI: 1.07–2.98]), high fasting plasma glucose levels , and nonalcoholic fatty liver disease  appeared to be significantly related to developing GSD.Among the 1296 participants who exhibited no GSD at the first screening, 23 patients developed GSD during 3640 person-years of follow-up. The incidence was 0.632% per year (95% CI: 0.292%–2.009%). After conducting a Cox regression, increased age , high body mass index (≥27 kg/mIncreased age is a well-established risk factor for developing GSD. The current findings indicated that high body mass index, elevated fasting plasma glucose levels, and nonalcoholic fatty liver disease were also associated with GSD. Gallstone disease (GSD), a digestive disorder with multifactorial origins, is a common gastrointestinal disorder throughout the world. The third National Health and Nutrition Examination Survey estimated that 6.3 million men and 14.2 million women ages 20–74 in the United States had GSD ), elevated FPG , and NAFLD.Table LD P = .0. In addiKnowledge of GSD epidemiology is crucial to managing this disorder when planning preventive programs and identifying the optimal therapeutic strategies . PreviouThe incidence of GSD appears to vary among test populations and differs among studies conducted in disparate countries ,12,13. IIn this study, the proportion of the population lost to follow up was in the higher risk categories for GSD. This deficiency is likely to have under recorded the overall incidence. In addition, the response rate was lower than that reported in other follow-up studies for GSD ,12,13 anThe estimated incidence of GSD in this study was higher in females compared with males, and increased as age increased. This finding is consistent with the reports of other studies conducted elsewhere ,13. GSD The findings indicated that FPG ≥ 126 mg/dL was highly associated with GSD incidence; that is, type 2 diabetes might be a positive risk factor for GSD. The association between diabetes mellitus and GSD is confounded by age, obesity, and family history of GSD . The posAfter using univariate and multiple Cox regression analyses, the findings indicate that obesity is a significant risk factor for GSD. This agreed with the results of other studies that employed extended screening intervals ,12,13. TCirrhosis is a predictive risk factor for GSD, particularly in the advanced stages of the disease ,21. The NAFLD includes a wide spectrum of liver diseases, ranging from simple fatty liver, simple steatosis, nonalcoholic steatohepatitis (NASH), and liver cirrhosis, which may eventually progress to liver damage, liver fibrosis, portal hypertension, and hepatocellular carcinoma . In mainAlthough we used a follow-up study design to clarify the temporal characteristics of the potential risk factors of GSD, certain limitations apply to this study. First, a major limitation was the potential self-selection bias caused by the hospital-based study design and those aged 60 or over were only 18% of the sample and yet developed 48% of the GSD; that is, the sample may not be representative of the general population. Second, the risk characteristics pertinent to the study participants significantly differed from those of non-respondents, indicating that the patients who did not complete the follow-up might have severe GSD. We assumed that all new GSD cases occurred during screening year; however, because additional GSD cases could occur at any time during the screening period, the incidence of GSD could be underestimated. Third, we did estimate the incidence of GSD formation, but rather investigated the incidence of newly screened GSD, focusing only on clinically relevant GSD. Fourth, we did not distinguish between cholesterol and pigment stones, potentially yielding measurement errors and distinct pathogenicity. Fifth, the incidence of GSD was higher  in female, however, there are no data regarding the number of pregnancies and the frequency of pill in this study. Finally, we further explored the 964 out of 2260 subjects who were lost to follow-up using telephone interview: 29 cases deceased, 106 cases moved out of the study township, 281 cases were refusals, 12 cases had presence of GSD or history of cholecystectomy. This might imply that the sample size was insufficient to estimate how potential risk factors affect the incidence of GSD. Further long-term research should be conducted to explore the morbidity of GSD and plausible biological mechanisms underlying its development.Increased age is a well-established risk factor for the development of GSD. The current findings indicate that high BMI, elevated FPG and NAFLD levels are also associated with GSD.The authors declare that they have no competing interests.T-HT and C-TH executed the study and drafted the manuscript. J-YC and T-HT participated in the study design and performed the statistical analyses. T-HT and J-HL envisioned the study and coordinated drafting the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/14/83/prepub


Ebolavirus and Marburgvirus, pose significant threats to public health and species conservation by causing hemorrhagic fever outbreaks with high mortality rates. Since the first outbreak in 1967, their origins, natural history, and ecology remained elusive until recent studies linked them through molecular, serological, and virological studies to bats. We review the ecology, epidemiology, and natural history of these systems, drawing on examples from other bat-borne zoonoses, and highlight key areas for future research. We compare and contrast results from ecological and virological studies of bats and filoviruses with those of other systems. We also highlight how advanced methods, such as more recent serological assays, can be interlinked with flexible statistical methods and experimental studies to inform the field studies necessary to understand filovirus persistence in wildlife populations and cross-species transmission leading to outbreaks. We highlight the need for a more unified, global surveillance strategy for filoviruses in wildlife, and advocate for more integrated, multi-disciplinary approaches to understand dynamics in bat populations to ultimately mitigate or prevent potentially devastating disease outbreaks.Filoviruses, including  Marburgvirus first emerged in humans, their importance as lethal pathogens causing hemorrhagic fever has been appreciated, but their origins, natural history, and ecology remained elusive for decades. In 2005, the first direct evidence from field studies that bats were reservoir hosts for Ebolavirus was reported . . Reston Miniopterus schreibersii, Family Vespertilionidae) in the Iberian Peninsula  prompted a wildlife disease investigation. Tissue microscopy from bats collected from a cave in Northern Spain (Cueva del Lloviu) suggested that the bats died from viral pneumonia, and subsequent pathogen screening found that individuals were infected with a novel filovirus, named Lloviu virus [Ebolavirus outside of Africa or Asia, and although causation was never proved, it has been speculated that mortality was from Lloviu virus infection. In 2002, widespread die-offs of Schreiber’s Bent-winged bats  and that virus could be isolated from feces [R. aegyptiacus bats of known serological and infection status Paweska et al. demonstrated that viremia could be induced and Marburg virus detected in multiple tissues 2 to 9 days post infection [R. aegyptiacus could not induce infection following oral or intra-nasal inoculation , nor could virus be isolated from secretions. Similarly, the study by Swanepoel et al. inoculated Tadarida spp. bats by sub-cutaneous injection; however, fecal shedding was observed in one individual. Thus, while these results are consistent with R. aegyptiacus being a reservoir host, they do not shed light on the potential mechanisms for bat-to-bat transmission [Rousettus colony housed at CDC Atlanta will likely shed more light on some of these unresolved issues [et al. was passaged almost 40 times in primate Vero cells prior to infecting bats [Marburgvirus strains from genome sequences obtained directly from bats, even in the absence of a viral isolate, and may be a useful tool more relevant than using human or vero-adapted viruses to understand viral dynamics in bats [Experimental studies supporting the role of bats as reservoirs are few, but two key studies have investigated the capacity for bats to become infected with filoviruses and to survive infection. As mentioned above, Swanepoel om feces . Using cnfection . Followismission . Additiod issues . Lastly,ing bats , and it  in bats .Rousettus aegyptiacus [Lloviu virus GP allows viral entry into bat cells more easily than other filoviruses, and thus may be an exceptionally bat-adapted virus [in vitro also using vesicular stomatitis virus expressing GP surface protein, found that Marburgvirus was able to infect 6 different bat cell lines from 4 divergent bat genera  [The recent establishment of bat cell lines , includiyptiacus , has beeed virus . This fied virus . Additioadarida) .Overall, filovirus ecology remains a neglected area of research, which is understandable as potential reservoirs are still being discovered and for many years remained elusive. Understanding zoonotic disease emergence and cross-species pathogen transmission require multi-disciplinary, process-based approaches that integrate ecological and evolutionary dynamics ,94. Seveet al., who looked at breeding cycles and their relationship to Marburgvirus prevalence [et al. were the first to test the hypothesis that birthing might be linked to increases in infection prevalence and ultimately spillover for Marburgvirus in bats. Prevalence of other bat derived viruses, including coronaviruses and rabies, are reported to show seasonal dynamics [R. aegyptiacus bats in Python Cave, Uganda discovered 2.5% of the bats were actively infected by PCR (and some yielding Marburgvirus isolates) [Marburgvirus infection occurred in distinct pulses in older juvenile bats (approximately 6 months old), coinciding with twice yearly birthing seasons. The authors also reviewed previous human infections and found that most  occurred during this same high prevalence/seasonal birth period. Relatedly, Pourrut et al. 2009 found that pregnant females bats were statistically more likely to be seropositive for Ebola virus [The most prominent study to test hypotheses regarding bat-filovirus ecology using field approaches and longitudinal sampling is by Amman evalence . Given tevalence ,103,104)dynamics ,106,107 dynamics . Their ssolates) . Their ala virus . As manyla virus ,111,112 la virus . Thus, sla virus ,95. et al. studied a maternal colony of Myotis myotis bats for three years and showed that RNA viruses (coronaviruses and astroviruses), but not DNA viruses (adenoviruses) were increasingly detected in greater numbers (by quantitative PCR) during colony formation and after parturition [et al. showed that Nipah Virus (NiV) in Pteropus lylei bats has seasonal dynamics, but with different dynamics for different strains, with a Bangladesh NiV strain more frequently observed April to June and a Malaysian NiV strain found from December to June [et al. who modeled the transmission dynamics of Hendra virus (HeV) in Australian Pteropid bats and found that their models fit the available data better when population connectivity and immunity  interact, suggesting more complex dynamics than a simple increase in susceptible juveniles providing enough young for persistence [Evidence from other bat-infection systems suggests that RNA virus shedding may be linked to host ecology and seasonality. Drexler turition . Wachara to June . These msistence . R. aegyptiacus) and other factors drive infection dynamics. Further still, it has recently been demonstrated that host population structure may be a useful tool to predict infection presence [Eidolon helvum, has been shown to have a high seroprevalence of antibodies against several RNA viruses, but not filoviruses compared to other species in the same locations [R. aegyptiacus , it poses the question as to whether the ecological differences prevent filovirus circulation  or if there are underlying genetic host restrictions. There remain, however, uncertainties about how strong the effects of seasonal birthing are for other filoviruses, and how much coloniality  with virus isolated from feces 21 days after infection [et al. showed that anti-Ebolavirus IgG-positive animals were not Ebolavirus PCR-positive, and vice versa, suggesting again that infection occurs and is followed by seroconversion [et al. showed that R. aegyptiacus bats were discovered to have Marburg virus PCR-positive lung, kidney, colon and reproductive tissues, which may suggest transmission by oral, urine, fecal, or sexual means [et al., in which a seropositive bat was known to be alive 13 months after release with a radio collar [There is little understood about filovirus shedding and persistence in bats, though several key studies ,45,85 sual means . The fino collar .versus incidental hosts and the relative contribution of each species to interspecific host dynamics is currently unknown. Multiple circulating pathogens can also change within-host and within-population dynamics and could confer cross-species immunity [et al. demonstrated numerous bats infected (detected by PCR) with similar Zaire ebolavirus species PCR fragments some years apart, but within the species, these short genomic fragments differed between species and collection time [Multi-species interactions are critical to understand in order to accurately model viral dynamics in bat populations. To date, there is evidence for filovirus infection in a total of 17 bat species for , but no currently known bat hosts for Bundibugyo, Sudan, or Tai Forest ebolavirus . Virus himmunity . For exaimmunity ,28. Thisimmunity . Leroy eion time .et al. provided evidence of direct movement between different caves for R. aegyptiacus and have found that there is genetic similarity between viruses detected in geographically distant locations [R. aegyptiacus exist as a large meta-population with virus circulation over broad geographic ranges. Population genetic studies using mitochondrial and microsatellite markers have confirmed that a congeneric species, Rousettus leschenaultia, is highly vagile and panmictic across large areas  [Another key aspect of ecological theory that must be investigated further is the role that meta-population dynamics may play in the ecology and evolution of filoviruses. Amman ocations . They sut China) . Furtheret al. showed that Pteropus vampyrus, the primary natural reservoir for NiV in mainland Southeast Asia, was highly vagile and panmictic using both host and parasite genetics, and was likely the primary player in NiV transmission and circulation [et al. suggested meta-population dynamics were necessary for HeV persistence in Australian Pteropid bats and they predicted reduced connectivity leads to larger epidemics within bat colonies due to a greater loss of herd immunity in colonies with lower levels of connectivity [et al. have used host panmixia to predict infection dynamics across sub-Saharan Africa and shown similar antibody prevalences against two viruses, Lagos bat virus (a lyssavirus) and an as yet undetermined henipa-like paramyxovirus [Several previous studies have investigated the relationship between host population structure and bat viral dynamics. Olival culation ,118,119.ectivity . More reyxovirus . This spyxovirus  and travyxovirus , suggestyxovirus . These mThere are over 1200 bat species globally and only a small fraction (~15%) have been targeted for viral discovery to date . That saWhile it is important to survey wildlife showing clinical signs of disease, most viruses are discovered in bats from asymptomatic animals, and a two-pronged approach of screening both healthy and diseased animals is required . Modelinand better detection methods for viral discovery [et al. demonstrated that Zaire ebolavirus could be detected in bat feces, but few studies to date have routinely screened bat excreta by PCR in the field. There are also limited data comparing viral detection from organ specimens with data from excreta collected from the same animals. Developing more sensitive assays to detect antibodies or virus from small quantities of blood [if transmission is occurring indirectly in bat habitat (caves or mines). However, if the risk interface is through bushmeat hunting and direct butchering of bats [As part of a more unified filovirus surveillance strategy in bats, it will also be necessary to develop non-invasive sampling protocols iscovery ,125. Folof blood  or bat eof blood , respect of bats , then unet al. demonstrated how accurate quantification of antibody responses using Luminex technology was able to demonstrate the potential effects of pregnancy on henipavirus transmission in a captive study of Eidolon helvum that would not have been possible with assays that used dilution series or provide binary responses [et al. have shown how similar data can be analyzed in the absence of validated gold standard assays from the appropriate species and population (and applied these methods to bat sera) [There is also a need for better studies of immunological responses in bats . Understesponses . These aat sera) . These aat sera) ,77,131 , as welin silica studies published in the past few years have used genomic data and identified filovirus genes  integrated into the genomes of several mammalian species, including bats [et al. found a strong association of endogenous filoviruses elements in both rodents and marsupials—pointing to these groups as potentially important reservoirs, although currently not known to harbor exogenous filoviruses [in silica analyses may be of use to help target which of the ~5000 mammals species to focus efforts for exogenous filovirus discovery and can be part of a more unified strategy for global filovirus surveillance. Several ing bats ,133,134.While we have a decent understanding of the progression of infection and immunity in individual humans , little While experimental studies with BSL-4 agents such as filoviruses can be challenging, captive studies can be used to understand infection and antibody dynamics in the absence of experimental challenge. Two studies of henipavirus infected or seropositive fruit bats have been undertaken and show the temporal dynamics of antibodies. Though both studies raise many additional questions, they allow researchers to better understand the results of field studies ,138. ExpReston ebolavirus [Zaire ebolavirus can be transmitted from pigs to cynomolgus macaques without direct contact [Experimental studies of other bat derived viruses and their non-bat hosts have been used to try to tease apart spillover transmission mechanisms. Horses can be infected with HeV through intranasal infection, suggesting inhalation may be a potential route of infection , but epiolavirus , pigs haolavirus ,144, how contact . The mecvs. those that did not. While there is evidence to support specific instances of viral spillover, the epidemiological links between bats, Ebolaviruses, and human and primate infection are not clear. Recent epidemiological surveys following an outbreak reported increased bat activity through bat migration and hunting prior to an outbreak of Ebola virus in DRC . One recEbolavirus-contaminated fruits that terrestrial mammals eat and become infected [Several authors have speculated that, like that suspected proximate cause for the NiV outbreak in Malaysia , bats mainfected . In Banginfected  and studinfected . The rolinfected , but it Models using the SIR structure have been used for human epidemic dynamics ,151,152 Collectively, these ecological studies will be critical to inform disease management options. For example, management options that reduce human–bat contact during seasonal periods of high risk viral shedding, or at key interfaces, will likely be the most effective approaches and can balance both conservation and human health needs ,95. The


N-Formyl peptides are common molecular signatures of bacteria and mitochondria and are known to play a role in the initiation of inflammation by activating the formyl peptide receptor (FPR). We have demonstrated that infusion of mitochondrial N-formyl peptides (F-MIT) leads to sepsis-like symptoms, including vascular leakage. We have also observed that F-MIT, via FPR activation, elicits changes in cytoskeleton-regulating proteins in endothelial cells. Therefore, we hypothesize that these FPR-mediated changes in cytoskeleton can cause endothelial cell contraction and, consequently vascular leakage. Here, we propose that endothelial FPR is a key contributor to impaired barrier function in SIRS and sepsis patients following trauma.The major pathophysiological characteristic of systemic inflammatory response syndrome (SIRS) and sepsis is the loss of control of vascular tone and endothelial barrier dysfunction. These changes are attributed to pro-inflammatory mediators. It has been proposed that in patients and rats without infection, cell components from damaged tissue are the primary instigators of vascular damage. Mitochondria share several characteristics with bacteria, and when fragments of mitochondria are released into the circulation after injury, they are recognized by the innate immune system.  Systemic inflammatory response syndrome (SIRS) and sepsis are the principal causes of death in intensive care units, with mortality rates between 30 and 70% –3 The divia adherens junctions. Vascular endothelial cadherin-regulated protein complexes that join adjacent endothelial cells and prevent leukocyte emigration and vascular leakage  kinases are a family of stress activated enzymes that initiate signaling cascades in response to several stimuli, including inflammation and injury. It has previously shown that p38 MAPK kinase leads to reorganization of the actin cytoskeleton to form stress fibers and increase in vascular permeability . FurtherDespite new information about the pathophysiology of molecular mechanisms regulating vascular permeability, this disruption continues to be treated inappropriately and this contributes to an unacceptably high mortality rate in patients with SIRS and sepsis. Therefore, understanding vascular function and the causes for this disturbance would ultimately provide starting points for therapies designed to treat these devastating diseases.N-formyl peptides are common molecular signatures of bacteria and mitochondria and are known to play a role in the initiation of inflammation by activating the formyl peptide receptor (FPR) , known to result in a prompt rise of the calcium, also induced a rapid decrease of G-actin content and increase of filamentous actin, F-actin, content  , 26, 27.1 and -2 . FPR act content . A reduc content . FPR act content . Recent  content .It is known though, that a major mechanism by which immune system causes organ damage is due to neutrophil-mediated increases in endothelial cell permeability. Nevertheless, in a recent study from Sun and collaborators  demonstrN-formyl peptides  and fMLP (bacteria derived) induce vascular leakage and exacerbated vasodilatation in resistance arteries, and that a FPR antagonist inhibits these responses . To answer this question, we treated HAEC with F-MIT  in the presence or absence of a cocktail of FPR antagonists . Treatment with F-MIT rapidly increased RhoA/ROCK , CDC42 , and phospho-MLC Thr/Ser19  in HAEC. These changes were abolished in the presence of FPR antagonist. These data suggest that F-MIT leads to changes in endothelial cell cytoskeleton independent of leukocyte activation. Given that we provided strong evidence FPR induced RhoA and CDC42 expression, and these proteins are linked with endothelial contraction via actin polymerization, we infer that F-MIT elicits endothelial contraction and subsequent vascular leakage not only via actomyosin interaction but also via actin polymerization.As described above, FPR activation leads to cytoskeleton rearrangement in immune and non-immune cells. Therefore, because FPR is present in endothelial cells and because we observed that F-MIT induces vascular leakage due to FPR activation , we quesOur new mechanistic hypothesis is that F-MIT released from trauma/cell damage activate FPR leading to changes in endothelial cell cytoskeleton which subsequently induces endothelial contraction and vascular permeability Figure .The inability to treat or prevent SIRS and sepsis in trauma patients may be due to our limited understanding of the molecular pathways that govern the disruption of normal vascular control and responsiveness. The proposed hypothesis is significant because it may offer a novel cellular and molecular mechanism underlying the development of arterial dysfunction in SIRS and extend our knowledge about how mitochondria-derived molecules mediate endothelial barrier dysfunction.via FPR activation, elicits changes in cytoskeleton-regulating proteins in endothelial cells. Therefore, we infer that this interaction can lead to endothelial contraction, increased vascular leakage, and attenuated barrier function as observed in SIRS patients following trauma. Corroborating this hypothesis, we previously observed that F-MIT induced vascular leakage in conduit and resistance arteries from Wistar rats (In the present study, we have observed that F-MIT, tar rats . Based oCW analyzed the data in HAEC and wrote, discussed, and reviewed the manuscript. CM performed the experiments in HAEC, discussed, and reviewed the manuscript. RW reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chenuda virus, Chobar Gorge virus, Wad Medani virus and Great Island virus . Nucleotide (nt) and amino acid (aa) sequence comparisons provide a basis for orbivirus detection and classification, however full genome sequence data were only available for the Great Island virus species. We report representative genome-sequences for the three other TBO species (virus isolates: Chenuda virus (CNUV); Chobar Gorge virus (CGV) and Wad Medani virus (WMV)). Phylogenetic comparisons show that TBOs cluster separately from insect-borne orbiviruses (IBOs). CNUV, CGV, WMV and GIV share low level aa/nt identities with other orbiviruses, in ‘conserved’ Pol, T2 and T13 proteins/genes, identifying them as four distinct virus-species. The TBO genome segment encoding cell attachment, outer capsid protein 1 (OC1), is approximately half the size of the equivalent segment from insect-borne orbiviruses, helping to explain why tick-borne orbiviruses have a ~1 kb smaller genome.The International Committee for Taxonomy of Viruses (ICTV) recognizes four species of tick-borne orbiviruses (TBOs):  Orbivirus within the family Reoviridae. The genus currently includes 22 species (representing 22 distinct virus serogroups) that have been recognized by the International Committee for the Taxonomy of Viruses (ICTV) [Orbivirus species, with ‘unclassified’ isolates from the genus, have led to proposals to ICTV for recognition of seven additional species [The orbiviruses are icosahedral, non-enveloped dsRNA viruses belonging to the genus s (ICTV) . Recent  species ,5,6,7,8.Culicoides, phlebotomines (sandflies), mosquitoes and ticks. The tick-borne orbivirus (TBOs) species include Chenuda virus, Chobar Gorge virus, Wad Medani virus and Great Island virus. It has been suggested that ‘Kemerovo virus’ (currently a sub-group within Great Island virus) could also be recognized as a separate species [St Croix River virus.The orbiviruses have a wide host range that collectively includes domestic and wild ruminants, equines, marsupials, sloths, bats, birds and humans ,10,11,12 species ,14. The Chenuda virus includes seven serotypes/strains: Chenuda virus (CNUV), Baku virus (BAKUV), Essaouira virus (ESSV), Huacho virus (HUAV), Kala Iris virus (KIRV), Mono Lake virus (MLV) and Sixgun city virus (SCV). CNUV was isolated in 1954 from ticks in Egypt, with serological evidence of infection in birds, camels, pigs, buffalo, dogs, donkeys and rodents [Chobar Gorge virus includes two serotypes/strains: Chobar Gorge virus (CGV) isolated in 1970 from Ornithodoros spp. ticks in Nepal; and Fomede virus (FV) isolated in 1978 from a bat in Kindia, Guinea. There is serological evidence for infection of cattle, horses, sheep, buffalo and humans [Wad Medani virus includes two serotypes: Wad Medani virus (WMV) isolated in 1952 from ticks collected at Wad Medani in Sudan; and Seletar virus (SELV) isolated in 1961 from ticks collected in the Seletar district, Singapore. There is serological evidence for infection of cattle, camel, pigs, buffalo and rodents [The species  rodents . The spe rodents .Orbivirus species, using either conventional or real-time RT-PCR [The orbivirus genome consists of ten linear segments of dsRNA (Seg-1 to Seg-10 in order of decreasing molecular weight), which are packaged within a triple layered icosahedral protein capsid ,18,19,20e RT-PCR ,24,25. Se RT-PCR ,28,29,30Culicoides-borne orbiviruses (CBOs), for one phlebotomine-borne orbivirus (PBO) and four of the six mosquito-borne orbiviruses (MBOs). Although full genome sequence data are also available for SCRV (a tick orbivirus (TO)), the genomes from isolates from only one species of TBOs have previously been fully sequenced (for isolates Great island virus (GIV), Kemerovo virus (KEMV) and Tribec virus (TRBV)). We report full genome sequences for representative isolates of the species Chenuda virus, Chobar Gorge virus and Wad Medani virus, providing a basis for further comparisons to other orbiviruses and the identification of novel TBO isolates and species (see Full-genome sequence data are now available for representative/reference strains of the ten established species of the cies see .Culicoides-borne orbiviruses have a much larger outer capsid protein 1 (OC1) encoding gene, resulting in a 3-3-4 migration pattern.Isolates of CNUV EGY1954/01), CGV (NEP1970/01) and WMV (SUD1952/01) were obtained from the Orbivirus Reference Collection (ORC) at The Pirbright Institute . These v954/01, CFull length nucleotide sequences for Seg-1 to Seg-10 of Chenuda virus (CNUV), Chobar Gorge (CGV) and Wad Medani virus (WMV)  have been determined and submitted to GenBank, with accession numbers KP268794 to KP268803; KP268784 to KP268793; and KP268804 to KP26813, respectively. The properties of the tick-borne orbiviruses genome segments and their encoded proteins are given in Culicoides-borne orbiviruses genome segments is between 39% in Warrego virus (WARV) to 45.9% in equine encephalosis virus (EEV). Changuinola virus (CGLV), which is a phlebotomine-borne orbivirus, has 41.7% GC, while the mosquito-borne orbiviruses have a more diverse GC content between 36.7% in Peruvian horse sickness virus (PHSV) to 45.1% in Corriparta virus (CORV). In contrast, the genomes of all of tick-borne orbiviruses that have been sequenced have a markedly higher GC content than the insect-borne orbiviruses, between 53% (CGV) and 58.1% (GIV) (The average GC content of the 1% (GIV) . St CroiOrbivirus species. Collectively, the terminal non-coding regions (NCR) represent 3.67%, 3.63%, 4.11%, 5% and 4.32% of the CNUV, CGV, WMV, GIV and KEMV genomes, respectively. Like most genome segments from other orbiviruses, RNAs of CNUV, CGV, WMV, GIV and KEMV all have shorter 5' than 3' NCRs  to 51.3% (between BTV-8w and EHDV-1w). NS4 of CGV, CNUV, WMV, GIV and KEMV is 238aa, 183aa, 189aa, 190aa and 151aa long, respectively. NS4 of CGV is approximately 20% larger than in the other TBOs and is larger even than the CGV NS3 protein. The insect-borne orbiviruses usually have a smaller NS4 (76 aa in EHDV to 152 aa in CORV), although this does not significantly affect the overall size of Seg-9, which also codes for the viral helicase, VP6.As previously reported for BTV and Great Island virus (GIV) ,19, Seg-Reoviridae, at both the species and genus level [Orbivirus species. In contrast, GIV and KEMV group more closely together, consistent with their current classification as different subgroups within the same Orbivirus species. As previously suggested [The orbivirus RNA dependent RNA polymerase (Pol) (encoded by Seg-1), is highly conserved and has previously been used in phylogenetic studies to classify viruses from the family us level ,22,26,33uggested , SCRV  of CNUV, CGV and WMV as the inner sub-core shell ‘T2’ protein. A phylogenetic tree constructed for orbivirus T2 proteins, separated the different isolates into groups that correlate with their different vectors, in a manner similar to the VP1 tree c. Three Orbivirus species  constructed for the aa sequences of VP7(T13) , is a strongly immuno-dominant serogroup-specific antigen  and is hVP7(T13)  exhibiteiviruses .Orbivirus serotype and is highly variable in both its aa sequence and size. OC1 is encoded by Seg-2 (VP2) in the PBO and CBOs (represented by BTV), by Seg-3 (VP3) in the MBOs (represented by PHSV) and by Seg-4 (VP4) in the TBOs [Outer capsid protein one (OC1) determines the TBOs ,11,13. OOrbivirus species) than any of the other viral proteins, thought to reflect immune-selective-pressure from neutralizing antibodies (targeting OC1) that are generated by the vertebrate host [The aa sequence of the OC1 protein is more variable  b, VP3 C all showIn general, the phylogenetic trees for all of the orbivirus proteins indicate that members of each virus species group closely together, while members of distinct species are branched separately, regardless of the protein selected.Orbivirus species, likely reflecting important functional and/or structural interactions and constraints on each of the RNAs and proteins. These functional interactions may restrict genome segment exchange/reassortment, to viruses within the same Orbivirus species, and suggests that any novel orbivirus isolate would be identifiable (at the virus-species level) based on a phylogenetic analysis of any of its proteins/genes.This reflects a relatively high level of conservation between homologous segments and proteins within each Orbivirus species. These similarities can result in low level or ‘one-way’ cross-reactions in serological assays ; Epizootic hemorrhagic disease viruses (EHDV); and Eubenangee viruses (EUBV)), making virus detection and conclusive identification more difficult, particularly if the viruses can co-circulate and can cause mixed infections [Orbivirus species/serogroups.Different orbivirus serogroups/species were originally identified and distinguished by a combination of their biological origins (host and vector), clinical signs and group-specific serological assays, including complement fixation (CF) and agar-gel immuno-precipitation (AGIP) tests and more recently serogroup-specific ELISA. However, significant similarities exist in the host ranges, clinical signs, arthropod vectors, distribution and serological properties between members of some different fections ,38,39,40Electrophoretic analysis of orbivirus genome segments on 1% agarose gels (AGE) usually shows highly conserved size distributions and consequently migration patterns (electropherotype) within individual serogroups/species . HoweverDifferences in the size of equivalent genome segments between the CBOs, MBOs and TBOs, have been previously reported by several authors ,26,32,43Orbivirus species [With the advent of more rapid and reliable sequencing methods, full genome sequence data have been generated for reference strains of many  species ,41,44,45 species ,27,33,43The intra-species genealogical and phylogenetic relationships of the CBOs  have been extensively studied, based on all ten genome segments/proteins ,48,49,50Orbivirus species and genus-specific RT-PCR assays for virus typing, diagnosis and virus discovery [The orbivirus polymerase ‘Pol’, sub-core-shell ‘T2’ and outer-core ‘T13’ proteins are all highly conserved. They have intra-species identity levels of >73%, >83%, and >73% aa identity (in BTV and EHDV) and maximum inter-species aa identity levels of 73%, 80% and 66%, respectively, between closely related virus species (such as BTV and EHDV) ,11,46,49iscovery ,52,53,54Orbivirus. However GIV and KEMV share 72.8%, 82.8% and 82.1% aa identity in Pol, T2 and T13 proteins, respectively, very close to, or just beyond the previous maximum levels of variation detected within the CBO species (BTV and EHDV). It was therefore proposed that these two viruses could be recognized as two distinct species [Reoviridae is the ability of the different viruses within the same species to exchange/reassort genome segments during co-infection of the same cell, leading to the production of viable progeny reassortant virus strains [Orbivirus species. Further sequence analyses of other virus isolates from the Great Island virus species may identify ‘intermediates’ between the different strains already analyzed, potentially filling in gaps, and confirming their inclusion within a single virus species.The tick-borne orbiviruses analyzed here, CNUV, CGV and WMV, share less than 65% aa identity in all three conserved proteins Pol, T2 and T13 confirming their classification as distinct species within the genus  species . However strains  and theyco-speciation’ with their arthropod vectors and that the TBOs provide an ancestral ‘root’ for the insect transmitted orbiviruses [Phylogenetic comparisons of most orbivirus proteins (VP1(Pol), T2, T13, CaP, OC1, OC2, NS1 and NS2) show three ‘clusters’ that correspond to the arthropod vectors that transmit each virus . These diviruses ,13,45. PSome of the differences/heterogeneity in the genome segments and their order of migration, of the CBOs/PBO, MBOs and TBOs are caused by large variations in the relative size of the highly variable outer capsid protein OC1. This heterogeneity is due to acquired point mutations, insertions and deletions, as well as inter- and intra-genic recombination and gene duplications (concatemerization) over a long time periods ,45.Culicoides-borne orbiviruses, OC1 is the second largest viral protein (VP2—Encoded by Seg-2: 110–120 kDa), while in the mosquito-borne orbiviruses it is slightly smaller  (VP3—encoded by Seg-3: 90–100 kDa) and is smallest in tick-borne orbiviruses  (VP4 encoded by Seg-4: 62–66 kDa) [In –66 kDa) . There a–66 kDa) . It is cAssuming that the tick associated orbiviruses (TBOs and TO) are ancestors of all other orbiviruses, duplication events may have led to the evolution of larger viral genes and proteins in the other groups ,45. ConcOrbivirus species. The full genome sequence reported here for reference strains of Chenuda virus, Chobar Gorge virus and Wad Medani virus, together with the earlier data for GIV and KEMV completes a genome data set for reference strains of the tick-borne Orbivirus species. This will not only help to identify novel tick-borne orbiviruses, but will also provide a useful tool for identification and study of other orbiviruses.Sequencing and phylogenetic analyses of virus genomes, provides a basis for classification, diagnosis and vaccine development and helps to identify recombinant/reassorted strains. This suggests that full genome sequencing will become an accepted standard for future molecular epidemiological studies. It will therefore be important to generate a full genome sequence database that includes representative members of all Orbivirus species recognized by ICTV. These data have provided a basis for proposals to ICTV to recognize seven novel Orbivirus species, the development, and testing (in silico) of relevant diagnostic assays, and provide support for molecular epidemiology/evolutionary studies to enhance our understanding of orbivirus diseases in vertebrates.Full genome sequences are now available for reference strains of twenty of the twenty-two The viruses used in this study EGY1954/01 (CNUV), NEP1970/01 (CGV) and SUD1952/01 (WMV) were obtained from the Orbivirus Reference Collection (ORC) at The Pirbright Institute. These viruses were originally taken from naturally infected animals by qualified veterinarians, as part of normal diagnostic testing procedures in the respective countries. CNUV and CGV were propagated in BHK-21 cells , while WMV was grown in BSR cells (a clone of BHK)  or BHK cet al. [® reagent (Invitrogen), mixed with 0.2 volume of chloroform vortexing and the mixture was incubated on ice for 10 min. Total RNA present in supernatant was separated from cellular debris and DNA by centrifuging at 10,000× g for 10 min at 4 °C. Removed single stranded RNA (ssRNA) by 2M LiCl precipitation at 4 °C overnight, followed by centrifugation at 10,000× g for 5 min. Equal volume of isopropanol and 750 mM ammonium acetate was added to supernatant and then viral dsRNA was allowed to precipitate for a minimum of 2 h at −20°C. The dsRNA was pelleted by centrifugation at 10,000× g for 10 min, washed with 70% ethanol, air dried and dissolved in nuclease free water (NFW). The RNA was either used immediately or stored at −20°C.Guanidinium isothiocyanate extraction procedure described by Attoui et al.  was usedet al. [The genome segments of CNUV, CGV and WMV were reverse-transcribed using a ‘full-length amplification of cDNA’ (FLAC) technique described by Maan et al. . Briefly®-Blunt’ vector supplied with the Zero Blunt® PCR Cloning Kit (Invitrogen). Recombinant plasmid vectors containing inserts were transformed into One Shot® TOP10 competent cells, supplied with the cloning kit. Clones containing relevant inserts were identified by colony PCR using M13 universal primers. Plasmids were extracted from the clones identified using the QIAprep Spin MiniPrep Kit (Qiagen). The plasmids and PCR products were sequenced using an automated ABI 3730 DNA sequencer (Applied Biosystems).Purified amplicons of CNUV, CGV and WMV were cloned into the ‘pCR‘Raw’ ABI sequence data were assembled into ‘contigs’ using the SeqManII sequence analysis package (DNAstar version 5.0). The ORFs of CNUV, CGV and WMV were identified and translated into aa sequences for further analysis using EditSeq (DNAstar version 5.0). The putative function of each protein was identified by BlastX comparisons to homologous orbivirus (BTV) proteins in GenBank . Multipl