Togaviridae. CHIKV is a reemerging virus for which there is no safe prophylactic vaccine. A live attenuated strain of CHIKV, CHIK181/25, was previously demonstrated to be highly immunogenic in humans, however, it showed residual virulence causing transient arthralgia.Chikungunya virus (CHIKV) is an arthropod borne alphavirus of the family In this study, we demonstrate the complete inactivation of CHIKV181/25 by 1,5 iodonapthyl azide (INA). No cytopathic effect and virus replication was observed in cells infected with the INA-inactivated CHIKV. However, a reduction in the INA-inactivated CHIK virus-antibody binding capacity was observed by western blot analysis.INA completely inactivated CHIKV and can further be explored for developing an inactivated-CHIKV vaccine. Togaviridae. It is a reemerging virus that has caused wide spread outbreak in the nations surrounding the Indian Ocean and Africa since 2004[Aedes albopictus, which is endemic to the previously CHIKV naïve regions[Earlier we have shown that 1,5 iodonapthyl azide (INA) and UV irradiation completely inactivated the Venezuelan equine encephalitis virus (VEEV). In thisnce 2004. Among t regions. Therefo regions. In the  regions. Several regions-13.INA is a photoactive hydrophobic azide molecule that sequesters in the hydrophobic domain of the biomembranes and binds to the membrane proteins upon irradiation with long wavelength ultra-violet (UV) light. It was initially used for labeling the biological membranes. InteracCHIKV181/25 was kindly provided by Drs. Parker MD and Glass PJ of the United States Army Medical Research Institute (USAMRIID), Frederick, MD. INA was obtained from Dr. Blumenthal RM, NCI, Frederick under a material transfer agreement. 0.5 mg of sucrose gradient purified CHIKV was diluted with 1X DPBS and vortexed to mix and disintegrate the virus clusters. 20 mM INA was then added to make a final concentration of INA to 50, 100 and 200 μM. The final volume of the reaction was set at 1.0 ml. Virus + INA suspension was then irradiated with UV radiation for a total of 5 min as described before[Our findings are important as CHIKV causes an ongoing outbreak affecting millions of people around the globe. An effective CHIKV vaccine is urgently needed to control the spread of CHIKV. Live attenuated CHIKV181/25 was shown to have an excellent immunogenicity; however, the clinical trial was halted due to issues with residual virulence causing transient arthralgia in the vaccine recipients. The INACHIKV: Chikungunya virus; INA: 1, 5 iodonapthyl azide; BHK cells: Baby hamster kidney cells; CPE: Cytopathic effect; DAPI: 4’,6-diamidino-2-phenylindole; PBS: Phosphate buffered saline; BSA: Bovine serum albumin; RT-PCR: Reverse Transcription-Polymerase chain reaction.The authors declare that they have no competing interests.AS and RKM were involved in the conceptualization and designing of the study. AS carried out the inactivation experiment and analyzed the experimental data. PG carried out the western blot experiments. AS, RKM and PG participated in writing the manuscript. All authors read and approved the final manuscript.


The significant economic burden associated with acute coronary syndromes (ACS) provides a need to evaluate both medical costs and productivity costs, according to evolving guideline-driven ACS treatment strategies, medical management (MM), percutaneous coronary intervention (PCI), or coronary artery bypass graft (CABG).Commercially insured individuals, aged 18-64, with an emergency room (ER) visit or hospitalization accompanied by an ACS diagnosis (index event) were identified from a large claims database between 01/2004 and 12/2005 with a 1-year follow-up period. Patients who had an ACS diagnosis in the 12 months prior to their index event were excluded. Patients were divided into 3 groups according to treatment strategies during the index event: MM, PCI, or CABG. A subset of patients was identified for the productivity cost analysis exploring short-term disability and absenteeism costs. Multivariate generalized linear models were performed to examine the ACS costs by 3 different treatment strategies.A total of 10,487 patients were identified for the medical cost analysis. The total 1-year medical costs (index event costs plus the 1-year follow-up costs) were lowest for MM patients , followed by PCI patients  and CABG patients . Of the 3,080 patients in the productivity costs analysis, 2,454 patients were identified in the short-term disability cohort and 626 patients were identified in the absenteeism cohort. Both the estimated mean total 1-year short-term disability and absenteeism costs were highest for CABG patients  compared to MM patients  and PCI patients .Both total 1-year medical costs and 1-year productivity costs are substantial for working-aged individuals with ACS. These costs differ according to the type of treatment strategy, with CABG having higher costs compared to either PCI or MM. Acute coronary syndromes (ACS), including unstable angina (UA), non-ST-segment elevation myocardial infarction (NSTEMI), and ST-segment elevation myocardial infarction (STEMI), are common and costly. In 2006 alone, it was estimated that 733,000 hospital discharges were due to ACS, increasing to 1,365,000 when adding secondary discharges [The substantial costs associated with ACS include treatment related medical costs, as well as costs due to loss of productivity . PreviouAnalyzing ACS costs by different treatment strategies should be of special interest to healthcare professionals as different treatment strategies are important drivers of ACS costs and may have different implications on healthcare resource utilization and productivity loss of patients. Depending on the different manifestations of ACS, UA/NSTEMI versus STEMI, the current 2007 American College of Cardiology/American Heart Association (ACC/AHA) guidelines allow conservative noninterventional medical management (MM) and/or invasive interventional management by percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) . There aOver the years, the ACC/AHA guidelines have been updated to reflect new data and clinical trial results. Previous studies investigating the cost of illness for ACS, however, have focused on data from the late 1990s or early 2000s, prior to the results of the Clopidogrel in Unstable angina to prevent Recurrent Events (CURE) trial for clopidogrel [® Research Databases. For the medical cost analyses, the MarketScan Commercial Claims and Encounters (CCAE) database was used. The CCAE database captures individual-level clinical utilization, costs, and enrollment across inpatient, outpatient and prescription drug categories from a selection of large United States (US) employers with contributing health plans. The MarketScan Health and Productivity Management (HPM) database was used for the productivity cost analyses. The HPM database is a subset of the CCAE database and contains information on short-term disability, absence, and workers' compensation experience that is linkable to the medical, pharmacy, and enrollment data in MarketScan databases.These retrospective claims database analyses were performed using two of the Thomson Reuters MarketScanMedical claims data were extracted from January 2003 to December 2006 for individuals, aged 18-64, that were seen in an emergency room (ER) or hospitalized between January 2004 and December 2005 with a diagnosis of ACS, based on ICD-9-CM codes 410.xx and 411.1. The first ACS-related ER visit or hospitalization was considered as the "index event." Patients who had an ACS diagnosis one year prior to the index event were excluded from the study. Patients were required to have a minimum 12 months of continuous insurance eligibility prior to and post the index event. Patients were categorized into three groups according to their ACS treatment strategy during the index event: MM, PCI, or CABG. For the subset productivity cost analyses, patients were required to have had a minimum of 12-months eligibility for incurring short-term disability and absence in the HPM database. Data for these individuals were linked to their employers' short-term disability and absenteeism records via unique encrypted personal identification numbers provided by the employers.a priori p-value of 0.05 was considered statistically significant. Descriptive analyses according to MM, PCI, and CABG were performed on patient demographic and clinical characteristics using chi-square tests for categorical variables and t tests for continuous variables. The demographic characteristics included age, gender, insurance plan type (see Appendix for description), and geographic region. The clinical characteristics included comorbidities, the Charlson comorbidity score [All statistical analyses were performed with Stata 9 software  and an ty score , primaryMedical costs were categorized into costs incurred during the index event and costs incurred during the 1-year follow-up period for MM, PCI, and CABG patients. For the 1-year follow-up costs, costs were further divided into inpatient, outpatient, and pharmacy costs. All costs were reported in 2005 US dollars. For the productivity cost analyses, short-term disability and absenteeism costs incurred during the 1-year follow-up period were estimated for the MM, PCI, and CABG groups. These productivity costs were further divided into two categories for each group: the costs incurred within the first 30 days of the index event and the costs incurred from day 31 through one year. The hourly value of $42.67 of productivity losses was estimated based on the average hourly wage and benefits rate reported in the 1999 Medstat Benchmark Survey of Hourly Compensation and inflated using the 2005 Bureau of Labor Statistics Employment Cost Index for Private Industry . DiffereA total of 10,487 ACS patients was identified and divided into three mutually exclusive groups according to treatment strategy during their index event: MM , PCI , and CABG (n = 864). Patient demographic and clinical characteristics for the total population are displayed in Table For the productivity cost analysis, 3,080 patients had available data for analysis with 2,454 patients in the short-term disability cohort  and 626 patients in the absenteeism cohort . In both cohorts, MM patients were more likely to be female, be younger, and have a higher mean Charlson comorbidity score compared to PCI and CABG patients.Table Table For the 1-year productivity costs, Table Tables This study contributes to the existing medical literature by utilizing more recent data than previously reported ACS cost studies -6 to exaThe total 1-year medical costs for patients treated conservatively by MM were $34,087, significantly lower compared to patients treated interventionally by PCI $52,673) or CABG . When controlling for patient characteristics, a similar trend was found: the total adjusted 1-year medical costs were $39,017 for MM patients, $58,313 for PCI patients, and $91,977 for CABG patients. All three treatment strategies had higher total 1-year medical costs compared to previous studies resulting from earlier data that reported estimates of $22,528 to $32,345 73 or CAB-18. BaseThese results show that PCI and CABG patients had higher total medical costs when compared to MM patients. For both interventional treatments strategies, the majority of the costs occurred during the index event: 59.6% for PCI patients and 73.5% for CABG patients, compared to 26.1% for MM patients. This finding is consistent with earlier studies showing the majority of medical costs resulting from the index event -6. DurinTo our knowledge, this is the first study examining productivity costs associated with ACS, and the results show the substantial impact on productivity costs. In this study, the productivity costs include short-term disability and absenteeism costs, which are true costs to employers-as employers have to either spend extra resources, such as overstaffing or hiring replacement, to compensate for work normally performed by absent employees or to experience the productivity loss with fewer goods and services provided, lower revenues, and lower profits. In both the short-term disability and absenteeism cohorts, CABG patients had the highest total productivity costs compared to both MM patients and PCI patients. For short-term disability costs, the CABG group had the highest costs within the first 30 days, but had the lowest costs after the first 30 days compared to the MM and PCI groups. In the absenteeism cohort, there was no difference in costs between the three treatment groups during the first 30 days. However, after the first 30 days, CABG patients had significantly higher costs. For all three treatment groups, the majority of the short-term disability costs occurred within the first 30 days of the index event, and the majority of the absenteeism costs occurred after 30 days from the index event.The treatment of ACS includes an inpatient diagnosis and treatment strategy during the index event, followed by long-term treatment of the underlying coronary heart disease condition. Therefore, it is important to understand the costs associated with different treatment strategies, not only during the index event, but also over time. This study was unique in dividing the costs according to the treatment strategy, MM, PCI, or CABG, employed during the initial ACS hospitalization. The results showed that for PCI and CABG patients, the majority of the costs were incurred during the index event. Costs across all three groups were similar during the 1-year follow-up period. A better understanding of the costs associated with different treatment strategies for ACS patients will help health care practitioners and policy makers in their decision-making process.Certain limitations must be considered when interpreting these results. Retrospective data analyses were performed; therefore, miscoding of medical claims and missing data, which could not be verified through medical chart reviews, are both possible. In addition, as patients in the study were required to have a minimum 12 months of continuous insurance eligibility after the index date, the results applied only to patients who survived and who did not switch or lose their jobs at least 1 year following their initial hospitalization for ACS. Using an alternative cohort definition, including those patients who lost follow-up during the 12 months after their index events, might have opposite effects on the cost estimation. First, incorporating those who died over the course of 1 year might increase the healthcare costs estimation due to higher end-of-life healthcare. On the other hand, incorporating patients, who experienced job changes or job loss during the one year period after their ACS index events, might lead to lower cost estimation due to loss of insurance eligibility. Mortality status is unknown in the database, unless a patient died in the hospital and the loss of follow-up reason is unclear due to death, job changes, or job loss, etc. As a result, it is unclear how this would impact the cost estimation; therefore, a clear defined cohort was chosen for the study to understand the 1-year medical and productivity costs of ACS to employers.The productivity cost analyses were limited to short-term disability and absenteeism, and did not include presenteeism  or long-term disability. The results from this study may not be generalizable to populations other than working-age populations with employer-sponsored private health insurance plans, such as retired patients or Medicaid beneficiaries.In conclusion, this study examined the total 1-year medical and productivity costs associated with short-term disability and absenteeism according to ACS index event treatment strategies, MM, PCI, or CABG. Both total 1-year medical and productivity costs were substantial for working-age individuals with ACS, and these costs differed according to the type of treatment strategy with CABG patients having the highest 1-year medical and productivity costs compared to PCI and MM patients. Treatment options that reduce resource utilization and improve productivity may potentially lower the costs of care for ACS.MarketScan is a registered trademark of Thomson Reuters  Inc.ZZ is employed by Eli Lilly and Company and is a stockholder.MRW is employed by i3 Innovus.This project was funded by Daiichi Sankyo Company, Limited and Eli Lilly and Company. The sponsors had no role in the design, methods, data collection, analyses, or preparation of the manuscript. The interpretation and reporting of these data are the sole responsibility of the authors.ZZ conceived and designed the study and performed the data analyses. MRW acquired the data and contributed to the study conception, design, and data interpretation. Both authors participated in writing the manuscript and have reviewed and approved the final version.Insurance plan type description:A variety of insurance plan types was available in the dataset used in this analysis, including fee-for-service (FFS), fully capitated, and partially capitated health plans, including preferred provider organizations (PPO), point of service (POS) plans, comprehensive plans, and health maintenance organizations (HMO). Specifically, in comprehensive plans there is no incentive for the patient to use a particular list of providers. Coverage is handled by one or two policies, with a deductible and coinsurance. Patients who have an HMO plan must choose from a particular list of providers for all non-emergency care. Referral from the patient care provider (PCP) is required for treatment by specialists. All services are paid for by the plan on a capitated basis. In POS plans, patients are offered financial incentives, through a lower copay or deductible, to use a particular list of providers. Referral from the PCP is required for treatment by specialists. Either no or some services are capitated and patients may seek treatment outside the network; however, this option usually involves a severe financial penalty. Patients who have a PPO plan have financial incentives, often through a lower copay or deductible, to use a particular list of providers. No PCP is required, nor are referrals necessary. No services are capitated. Patients may seek treatment outside the network, but this option is usually at a higher cost to the patient. The financial incentives may be offered only through discounted rates in-network.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/11/35/prepub


Equine encephalosis virus (EEV) distribution was thought to be limited to southern Africa until 2008 when we reported EEV in Israel. It was then assumed that the clinical presentation resembled the initial incursion in Israel. To investigate further we conducted a retrospective analysis of equine sera, which had been collected for diagnosis of other suspected diseases, via serum neutralisation test. The data demonstrated that EEV was circulating as early as 2001 with incidence ranging from 20–100% for time period 2001–2008. As the symptoms of EEV can be similar to other equine notifiable diseases this is a significant finding which highlights the need for vigilance and education to accurately diagnose new and emerging diseases. Reoviridae. The genome consists of 10 double stranded RNA segments of which the second segment (VP2) can used to determine serotype Culicoides species. It was first isolated in South Africa in 1967 and is thought to be endemic in this region. Until 2008 and its appearance in Israel Equine encephalosis virus (EEV) is an Orbivirus, family Some of the symptoms observed in cases of EEV are common to cases of other notifiable diseases, particularly African Horse Sickness (AHS) and Equine Viral Arteritis (EVA) making EEV important as a differential diagnosis Samples were selected from archived material which was submitted to the Kimron Veterinary Institute during the period 2001–2008 for diagnosis of other suspected diseases, but for which a laboratory diagnosis was not reached. The samples were selected on basis that clinical examination suggested symptoms associated with notifiable diseases. The investigated samples were from foals (9.2%), geldings (45.4%), mares (42.9%) and stallions (2.5%). Over 50% of the samples were from horses housed along the Mediterranean Coast with the remainder originating from various in-land regions across northern Israel. Symptoms observed in the sample set included respiratory, neurological and reproductive (abortions). None of the sampled animals had travelled outside of Israel. Sample collection was conducted according to the animal welfare committee guidelines of the Kimron Veterinary Institute, which also approved the use of the samples for this study.2 flask of Vero cells (ATCC) at 90% confluent monolayers. The flasks were incubated for 1 hour at 37°C in 5% CO2 to allow the virus to adsorb and then the inoculums washed off and overlaid with 10 ml of MEM supplemented with 5% fetal bovine serum . The cultures were examined daily for up to 5 days incubation, before being harvested by freeze/thawing and passaging onto fresh cell cultures.1 ml of blood was inoculated onto 1 day old 25 cmet al.50 per ml. Serial twofold dilutions of the test sera (heat treated at 56°C for 30 minutes for complement inactivation) were made in duplicate in serum free cell-culture medium using a 96-well microtitre plate starting at a 1∶2 serum dilution. Using serum-free cell culture medium as a diluent, a dilution of stock virus was prepared containing 100 TCID50/ml. A virus back titration of the working dilution of stock virus was included with each test. The plates were incubated for 1 hour at 37°C in a humid atmosphere of 5% CO2 in air. A suspension of Vero cells was added and the plates were returned to the incubator for 3–5 days before reading for cytopathic effect. To obtain a more accurate reading of the titre end-points an immunoperoxidase stain was performed as described by Smith et al., The virus neutralisation assay was essentially that described by Senne et al., 2008 and 2010 The Kimron1 isolate was used as the starting material for next generation sequencing via the Roche 454 FLX platform following protocols described by Ramussen The serological analysis revealed antibody positives in each year analysed back to 2001 . The peret al.et al.The resultant full genome sequence of Kimron1 (accession: AB811630-9) was compared to other publicly available EEV sequences. Here, we were limited as full genome data is only available for one other EEV strain HS103/06 Bryanston (accession FJ183384-93). In comparison to HS103/06 the greatest diversity was observed in comparison of the VP2 gene (54% at the nucleotide level) and greatest similarity was observed in comparison of the VP7 gene (96% at the nucleotide level). Analysis of other VP2 sequences revealed closest similarities to Kaalpaas serotype 3 strain (92% at a nucleotide level), Israeli isolates from 2008–9 outbreak described by Aahronson-Raz The similarity in the clinical presentation of EEV with other notifiable diseases, particularly EVA, is demonstrated by the symptoms and history of the 2008–9 outbreak. The detection of EEV outside of Africa highlights the risk of its spread to other regions where susceptible animals and suitable vectors are present, it is thus essential to understand the distribution of EEV. Here we demonstrate that a very similar strain of EEV, belonging to the same serotype, was already circulating in the Mediterranean region during a time period in which it was thought that the virus circulation was limited to southern Africa. None of the animals investigated had travelled outside of Israel therefore infection must have been acquired in country. From 2001 onwards at least, EEV infection was either overlooked or mis-diagnosed: demonstrating both the importance of vigilance and education to diagnose new and emerging diseases with the assistance of laboratories equipped to carry out investigative virology.Full genome sequence data will be of interest to those wishing to undertake further investigation of this disease. As EEV has a segmented genome it would be of interest to investigate whether recombination events contributed to the virulent strain observed in the 2008–9 Israeli outbreak. The similarity of the Israeli VP2 sequences to the 2009 Gambian isolate


Inactivated severe acute respiratory syndrome–associated coronavirus samples were used for an external quality assurance study within the World Health Organization SARS Reference and Verification Network and other reference institutions. Of 58 participants, 51 correctly detected virus in all samples >9,400 RNA copies per milliliter and none in negative samples. Commercial test kits significantly improved the outcome. The first epidemic of SARS began in November 2002 in southern China, spread to all five continents, and was interrupted in July 2003. It caused 774 deaths among the 8,098 cases. Two laboratory-associated infections and four new isolated cases have since occurred . Aliquots of the inactivated virus stock solutions were lyophilized and redissolved, and the virus RNA was quantified by two different noncommercial real-time RT-PCR assays  of 58 laboratories passed the minimum requirements for successful participation. Failure in three laboratories was due to lack of sensitivity, in three due to false-positive results, and in one due to both. Thirteen of 51 successful laboratories  could also detect the virus in all three weakly positive samples , and another 17 missed only one positive sample. Ten of the 58 laboratories issued indeterminate results in one or more samples.Whether common technical factors would influence the performance of laboratories was also assessed. We subjected cumulative results from low concentration samples  to analysis of variance (ANOVA) analysis. The overall positivity rate in these samples was 65.6% (95% CI 56.1%–75.0%). Seven technical factors  were uset-test).We finally assessed whether laboratories belonging to the international WHO SARS Reference and Verification Network (The results of this first external quality assurance study on SARS-CoV molecular detection are assuring. Compared to an earlier study on molecular testing for filoviruses, Lassa virus, and orthopoxviruses, using very similar proficiency criteria (Commercial tests clearly were the preferred way of achieving good diagnostic performance, possibly because SARS-CoV is a pathogen with which relatively few laboratories have had experience. However, developing and approving commercial tests is a lengthy process and high costs limit their application. Other approaches have to be adopted for efficiently providing good diagnostic tools in immediate response to an infectious disease outbreak. WHO's strategy of disseminating essential information through a public Internet resource before publication has proven successful. Laboratories have willingly shared protocols and positive control material with other institutions, enabling qualified diagnostics within weeks after the primary description of the new virus. The benefit is proven by good overall results in this study.International strain collections should be complemented with noninfectious reference material of rare pathogens. Until now, such material has been available only for highly prevalent agents like HIV-1, herpes viruses, or hepatitis viruses. For SARS-CoV, reference material has been created in this study for the first time. All samples described can be obtained for a nonprofit charge through the WHO SARS Reference and Verification Laboratory Network.


The geno2pheno algorithm (g2p) can give dichotomous tropism based on a selectable "false positive rate" (FPR), reflecting the proportion of individuals inappropriately called "non-R5" and falsely excluded from taking CCR5 antagonists. The effect of replicate genotype measures and different FPR values remains controversial. Here we characterize different FPR "cut-points" in predicting tropism for single vs multiple replicates for interpreting V3 genotype based on data from the clinical trials of Maraviroc (MVC) in experienced patients.The first study population comprised all patients screened for MOTIVATE 1  for whom both triplicate and single V3 genotypes were available. We also examined virological response (defined as a week 8 decrease ≥2 logs and/or to <50 copies/ml) in an outcome dataset of 547 patients who received MVC+optimized background therapy in the MOTIVATE-1, 2 or A4001029 studies with very limited background antiviral activity from other agents (wSS <1).Triplicate sequence analyses typically identified 10-25% more individuals with non-R5 virus compared to single replicates. A comparison of the predicted FPR by g2p to the virologically defined FPR at different g2p cut-points showed an excellent correlation (r2 =0.99; see Table The g2P algorithm shows the expected association with observed virological response, but this testing procedure may be more conservative than expected from the nominal FPR values, particularly for triplicate sequence analysis. A g2p FPR value above 10 likely excludes too many individuals who could respond to therapy if this cut-off is employed to screen individuals for maraviroc.


Novirhabdovirus, Aquabirnavirus, Alphavirus and Betanodavirus genera. The recent establishment of reverse genetics systems to recover infectious fish RNA viruses entirely from cDNA has made possible to genetically manipulate the viral genome. These systems have provided powerful tools to study all aspects of the virus biology and virus-host interactions but also gave the opportunity to use these viruses as live vaccines or as gene vectors. This review provides an overview on the recent breakthroughs achieved by using these reverse genetics systems in terms of viral protein function, virulence and host-specificity factor, vaccine development and vector design.Aquaculture has expanded rapidly to become a major economic and food-producing sector worldwide these last 30 years. In parallel, viral diseases have emerged and rapidly spread from farm to farm causing enormous economic losses. The most problematic viruses encountered in the field are mainly, but not exclusively, RNA viruses belonging to the  Novirhabdoviruses which can coexist in the same fish farms: the viral hemorrhagic septicemia virus (VHSV) and the infectious hematopoietic necrosis virus (IHNV). Other viruses have also a serious impact in the field such as the infectious pancreatic necrosis virus (IPNV), an Aquabirnavirus infecting for instance salmonid and viruses belonging to the Betanodavirus genus which cause serious diseases in several marine fish species. The genome of these viruses consists of either a negative-sense single-stranded RNA molecule for the Novirhabdoviruses of about 11 kilobases (kb) [Aquabirnavirus [Betanodaviruses [Fish farmers are faced worldwide to viral infections which destroy each year a significant part of the production and thus induce important economic losses ,2. In adses (kb) ,4, two srnavirus ,6 or twoaviruses ,8.Oncorhynchus mykiss) and the pancreas disease in marine-reared Atlantic salmon . These diseases are known since the 80s and have now spread in most of the European countries and in North America. For instance, the sleeping disease may affect 30 to 40% of the French fish farms. Viruses responsible for these diseases, the sleeping disease virus (SDV) and the salmon pancreas disease virus (SPDV) belong to the Alphavirus genus, with a genome which consists of a positive-sense single-stranded RNA molecule of about 12 kb [These viral diseases are the most frequent but due to the expansion of the salmonid aquaculture and the development of new fish species production, new diseases have emerged in the recent years . One of ut 12 kb ,10.Picornaviridae family), was in 1981 [Since many years, goals of different laboratories around the world have been mainly but not exclusively devoted to the development of new vaccine approaches based in part on live attenuated viruses to prevent viral diseases in aquaculture ,12 and t in 1981 . Thirtee in 1981 . The abiNovirhabdoviruses IHNV and VHSV [Alphavirus SDV [Aquabirnavirus IPNV [Betanodavirus species such as for the striped jack nervous necrosis virus (SJNNV) [Reverse genetics systems have been now established for several fish RNA viruses such as the and VHSV -18, the irus SDV , the Aqurus IPNV  and also (SJNNV) . A largeAlphaviruses are recognized as serious pathogens of farmed Atlantic salmon and rainbow trout in Europe [Alphavirus genus of the Togaviridae family [Alphavirus genus, is transmitted by mosquitoes, and its alternate vertebrate host is usually a bird or a mammal. Like all Alphaviruses, SDV and SPDV virions are spherical (70 nm in diameter), with a lipid envelop containing heterodimeric glycoprotein spikes composed of two virus glycoproteins (E1 and E2). Some alphaviruses may contain a third envelop protein (E3). The envelope is tightly organized around an icosahedral nucleocapsid which contains the genomic RNA. The viral genome consists of a positive-sense single-stranded RNA molecule of about 12 kb in length, which is capped at the 5'-terminus and polyadenylated at the 3'-terminus, and contains two open reading frames (ORF). The 5' two-thirds of the genome encode four non-structural proteins (nsP1 to nsP4) that are translated directly from the genomic RNA as a polyprotein precursor, which undergoes subsequent proteolytic cleavage. The four non-structural proteins are involved in genome replication and the synthesis of a subgenomic RNA from the negative-strand copy of the genome that corresponds to the 3' third of the viral genome and encodes the second ORF. This second ORF is translated as a polyprotein, which is processed to produce the viral structural proteins: the capsid, the glycoproteins E3, E2, E1 and the 6K protein. In contrast to the other Alphaviruses, an arthropod-independent transmission has been demonstrated for salmonid Alphaviruses by cohabitation experiments [Salmonid n Europe . Sleepine family ,22. Sinderiments .Alphavirus genus was established for SINV few years later by Rice et al. [Based on the observation that genomic RNA from most of the positive-stranded RNA viruses could serve as messenger RNA (mRNA) and initiate infection upon transfection in a permissive cell line, the recovery of infectious virus from complementary DNA (cDNA) was first described for poliovirus by Racaniello and Baltimore, almost 30 years ago . The fire et al. . The rece et al. ). Similae et al. ,25. The e et al. . Indeed,e et al. ) expresse et al.  and secte et al. . A simile et al. .Aquabirnavirus genus of the Birnaviridae family. Infectious bursal disease virus (IBDV), another member of Birnaviridae family that has been studied in the greatest detail is the causative agent of highly immunosuppressive disease in young chickens and belongs to another genus, Avibirnavirus [Infectious pancreatic necrosis (IPN) is a serious disease of salmonid and a number of other fish species that can lead to high mortality (70%) in first feeding fry and fingerlings of most salmonids. Furthermore, the aetiological agent responsible for the disease (IPNV) can be vertically transmitted via the egg and broodstock carrier fish facilitating its rapid spread to new geographic area and its expansion to other farmed fish species . IPNV isrnavirus . IPNV isrnavirus . Segmentepithelioma papulosum cyprinid) cells) with segment A and B cDNA copies . Segments A and B were cloned under the control of a T7RNAP promoter and upstream the antigenomic sequence of a self-cleaving ribozyme derived from the hepatitis delta virus (δ) allowing the expression by the T7RNAP of RNA molecules displaying a precise 3'-end without any additional nucleotide [The first description of segmented double-stranded RNA virus recovery entirely from cDNA was given by Mundt and Vakharia for IBDV . Few yeacleotide .Betanodavirus genus, within the Nodaviridae family. Betanodavirus genus is further divided in four species based on similarities in the variable region of the RNA2 and designated as Barfin flounder nervous necrosis virus (BFNNV), Redspotted grouper nervous necrosis virus (RGNNV), Striped jack nervous necrosis virus  and Tiger puffer nervous necrosis virus (TPNNV) [Nodaviridae family, infects insects and belongs to the second genus, Alphanodavirus. Fish nodaviruses are icosahedral, non-enveloped viruses with a commonly reported diameter of about 25 nm. The virus contains two single-stranded positive-sense RNA molecules, namely RNA1 (3.1 kb) and RNA2 (1.4 kb). Both RNA molecules are capped at their 5'-ends and lack poly(A) tails at their 3'-ends. The larger genomic segment, RNA1, encodes the RdRp (or protein A) and the smaller genomic segment, RNA2, encodes the coat protein. In addition, subgenomic RNA3, which is synthesized from RNA1 during RNA replication and not packaged into virions, encodes the two non-structural proteins B1 and B2.Viral nervous necrosis (VNN) and viral encephalopathy and retinopathy (VER) have emerged as major constraints on culture of larval and juvenile and sometimes older marine fish worldwide leading to behavioural abnormalities and high mortalities ,7. The a (TPNNV) . Black BNodaviridae family was reported by Kaesberg's group for BBV [The first recovery from cDNA of a virus belonging to the  for BBV . Because for BBV . This sy for BBV . In that for BBV ,36. Reve for BBV ,35-37.Rhabdoviridae family within the Mononegavirales order. The most extensively studied mammalian rhabdoviruses are the rabies virus (RV) that belongs to the Lyssavirus genus and the vesicular stomatitis virus (VSV) that belongs to the Vesiculovirus genus. Similar to the mammalian rhabdoviruses, the IHNV and VHSV genomic RNA which encodes five structural proteins, is tightly encapsidated with a nucleoprotein (N), a polymerase-associated phosphoprotein (P) and the large RdRp (L) to form the helical ribonucleoprotein complex (RNP). The two other structural proteins are the matrix protein (M), which interacts with the RNP and the viral envelope and is involved in the budding step, and the unique viral surface glycoprotein (G) which is implicated in the entry step. In contrast to other rhabdoviruses, IHNV and VHSV genomes encode a small non structural NV (Non Virion) protein. Due to the presence of the NV gene, localized between the G and L genes, IHNV and VHSV are separated from the other rhabdoviruses and belong to the Novirhabdovirus genus [IHNV and VHSV are causative agents of acute systemic diseases leading to high mortality, mainly, but not exclusively, in young cultured and wild salmonid fish all over the world and are listed as notifiable by the World Organisation for Animal Health (OIE) ,38. IHNVus genus .The recovery of rhabdoviruses from cDNA presents a unique challenge because neither their genomic RNA nor their antigenomic complements serve as mRNA, and thus cannot be used directly to recover infectious viruses. In other words, the RNA of negative-stranded viruses (NSV) are not infectious upon transfection into a permissive cell line in contrast to those of positive-stranded viruses. The minimal infectious unit of NSV is the RNP complex which is able to drive both the sequential transcription of each viral gene and the replication of the full-length RNA genome. Based on this observation, the feasibility of producing infectious NSV entirely from cDNA was demonstrated by Conzelmann's group for RV, 15 years ago . The goaMononegavirales order [This system was then adapted to numerous viruses belonging to the es order  and was es order ,41). Cones order . This syes order ,18,27. Tes order ). Two kies order   [VHSV, in which the IHNV NV ORF was replaced by that of VHSV, was shown to replicate efficiently in fish cell and to induce similar cumulative percentage of mortality in vivo in trout compared to the wild-type IHNV [-4 mutations per nucleotide copied, which is almost equivalent to one mutation per rhabdovirus genome copy, and thus exist as quasispecies [Novirhabdovirus genus indicates that the NV protein by itself has a function. It could be of interest to replace the VHSV and IHNV NV gene by that of SHRV  to evaluate the potential of complementation of this NV protein on both virus phenotypes, and thus evaluate whether these three NV proteins that share less than 30% of homology have a common function.The ability to generate recombinant viruses by reverse genetics allows the opportunity to examine the role of the non-structural proteins in virus biology and pathogenic mechanisms. For instance, the NV proteins (111 to 122 amino acids), expressed by IHNV and VHSV in infected cells , were shvescens) . Indeed,vescens) . The obsype IHNV . Thus, aype IHNV ,45. Thisispecies . This prAlphanodavirus genus, a subgenomic RNA3, corresponding to the 3'-end of RNA1, is synthesized by the viral RdRp during the replication. This subgenomic RNA3 encodes two small non-structural proteins, B1 and B2  during their replication in infected cells and fish [Mx gene induction which is proportional to RNA1 accumulation in fish cells. Thus it seems that for betanodaviruses, which cause high mortalities in larval and juvenile marine fish, the most important host response at these fish developmental stages is RNA silencing [In r review ). B2 pror review -53. The and fish -56. The and fish , and greand fish . Fenner and fish . Indeed,and fish ,59. In cilencing . To furtilencing . The exiilencing , Chen anilencing . RecoverThe reverse genetics has also been used to investigate the potential role of VP5 on IPNV replication and virulence. Indeed, studies on recombinant IBDV lacking expression of VP5 showed that VP5 was nonessential for productive virus replication in cell culture, but played a key role in pathogenicity of IBDV . SeveralWith efficient reverse genetics systems available for several major fish pathogens, the degree of flexibility of the genome of these viruses to genetic engineering and their ability to express foreign genes of interest were verified by several groups. For this purpose, negative-stranded viruses (NSV), such as rhabdoviruses, appear to be good candidates as vectors for transient gene expression compared to positive-stranded viruses (PSV) for many reasons . Novirhabdovirus, 51% homology between both proteins) and the spring viremia of carp virus  [An attractive method to express foreign antigens by a recombinant virus is to replace its major protective surface antigen with that of another virus of interest. Such constructs have been generated by exchanging the unique surface glycoprotein of different distantly-related and antigenically-distinct fish rhabdoviruses ,69. The  both G) ,71. Inte both G) . The eff both G) ). In con both G) . The rec both G) . Finally both G) . Moreove both G) .As explained above (see section 3.2), the strategy used to insert an additional ORF in RNA viruses depends on the genome organization. Concerning NSV, such as rhabdoviruses, the sequential transcription is initiated by the RdRp upstream of each ORF by the recognition of a gene start signal and ended at a gene end signal that serve as transcription stop and polyadenylation signals . A fRenilla luciferase (LUC), the VHSV G and different other virus-antigen encoding genes ([7RCCGUG(N)4CACR versus UCUAUC(U)7RCCGUG, respectively). These signals were successfully used to express a large panel of reporter genes: LUC, GFP and red fluorescent proteins such as Cherry and Tomato [Both strategies were tested for IHNV and VHSV -17,47,72g genes , large inserts (> 2 kb) are not stable upon passaging and rapidly removed by recombination which is observed at high frequency with PSV . TherefThe reverse genetics is a powerful tool to map in the virus genome virulence and host specificity factors based on previous putative nucleotide changes determined after genome sequence comparisons of virus strains displaying different phenotypes. For instance, amino acid sequence comparisons of IPNV VP2 protein from various field isolates belonging to the Sp serotype, which exhibit different mortality rates in Atlantic salmon fry, identified putative residues responsible for modulating virulence between strains ,82. The Pseudocaranx dentex), whereas members of RGNNV type, such as sevenband grouper nervous necrosis virus (SGNNV), has a broad host range and causes diseases for a variety of warm-water fish species, particularly for sevenband grouper (Epinephelus septemfasciatus). Recombinant reassortant and chimeric viruses were generated by using reverse genetics systems established for SJNNV, RGNNV and SGNNV [Similar approaches were undertaken to study betanodavirus host specificity and temperature restriction. Although a high overall percentage of homology is found between the four types of betanodavirus isolates, some isolates have marked host specificity. For example, SJNNV causes disease only in the striped jack  of rSDV could be harvested from sacrificed fish up to three weeks post-infection. Immunized rainbow trout acquired long-lasting (at least seven months) and complete protection against challenges with a highly virulent SDV strain by bath immersion or injection. In addition, rSDV-vaccinated fish were also protected from a challenge with the wild-type SPDV, a closely related but distinct salmonid alphavirus. It has to be mentioned that in addition to the genome sequence mutations, the temperature of virus adaptation to the cell culture seems to play a role in the SDV attenuation. Indeed, naïve trout infected with the wild-type SDV adapted to 14°C (SDV14) or with that adapted to 10°C (SDV10) exhibited different cumulative mortality rates two months postinfection, roughly 80% and 8%, respectively. The rSDV was also recovered at 10°C which could explain in part its in vivo attenuated phenotype. The effect of the temperature at which the viruses were produced on the virulence was established by adapting rSDV and SDV10 to grow at 14°C. Indeed, the resulting viruses induced higher cumulative mortality rates in trout: from 0 to 24% for rSDV and 7% to 30% for SDV10, respectively. The comparison of the nucleotide sequences encoding the structural proteins pinpointed six major amino acid changes  between rSDV and its 14°C-adapted derivative. Moreover, as SDV10 was derived from SDV14 after plaque purification, it could be assumed that SDV14 inoculum represents a quasispecies population pathogenic for trout and that the viral heterogeneity was greatly reduced for SDV10 and even more for the rSDV derived from a single cDNA clone. This hypothesis was in part demonstrated by the partial sequencing of the three virus genomes which pinpointed a large number of amino acid changes, roughly 60 and 20 in the non-structural genes of SDV14 and SDV10 compared to the counterpart region in rSDV [For alphaviruses, incidental mutations introduced during the RT-PCR steps needed to construct infectious cDNA for SDV have also contributed to identify potential attenuating position in their genomes. Concerning SDV, several nucleotide substitutions have been mapped in the structural and non-structural genes of the infectious cDNA compared to the published sequence . AltOncorhynchus nerka) . The U and M IHNV strains differ in host specific virulence: U strain is virulent in sockeye salmon but not rainbow trout, and the M strain is virulent in rainbow trout but not sockeye salmon. These studies revealed that the G and NV genes do not contain the determinants of host-specific virulence for these IHNV strains, suggesting that the molecular mechanism(s) of host-specificity in this case must be different from the viral coat protein host cell attachment mechanism described for IPNV and betanodaviruses. Finally, in recent decades, several VHSV and IHNV strains have been isolated and fully sequenced. For instance, several VHSV strains have been isolated from several marine fish species . Several nearly complete genome sequences from marine and freshwater isolates displaying variable level of virulence in rainbow trout are now available in GenBank. From the analysis of these sequences, several groups have already pinpointed putative amino acid residues that could be involved in VHSV strain virulence [Concerning IHNV, the introduction of four incidental mutations during the construction of the full-length cDNA construct was enough to highly attenuate the recovered virus. This recombinant IHNV, containing 2 amino acid substitutions in the N, one in the P and one in the M, induced less than 10% of cumulative mortality in trout by bath immersion and was found to be a strong inducer of IHNV-specific antibodies ,71. Surpview see ). The inirulence ,89. Inteirulence , these as could be used to generate single-replication cycle virus-based vaccine vectors such as alphavirus replicons. Although this kind of vaccine was found safe and protective in mammals after injection, the immunogenicity in fish of such vaccines should be demonstrated when administrated by bath immersion. The next several years will clearly be very exciting time in fish RNA virus research since advances in virus genome manipulation, fish genome sequencing and fish immune system characterization have greatly increased these last years.Since the description of the first reverse genetics system for a fish RNA virus in 1998 , severalThe authors declare that they have no competing interests.


Corriparta virus (CORV), within the genus Orbivirus, family Reoviridae, currently contains six virus strains: corriparta virus MRM1 (CORV-MRM1); CS0109; V654; V370; Acado virus and Jacareacanga virus. However, lack of neutralization assays, or reference genome sequence data has prevented further analysis of their intra-serogroup/species relationships and identification of individual serotypes. We report whole-genome sequence data for CORV-MRM1, which was isolated in 1960 in Australia. Comparisons of the conserved, polymerase (VP1), sub-core-shell ‘T2’ and core-surface ‘T13’ proteins encoded by genome segments 1, 2 and 8  respectively, show that this virus groups with the other mosquito borne orbiviruses. However, highest levels of nt/aa sequence identity  were detected between CORV-MRM1 and California mosquito pool virus (CMPV), an orbivirus isolated in the USA in 1974, showing that they belong to the same virus species. The data presented here identify CMPV as a member of the Corriparta virus species and will facilitate identification of additional CORV isolates, diagnostic assay design and epidemiological studies.The species  Orbivirus, family Reoviridae. Currently there are also 15 ‘unclassified’ orbiviruses in the genus, which may represent additional species Culicoides, mosquitoes or sand flies) and collectively have a wide host-range that includes both domesticated and wild ruminants, equids, camelids, marsupials, sloths, bats, birds, large canine and feline carnivores, and humans Corriparta viruses are mosquito-borne arboviruses, classified within one of the 22 virus species currently recognised within the genus Corriparta virus currently contains six distinct viruses, that are identified as: corriparta virus MRM1 (CORV-MRM1); CS0109; V654; V370; Acado virus; and Jacareacanga virus The species Corriparta virus species/serogroup have been detected in Australia, Africa and South America Culex mosquitoes, as well as from Aedeomyia catasticta, a rare mosquito species collected near Mitchell River in North Queensland, Australia. Subsequently, strains CS0109, V654 and V370 were also isolated in Australia Culex mosquitoes collected in Ethiopia and Brazil during 1963 and 1975 respectively Members of the Orbivirus species The International Committee on Taxonomy of Viruses (ICTV) has agreed ‘polythetic’ definitions for individual virus species Orbivirus species were originally identified as belonging to distinct ‘serogroups’, based on their cross-reactivity in ‘group-specific’ serological assays that include complement fixation (CF) tests, group-specific ELISA, or agar-gel-immuno-diffusion (AGID) tests, most of which target outer-core protein VP7(T13) The members of the different Orbivirus species, including Bluetongue virus (BTV); African horsesickness virus (AHSV) and Epizootic hemorrhagic disease virus (EHDV) Orbivirus species Orbivirus species, including Warrego virus (WARV), Wallal virus (WALV), Wongorr virus (WGRV) and CORV Recently, full genome sequencing and phylogenetic analyses have been used to determine the genetic relatedness and taxonomic status of individual isolates belonging to different Culex tarsalis mosquitoes collected as part of an infectious agent surveillance program conducted by The California Department of Public Health Orbivirus species, made it impossible to confirm the taxonomic status and species identity of CMPV at that time.‘California mosquito pool virus’ (CMPV) was isolated in 1974 from pooled Corriparta virus.We report the full genome sequence of CORV–MRM1 (AUS1960/01). Comparisons of nucleotide (nt) and deduced amino acid (aa) sequences for the conserved polymerase ‘VP1(Pol)’, subcore-shell ‘T2-protein’, and outer-core ‘T13-protein’, to data published for other orbiviruses, indicate that CORV and CMPV belong to the same species - Culicoides borne orbiviruses (as illustrated for BTV-1w [LIB2007/07] and EHDV-8e [AUS1982/05]).CORV–MRM1 induced severe cytopathic effect (CPE) in BHK cell monolayers, by 48–72 hours post infection (pi). The viral dsRNA was purified from infected cell-cultures and analysed by 1% agarose gel electrophoresis (AGE) . CORV Se5′-GUAUAG………..CAAAGGAUAC-3′). Two terminal nucleotides at the 5′ end and three nucleotides at the 3′ end (5′-GU……UAC-3′) are characteristic of the genus Orbivirus and the first and last two nucleotides represent inverted complements (http://www.reoviridae.org/dsRNA_virus_proteins/CPV-RNA-Termin.htm).Sequences for Seg-1 to Seg-10 CORV-MRM1 (AUS1960/01) have been deposited in the GenBank with accession numbers KC853042 to KC853051, respectively. They range from 3,925 bp to 790 bp  with a total length of 19,093 bp, . The difCulicoides-borne orbiviruses (CBOs) show similar or lower values  represent 4.99% of the CORV-MRM1 genome . Comparir values .Like other orbiviruses, most genome segments of CORV-MRM1 (AUS1960/01), have shorter 5′ than 3′ NCRs, except for Seg-6 ) which has a longer 5′ NCR and Seg-9 (encoding VP6) which has 3′ and 5′ NCRs of equal length . ExceptiSeg-5  of CORV-MRM1 which encodes the ‘tubule’ protein NS1(TuP) (one of the most abundantly expressed orbivirus proteins) is longer than the homologous Seg-5 of the CBOs or TBOs  with a lThe ‘highly conserved’ subcore-shell VP2(T2) protein and the ‘highly variable’ outer-capsid/cell-attachment protein VP3(OC1) of CORV-MRM1, are encoded by Seg-2 and Seg-3, respectively. A similar coding pattern is seen in other MBOs, but the presence of a larger OC1 in the CBOs  results in a reversed coding assignment for these two genome-segments .The size of the highly conserved T13 core-surface protein (VP7 of BTV) is also relatively consistent in most orbiviruses, while the viral inclusion-body matrix-protein, ]) is more variable in size . As a reAUGG) AUGU), possibly enhancing read-through and initiation of translation from the second ‘in frame’ initiation site (at 407 bp). This has a strong Kozak context (GUUAUGG), but would express the smallest NS3a in any of the orbiviruses characterized to date.Most of the genome segments of CORV-MRM1 (AUS1960/01) except Seg-9 and Seg-10 are monocistronic, encoding a protein from a single large ORF, starting from an initiation codon with a strong Kozak sequence (RNNAUGA) and a second down-stream ORF. However, this is in the +2 reading-frame (at 143–598 bp), encoding the 152 aa NS4 protein. NS4 has previously been identified in several other orbiviruses and has been characterised in BTV and GIV AGGAUGG) and is expected to produce a protein in infected cells. Weak or moderate Kozak sequences have also been observed in several of the genome segments of other orbiviruses, but they still appear to be translated effectively The first start codon of CORV-MRM1 (AUS1960/01) Seg-9 also has a moderate Kozak sequence (UUGCulicoides)]. However, it is lower than that of the tick-borne or tick-associated orbiviruses [57.29% in GIV and 51.93% in St Croix River virus (SCRV)].The G+C content of the CORV genome is 45.16%, which is considerably higher than that of other MBOs [Peruvian horse sickness virus (PHSV) and YUOV with 36.66% and 41.59% respectively] but within the overall G+C range of the insect-borne orbiviruses , the MBOs [VP2(T2) and OC1(VP3) - encoded by Seg-2 and Seg-3 respectively], and the CBOs [VP3(T2) and VP2(OC1) - encoded Seg-3 and Seg-2 respectively] Previous studies have suggested that the MBOs have evolved from tick-borne ancestors, with CBOs being last to evolve Previous studies also indicate that the orbiviruses have evolved through a process of ‘co-speciation’ with their vectors Corriparta virus. The occurrence of these closely related viruses in the Americas and Australia indicates that there has been spread of viruses between these regions, which could be due to movement of infected hosts or vectors. Similar movements are also suggested by the detection of other orbiviruses  in more than one continent, e,g, in Australia, Africa and the Americas Corriparta viruses have been isolated in Australia, Africa and South America Corriparta virus species, as well as helping to identify the arthropod vectors involved in their transmission. Further studies are still needed to define the different serotype and topotypes of CORV.The sequences and relative sizes of the VP1(Pol), T2 and OC1 proteins are important evolutionary markers that can help differentiate/group orbiviruses by species, serotypes and topotype. The sequence data generated in this study will facilitate the use of phylogenetic analyses to identify other novel isolates belonging to the CORV-MRM1 (AUS1960/01), obtained at passage level MB6/BHK2 from the Orbivirus Reference Collection at The Pirbright Institute, was propagated in BHK-21 cell monolayers [clone 13 obtained from European Collection of Animal cell Cultures (ECACC – 84100501)], in Dulbecco's minimum essential medium (DMEM) supplemented with antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) and 2 mM glutamine. Infected cell-cultures were incubated at 37°C until they showed widespread (100%) cytopathic effects (CPE). Then viruses were harvested, aliquoted and used for the extraction of viral dsRNA. All virus isolates used in these studies were obtained from diagnostic samples of naturally infected animals and were taken as a part of normal diagnostic investigations by qualified veterinarians in the individual countries.g for 5 min. The viral dsRNA was released and purified using TRIzol® reagent (Invitrogen) as described by Attoui et alg for 10 min before 900 µl of isopropanol was added prior to incubation at −20°C for 2 hours. The RNA was pelleted at 10,000 g for 10 min, washed with 70% ethanol, air dried and dissolved in 100 µl of nuclease free water (NFW). Single stranded RNA (ssRNA) was removed by precipitation with 2M LiCl (Sigma) at 4°C overnight, followed by centrifugation at 10,000 g for 5 min. An equal volume of isopropanol, containing 750 mM ammonium acetate, was mixed with the supernatant. After precipitation at −20°C for 2 hours, the viral dsRNA was pelleted at 10,000 g for 10 min, washed with 70% ethanol, air dried and suspended in 50 µl of NFW. The RNA was either used immediately or stored at −20°C.Cell monolayers showing 100% CPE after infection with CORV-MRM1, were harvested and pelleted at 3000 et al.CORV-MRM1 genome segments were reverse-transcribed into cDNA using the full-length amplification (FLAC) technique described by Maan cDNA amplicons were purified and cloned into the ‘pCR®-Blunt’ vector supplied with the Zero Blunt® PCR Cloning Kit (Invitrogen). Recombinant plasmid-vectors containing CORV-MRM1 inserts were transformed into One Shot® TOP10 competent cells supplied with the cloning kit. Clones containing the desired inserts were identified by colony touch 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).http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome). Multiple alignments of consensus sequences were performed using Clustal X (Version 2.0) http://www.ebi.ac.uk/Tools/msa/clustalo/) and MAFFT http://www.cbs.dtu.dk/services/RevTrans/) for further nucleotide analysis. Pairwise distance (aa and nt) calculations and phylogenetic trees constructions were done using MEGA 5 software ‘Raw’ ABI sequence data was assembled into ‘contigs’ using the SeqManII sequence analysis package (DNAstar version 5.0). The ORFs of CORV-MRM1 genome segments were identified and translated to aa sequences for further analysis using EditSeq (DNAstar version 5.0). The putative function of each protein was identified by Blast X comparisons to homologous orbivirus (BTV) proteins in GenBank Click here for additional data file.


Plant viruses cause extensive remodeling of infected cells. These structural alterations include reshaping of large organelles , proliferation of membranes and membranous vesicles and modification of the plasmodesmata (PD) structure. These alterations have a profound impact on plant physiology and development. However, it is only recently that studies have deeply investigated the biogenesis of virus-induced structures and their biological function(s).Novel techniques that allow easy expression of viral proteins or modified viral genomes in plants combined with powerful visualization tools  offer a new perspective on cellular remodeling in virus-infected plant cells. We now know that some of these novel sub-cellular structures are virus-induced organelles or “factories” that house the RNA replication complex. Other morphological changes  are related to the intracellular and intercellular movement of the virus. Finally, induction of autophagosomes and modification of large organelles have been observed in association with the innate immune response.This e-Book on Plant Virus Infection—a Cell Biology Perspective aims at providing the latest information on the molecular and cellular requirements that underlie the biogenesis of these virus-induced structures.First, we have a look at a non-virus pathogen: the viroid. In contrast to a virus, the viroid genome is composed of a tiny circular RNA (250–400 nt) that does not code for proteins. Nevertheless, viroid infections are accompanied by cellular and developmental disorders that sometimes have dramatic economic consequences, for instance the cadang-cadang disease of coconut palms. Di Serio et al.  look at “Hijack it, change it” is applicable to the description of many viral processes, given the propensity of viruses to commandeer the host machinery for their own purposes. In their review, Patarroyo et al.  discuss Secoviridae, She further reviews and discusses literature demonstrating the ability of these proteins to induce membrane proliferation and perhaps alter membrane structure  and integrity (formation of pores through oligomerization) in the host cell. The membrane often targeted by these viral proteins is the endoplasmic reticulum.Sanfaçon  summarizTurnip vein clearing virus is presented.In reviewing the latest cell biological studies describing tobacco mosaic virus (TMV) replication and movement, Liu and Nelson  highlighThe triple gene block (TBG) proteins are the focus of several contributions. First, Solovyev et al.  introducIndeed, TGB proteins may have multiple functions in the viral infection process. Linnik et al.  contribuPotexvirus genus member, alternanthera mosaic virus (AltMV), provides insight into how its TGB3 protein may function to modify its target, the chloroplast and its membranes. In this study, Jang et al. , genomic RNA, and labeled TGB2 were colocalized to chloroplasts in PTMV-infected tissues.In addition to TGB3, TGB2 seems also to play a role in targeting the virus to chloroplasts. In another research paper, Cowan et al.  demonstrTenunivirus genus. They showed that NSvc4 traffics on the actin filament and ER network and that targeting of NSvc4 to PD requires the functional cytoskeleton. They also found that NSvc4 contains a chloroplast-targeting signal and localizes to chloroplasts in infected cells, suggesting NSvc4 may also be a multi-functional protein.Viral cell-to-cell movement remains a hot topic for plant virologists. Xu and Zhou  studied In their hypothesis and theory article, Krenz et al.  discuss Cellular remodeling is also the consequence of molecular pathways being overpowered by viruses. Verchot  exploresZhang and Wang  summarizBujarski  gives an


Human gamma-herpesviruses, Epstein-Barr virus (EBV or HHV-4) and Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8), are associated with several malignancies, especially in AIDS patients. Although highly active antiretroviral therapy (HARRT) has significantly reduced the incidence of EBV- and KSHV-associated tumors, it does not eliminate EBV or KSHV infection, and the tumor risk remains high for HIV-1-infected individuals who are also carriers for EBV and KSHV. Tumorigenesis of gamma-herpesviruses is associated with the persistence of infection. Thus, vaccination to elicit protective immunity that inhibits the establishment of viral persistence will prevent the occurrence of virus-associated cancers. However, currently there are no effective vaccines available for KSHV or EBV.in vivo lytic replication capacity of a live gamma-herpesvirus needs to be significantly weakened without losing immunogenicity. We hypothesize that removal of the viral genes that inhibit the host immune responses will reduce the fitness of the virus but potentially increase its immunogenicity. For this purpose, we have removed immune evasion genes from AC-RTA along with other modifications.For proof of concept vaccination experiments, we utilize a mouse gamma-herpesvirus infection model. Previously, we have shown that vaccination with a non-persistent highly lytic live attenuated virus (AC-RTA) provides effective protection against a challenge infection by the wild type virus. To increase the safety of vaccination, the Deficient in Immune evasion and Persistence), replicates in cell culture but is severely attenuated in mice, deficient in acute productive infection and latency. However, immunization of the DIP virus prevents latency establishment by the challenge wild type virus. Next, we aim to test strategies to improve the immunogenicity in immunocompetent and CD4-deficient hosts by incorporating expression of co-stimulatory molecules into the DIP virus.The resultant virus named DIP (in vivo viral replication provides us a novel vaccine strategy for preventing infection of human gammaherpesviruses. The “proof of concept” study in the mouse infection model is necessary to provide fundamental insights into the development of vaccines for the tumor-associated human herpesviruses.The non-persistent DIP virus that undergoes limited


We will also discuss implications of the ubiquity of viruses in insects and in insect cell lines. All of the methods described in this article can also apply to the discovery of viruses in other organisms.Insects are commonly infected with multiple viruses including those that cause sublethal, asymptomatic, and latent infections. Traditional methods for virus isolation typically lack the sensitivity required for detection of such viruses that are present at low abundance. In this respect, next generation sequencing technologies have revolutionized methods for the discovery and identification of new viruses from insects. Here we review both traditional and modern methods for virus discovery, and outline analysis of transcriptome and small RNA data for identification of viral sequences. We will introduce methods for  In adifera L. ; silk mo(Hübner) , (c) ide(Hübner) , and (d)(Hübner) ,8. Contr(Hübner) . For exa(Hübner) . A denso(Hübner) . A bactesp larva ,13.i.e., in a chronic infection), or become latent, such that virus production ceases altogether. These viruses would not readily be detected by use of traditional protocols for virus discovery.Traditionally, viruses were isolated from insects that displayed an abnormal phenotype as a result of virus infection. While infection with some insect viruses, such as baculoviruses, results in clear symptoms and ultimately death of the host, many virus infections are asymptomatic. In recent years, with the development of the Next Generation Sequencing (NGS) technologies, it has become evident that asymptomatic or covert virus infections are ubiquitous. These viruses may accumulate to relative low titers in the host organism  discovered by Dmitri Iwanowsk, a Russian botanist in 1882. He showed that extracts from diseased tobacco plants could transmit disease to other plants after passage through ceramic filters that were sufficiently fine to retain bacteria. The first virus particles (TMV) were observed following the invention of the electron microscope in 1931. Although it was known from the 1930s that viruses consisted of a protein shell and nucleic acids, methods for detection of viral protein and viral genomic RNA or DNA were not developed until the late 1970s and early 1980s. Conventional approaches for virus discovery are to collect insects from multiple populations from multiple locations, and use several micrograms of material at minimum for virus purification. Virus purification protocols vary, but material is typically homogenized, centrifuged and filtered under sterile conditions. Further purification may include ultracentrifugation and sucrose or cesium chloride gradient steps. The sample may then be used for visualization of virus particles by electron microscopy, infection of cultured insect cells and observation for cytopathic effects –17, and 2.1.Electron microscopy (EM) has been and continues to be one of the most important techniques for virus discovery . Indeed Samples used for EM analysis for virus observation range from crudely extracted samples to viruses purified via ultracentrifugation and sucrose or cesium chloride gradients. Thin sectioning of tissues is also common for observing the tissue tropism of insect viruses. For examination of virus particles, specimens are placed on to grids and typically subjected to negative staining . CommonlImmunolocalization is used for observing virus in thin sectioned tissue specimens and for specifically identifying known viruses. The viruses are coated with viral antibodies followed by secondary antibody conjugated with colloidal gold (gold labeling) in the grid. The grid is then negatively stained.2.2.Two serological methods that are commonly used for virus detection are enzyme-linked immunosorbant assay (ELISA) and western blot. Both methods employ antibodies that recognize viral coat or other viral proteins to detect the presence of a given virus either from samples of purified virus or from total proteins extracted from infected insects or tissues. ELISA and western blot are widely used for detection of known viruses and may also be used to assess the serological relationship of a new virus to known viruses within the same family.2.3.Nucleic acid hybridization  are also useful for identification of viruses, but these methods also rely on prior knowledge of the target virus; Viral specific nucleic acid probes are labeled for hybridization to the target viral DNA or RNA to demonstrate the presence of the virus.Polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) are used to amplify viral DNA or RNA respectively, either full length genomic sequences, or parts thereof. The resulting DNA or cDNA fragments are cloned into vectors and used for sequencing, and in the case of full length sequences may also be used for screening for infectious virus clones . Compari2.4.Several insect viruses were discovered through analysis of expressed sequence tag (EST) libraries. EST libraries are produced by isolating total RNA from insects, purifying mRNA, and generating and sequencing a cDNA library. Hence ESTs are sequences  that represent the sequences of transcribed genes. Purification of mRNA to generate the cDNA library requires selection of RNAs that include polyA tails on a polyT column, thereby limiting sequence representation to viral RNAs with polyA tails. Although relatively little viral sequence with low coverage is provided by ESTs, there may be sufficient sequence for 5′ RACE to acquire the full genomic sequence. Given the low sequence coverage provided by EST libraries, virus detection via EST sequences is likely to be limited to viruses that are present at high titers in the host insect.et al. [Solenopsis invicta, which causes significant economic damage in the U.S. Three of these ESTs exhibited significant homology to Acute bee paralysis virus (Dicistroviridae) and 5′ RACE was used to delineate the entire genome sequence of the virus, Solenopsis invicta virus 1 (SINV-1).Valles et al.  detectedet al. [H. vitripennis  [H. vitripennis vectors the bacterium Xylella fastidiosa, which negatively impacts numerous plant species. Viruses isolated from this insect may have potential for use in its management. Sequences derived from a phytoreovirus (plant virus) were also detected from an H. vitripennis salivary gland cDNA library [Hunnicutt et al.  isolatedagulata) . This in library .et al. [i.e., series of overlapping DNA sequences used to reconstruct the original sequence) of 2789 nt  from the parasitoid wasp, Nasonia vitripennis, by data mining of EST libraries.Oliveria et al.  discover2.5.The use of microarrays was proposed and tested for detection and genotyping of pathogens . The DNA3.Next Generation Sequencing is a non-Sanger-based and high-throughput methodology  which al3.1.Viral sequences can be extracted from either total DNA (for DNA viruses only) or RNA isolated from insects . AlternaMethods used for library preparation vary according to the platform used for sequencing. Reagents, kits and methods for preparing libraries can be obtained from the corresponding companies. In general, there are three types of libraries that are most useful in the context of virus discovery: DNA, RNA (including transcriptome) and small RNA libraries. For transcriptome sequencing, mRNA is extracted from total RNA by polyT treatment or by methods for ribosomal RNA depletion  before b3.2.Basic Local Alignment Search Tool) searches .Following detection of viral sequences by NGS technologies, the presence of viral sequences in the sample must be confirmed by PCR (DNA viruses) or RT-PCR (RNA viruses). Real time PCR/RT-PCR can be used to quantify the amount of virus present, and provide validation for the number of observed reads in the NGS datasets. To confirm whether the identified virus replicates in the host insect, RT-PCR for detection of viral transcripts, or negative strand-specific RT-PCR for ssRNA viruses can be performed. The use of tagged primers for enhanced specificity is recommended . Detecti4.Next Generation Sequencing has been widely applied ,48–50 in4.1.Apis mellifera L., conducted to elucidate the causes of colony collapse disorder (CCD) [The first application of NGS technology that demonstrated the potential use of this approach for virus discovery was a metagenomic analysis of the honey bee, er (CCD) ,55. The er (CCD) , have beer (CCD)  against er (CCD) . The preer (CCD) .11 viruses per bee. High frequency sampling along with molecular detection methods including a custom arthropod pathogen microarray, qPCR, and deep sequencing were used for episodic viral detection throughout the year. Total nucleic acids from 20 monitor hives (3 μg nucleic acids per hive) were pooled and three sequencing libraries prepared . The RNA libraries were constructed with various modifications  to optimize the detection of viruses, bacteria, fungi/protists, mites, and nematodes. Sequencing was performed with paired-end 65 nt reads by using an Illumina GAII. To analyze the sequencing data, a database was created that included all of the complete arthropod virus genome sequences available at the time. The entire sequencing dataset was queried against the arthropod virus library by using blastn and tblastx, and hits with a minimum e-value of 1 × 10−3 used for further analysis. Hits were assembled using the Geneious sequence analysis package. Contigs (>250 nt) were queried again against the dataset by tblastx with an e-value ≤ 1 × 10−5. The positive hits were then queried against the nr database with the same parameters to eliminate spurious hits. For the contigs that appeared divergent or that hit non-honey bee associated viruses, extension of the contigs was performed using the entire read dataset with a paired-end contig extension program. From this analysis, it appeared that overall virus incidence was sporadic, although the use of only five bees per sample from each hive and a virus detection limit of 1.9 × 105 virus genome copies may explain the apparent disappearance of some viruses over time. Four new viruses were discovered from the honey bees, including two dicistroviruses , and two viruses for which the complete genome sequence was acquired  approach was used to examine the diversity of DNA viruses present in multiple species of mosquitoes from California, U.S. . In thisThe first insect nidovirus, Cavally virus (CAVV), was discovered following virus isolation from mosquito heads, and amplification in the C6/36 mosquito cell line. The virus was titrated on insect cells and cell culture supernatant used as a source of pure virus . Virions4.3.RNA interference (RNAi) plays a vital role in defense against RNA viruses in a wide range of organisms including insects –72. The The first report of the use of sRNA sequencing for virus identification was for analysis of the sweet potato . In thisDrosophila cell line, and from published sRNA datasets for mosquitoes and nematodes [Drosophila S2-GMR cell line. In addition, two viruses, including one new virus, were identified from the mosquito. However, full length genomes of the viruses could not be assembled from the sRNA datasets. RT-PCR, RACE-PCR and sequencing were used to fill gaps in the viral sequences.A similar strategy was used to identify viruses present in a ematodes . Four vi4.4.The traditional approach for the sequencing of viral genomes involves PCR or RT-PCR amplification of DNA or cDNA fragments, and cloning prior to Sanger sequencing for DNA or RNA viruses respectively. For the large DNA viruses such as baculoviruses which have genomes of 80–180 kb, fragments of genomic DNA are generated using restriction enzymes rather than PCR, prior to cloning and sequencing. NGS now provides an alternative approach for the sequencing of large DNA viruses and was used successfully for sequencing of the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infects tsetse flies, and has been detrimental to laboratory colonies established for use in a sterile male release program for management of tsetse flies, which transmit the agents of both human and animal trypanosomiasis . GpSGHV C. vestalis. Genome sequencing was performed using 454 GS FLX [Cotesia plutellae bracovirus (CpBV) using blastn, and then validated by PCR. The relationship of the remaining contigs was determined by multiplex PCR [NGS was also applied to sequencing of the genome of the polydnavirus, Cotesia vestalis bracovirus (CvBV) . Polydna4 GS FLX . Assemblplex PCR  and the 4.5.We sequenced the soybean aphid transcriptome using Illumina GAII. Total RNA was extracted from aphids using TRIzol reagent (Invitrogen) and transcriptome libraries prepared for single read analysis according to Illumina protocols. Three samples were prepared, one of which had a single polyT purification step, as compared to the others that were subjected to two of these steps. The resulting reads of 75 nt were assembled using Velvet  and ABySAcyrthosiphon pisum), the green peach aphid (Myzus persicae) and the soybean aphid (Aphis glycines). Total RNA was extracted from aphids using TRIzol and the Illumina Small RNA Sample Prep Kit used for production of sRNA libraries. For the small RNA sequencing reads, 3′-adaptors were removed and the reads were then de novo assembled using Velvet [We then used small RNA sequencing using Illumina GAII for detection of RNA viruses in apparently healthy laboratory colonies of the pea aphid  will likely further improve our ability to discover new insect viruses –86.It is evident from this review that there are multiple approaches for identifying novel insect viruses and for assembly of viral genome sequences . DiffereThe examples of novel insect viruses discovered by use of NGS technologies illustrate the ubiquity of viruses in field populations, laboratory colonies of insects and in insect-derived cell lines. These viruses may cause covert or overt infection, or be vectored by the insect to their primary plant or animal hosts. The ubiquity of viruses in laboratory colonies of insects and in cell lines has implications for the use of these tools for analysis of insect-virus interaction. Insects have several anti-viral defense pathways , includiWhile there is increasing interest in gene silencing approaches for insect pest management, with successful demonstration of the approach for beetles , results


To the Editor: We report maternal–fetal transmission of Cryptococcus gattii and death in a wild porpoise. Cryptococcus neoformans and C. gattii are 2 environmental, encapsulated yeasts that cause invasive, potentially life-threatening infections in humans and animals  and nearby surrounding areas  was reported on February 22, 2007, on western Whidbey Island, in Puget Sound, Washington State . The carcass was iced and necropsy was performed on February 24. Sampled tissues from the adult and fetus were divided: half fixed in 10% formalin for histopathologic analysis, and half frozen for ancillary studies.A pregnant, dead, stranded, harbor porpoise (www.cdc.gov/EID/content/17/2/304-appF.htm). The first stomach chamber contained two 3.5 cm × 2.5 cm raised, centrally umbilicated ulcers and several embedded anisakid nematodes. The uterus was gravid in the right horn with a mid-term fetus. No other gross lesions were identified. Microscopically, the lung lesions correlated with granulomatous to pyogranulomatous infiltrates, often with a myriad of yeasts.For histologic analysis, tissues were embedded in paraffin, sectioned to 3–5 μm, and stained with hematoxylin and eosin. Selected sections were stained with mucicarmine. The adult porpoise  was in poor condition (reduced blubber layer). Both lungs were exposed, extensively scavenged, firm, and nodular; a sectioned surface exuded clear to slightly opaque gelatinous to mucinous discharge. Mediastinal lymph nodes were grossly enlarged, multinodular, and firm with large numbers of yeasts visible by microscopy , was examined separately at a different facility than the dam. It appeared grossly normal externally and was at a gestation of ≈5–6 months. Mediastinal lymph nodes had mild granulomatous inflammation and contained numerous yeasts morphologically consistent with cus spp. , panel BYeasts were found in the amniotic fluid and interspersed within the chorioallantoic villi and submucosal vasculature of the placenta. Mild multifocal nonsuppurative myocarditis was detected. However, no yeasts were seen in inflamed areas. There were no overt lesions in the remaining organs.C. gattii and C. neoformans  and fetus . Genotyping of primary isolates identified VGIIa C. gattii in both animals. Test results for enteric pathogens, intestinal nematodes, morbillivirus, and Brucella spp. were negative.Maternal and fetal tissues were cultured for fungi, and diagnosis was based on Gram stain (budding yeast-like cells), India ink stain (positive for encapsulated cells), hydrolysis of urea (positive), and final confirmation by using API 20C Aux V3.0 . Canavanine-glycine-bromthymol blue agar was used to differentiate between Fetal infection was most likely hematogenous, disseminated from a primary maternal pulmonary source to the uterus and subsequently to placental vasculature and internal fetal tissues. Infection by aspiration or ingestion of contaminated amniotic fluid was also possible. Although close evaluation of the lung did not show any discernible yeasts, the organism may have been present in an area other than that sectioned.Phocoenoides dalli) and Pacific white-sided dolphins (Lagenorhynchus obliquidens) along the Pacific Northwest Coast were recovered and subjected to necropsy. Disseminated cryptococcosis caused by C. gattii since 2000 was diagnosed in 15 harbor porpoises, 10 Dall’s porpoises, 2 adult Pacific white-sided dolphins, and 3 unrecorded species .During 1998–2007, ≈450 harbor and Dall’s porpoises (C. gattii at the air–water interface or ingest seawater containing yeasts while feeding (Pseudotsuga menziesii) and Western hemlock (Tsuga heterophylla) may play a role in the epidemiology of C. gattii because these trees have been associated with cases of C. gattii (,Wild porpoises in the Pacific Northwest Region, being near shore inhabitants in waters surrounding Vancouver Island, may come into contact with air containing This fetal case of cryptococcosis may have major human and animal health implications. Further studies should be undertaken to assess possible fetal involvement, identify infections in pregnant females, and provide information on risk reduction and improving diagnosis and treatment.


Concentration and prevalence of coronaviruses and astroviruses increase when bats form maternity roosts and bear young. Myotis myotis bats in the attic of a private house in a suburban neighborhood in Rhineland-Palatinate, Germany, during 2008, 2009, and 2010. One coronavirus, 6 astroviruses, and 1 novel adenovirus were identified and monitored quantitatively. Strong and specific amplification of RNA viruses, but not of DNA viruses, occurred during colony formation and after parturition. The breeding success of the colony was significantly better in 2010 than in 2008, in spite of stronger amplification of coronaviruses and astroviruses in 2010, suggesting that these viruses had little pathogenic influence on bats. However, the general correlation of virus and bat population dynamics suggests that bats control infections similar to other mammals and that they may well experience epidemics of viruses under certain circumstances.Bats host noteworthy viral pathogens, including coronaviruses, astroviruses, and adenoviruses. Knowledge on the ecology of reservoir-borne viruses is critical for preventive approaches against zoonotic epidemics. We studied a maternity colony of  Available studies have focused on lyssaviruses, hennipaviruses, and filoviruses, which have extremely low detection frequencies, thus causing viruses to be encountered too rarely to enable the characterization of virus frequency and concentration over time . The equivalent of ≈100 mg was purified by the Viral RNA kit (QIAGEN) according to manufacturer’s instructions.Permission for this work on protected bats was obtained from the environmental protection authority (Struktur-Und Genehmigungsbehörde Nord Koblenz) of the German federal state of Rhineland-Palatinate. Sampling took place over 3 consecutive years: 2008, 2009, and 2010. The sampling site was the attic of a private house in a suburban area in the state of Rhineland-Palatinate, western Germany . The stuCoronavirinae (Astroviridae (Mastadenovirus (M. myotis bats or the Platinum Taq DNA Polymerase Kit (Invitrogen) for detecting AdVs in M. myotis bats. Reactions were generally composed as follows: 400 nmol/L of the respective primers, 200 nmol/L of the respective hydrolysis probe, 0.5 µL enzyme mix or 0.1 µL Platinum Taq, 1 µg bovine serum albumin, and 5 µL RNA/DNA extract. For AdV DNA PCR, supplements of 0.2 mmol/L of each dNTP and 2.0 mmol/L of MgCL were added. Amplification involved 15 min at 55°C for reverse transcription of RNA viruses and 3 min at 95°C, followed by 45 cycles of 15 seconds at 94°C, and 25 seconds at 58°C for all viruses. Fluorescence was measured at the 58°C annealing/extension step.Five microliters of RNA/DNA eluate were tested by broad range reverse transcription–PCR (RT-PCR) assays for the whole subfamily enovirus . All 4 dFor quantification, PCR amplicons from the initial screening assay were TA cloned in a pCR 4.0 vector (Invitrogen). Plasmids were then purified and reamplified with vector-specific oligonucleotides, followed by in vitro transcription with a T7 promotor-based Megascript kit . The in vitro–transcribed RNAs or, in the case of AdVs, the photometrically quantified plasmid alone, were used as calibration standards for virus quantification in bat fecal samples, as described previously (www.megasoftware.net). Neighbor-joining phylogenies used an amino-acid percentage distance substitution model and 1,000 bootstrap reiterations. All sequences were submitted to GenBank under accession nos. HM368166–HM368175. All analyses were performed with Epi Info 3.5.1 (www.cdc.gov/epiinfo) and with SPSS 17 .Sanger sequencing of PCR products was done by using dye terminator chemistry (Applied Biosystems). Nucleic acid alignments with prototype virus sequences were done based on amino acid code by the BLOSUM algorithm in the MEGA4 software package  that was clearly separated from a clade of AdV recently reported in a M. ricketti bat in China and a Pipistrellus pipistrellus bat in Germany . A second and more significant amplification occurred ≈1 month after parturition, with 100% of collected fecal samples testing positive for CoV RNA (sampling dates 4 and 5). The second peak was characterized by an increase in median RNA concentration by ≈2 orders of magnitude .For all 3 viruses, strain-specific real-time RT-PCR assays, including cloned, in vitro–transcribed RNA or plasmid DNA quantification standards, were generated . For Astuly 2008 ; 195 pooagnitude . Peak co2 7.4, p = 0.006); ANOVA, F = 4.4, p = 0.03). This pattern resembled the amplification after formation of the colony as also observed in CoV. For AstV, no amplification was associated with parturition in the same samples. Total detection rate of astroviruses was 51.2% before birth of the first pup and 40.5% thereafter. However, prevalence and virus concentration significantly increased in the second sampling than in the first and fourth samplings, respectively , this was exclusively contributed by slightly lower virus concentrations in the first sampling than in the succeeding samples , while no significant variation in prevalence or virus concentration was visible for the other viruses (data not shown).Because of the diverging pattern of amplification of the RNA viruses  against the DNA virus (AdV), the investigation was repeated the next year (2009). All viruses were detected again . UnfortuF = 22.0, p<0.001). The detection rate during the first peak was 100.0%, followed by 2-fold and 5-fold decreases 3 and 6 weeks later , and an augmentation to 97.5% after parturition . The maximal CoV concentration in 2010 was higher than in 2008, at 50,495,886,830 RNA copies/g of feces. The amplification pattern of AstV showed clearer similarities to that of CoV in 2010. An initial peak of detection rate was 97.5%, followed by a detection rate of 22.2%–22.4% in subsequent samples and 97.5%–100% after parturition . Virus concentrations were significantly increased in these amplification peaks . The amplification was almost completely contributed by one of the AstV lineages (represented by BtAstV/N58–49), while the other lineages were constant .A repetition of the full sampling scheme was attempted again in 2010. All 5 sampling dates could be completed, yielding a sample of 187 pools in total, equivalent to 935 individual fecal pellets. As shown in constant , panel Bconstant , panel B2 18.3, p<0.001).CoV, AstV, and AdV are clearly pathogenic for other mammals. To determine whether the presence of these viruses had any influence on bats’ health, the reproductive success of the maternity colony was evaluated in 2008 and 2010. The data are summarized in Viral host switching is probably determined by the chances of interspecies contact, as well as by the concentration and prevalence of virus in the donor species. To judge zoonotic risks associated with bats, when and where these 2 variables would favor transmission must be determined. In this study, we found that strong and specific amplification of the RNA viruses, but not of the DNA virus, occurred upon colony formation and following parturition.,Pteropus spp. bats depended on seasonal factors, which was interpreted as evidence for season-dependent variation of virus concentration or prevalence , filoviruses (Ebola and Marburg viruses) and henipaviruses (Hendra and Nipah viruses). However, because these viruses are found rarely, only vague conclusions have so far been made. For instance, increased contact between bats and humans through bat migratory events or fruit harvesting periods have been temporally linked with individual human cases of Ebola and Nipah virus infection (,A common, but unproven, assumption is that bats are resistant to even highly pathogenic viruses (,Another intriguing finding of our study was the difference in the amplification pattern of the RNA viruses and that of the DNA virus. We selected these viruses because, in humans, AdVs are typically capable of persisting in tissue (9–1010 copies/g were observed, which is tremendously higher than CoV concentrations observed in earlier studies outside the parturition period (,,,For CoV, our study indicates clearly that virus amplification takes place in maternity colonies, confirming our earlier statistical implications from studies in a different region and on a different species (