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Background: Kadipiro virus (KDV) is a species of the new 12 segmented RNA virus grouped under the genus Seadornavirus within the Reoviridae family. It has previously been isolated or detected from mosquito , Odonata , and bat feces in Indonesia, China, and Denmark, respectively. Here, we describe the isolation and characterization of a viral strain from mosquitoes in Yunnan Province, China. Methods: Mosquitoes were collected overnight using light traps in Shizong county, on July 17, 2023. Virus was isolated from the mosquito homogenate and grown using baby hamster kidney and Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by agarose gel electrophoresis (AGE). The full-genome sequences of the strain were determined by full-length amplification of cDNAs and sequenced using next-generation sequencing. Results: We isolated a viral strain (SZ_M48) from mosquitoes ( Culex tritaeniorhynchus Giles ) that caused cytopathogenic effects in C6/36 cells. AGE analysis indicated a genome consisting of 12 segments of double-stranded RNA that demonstrated a "6-5-1" pattern, similar to the migrating bands of KDV. Phylogenetic analysis based on the full-genome sequence revealed that SZ_M48 is more clustered with KDV isolates from Hubei and Shangdong in China than with Indonesian and Danish strains. The identity between SZ_M48 and SDKL1625 (Shandong, China) is slightly lower than that of QTM27331 (Hubei, China), and the identity with JKT-7075 (Indonesia) and 21164-6/M.dau/DK (Denmark) is the lowest. Conclusion: The full-genome sequence of the new KDV strain described in this study may be useful for surveillance of the evolutionary characteristics of KDVs. Moreover, these findings extend the knowledge about the genomic diversity, potential vectors, and the distribution of KDVs in China.

Reovirus designated as Mud crab reovirus (MCRV) is associated with the mass mortalities of mud crabs resulting in significant economic loss to crab and shrimp-mud crab polyculture farmers in the Nagayalanka, Krishna district, Andhra Pradesh. The 100 % chronic mass mortalities have been attributed to the outbreak of Mud crab reovirus (MCRV) in the polyculture farms. The moribund crabs showed autotomy, discoloration of carapace, loss of appetite, slow movement and loose gills. Histopathological observations of the infected mud crabs showed an atrophied hepatopancreas, complete degeneration of tissues along with viral inclusions in hepatopancreas, gills and muscles. Further analysis using Transmission electron microscopy (TEM), showed that the viral particles had a diameter of 70 nm and exhibited a non-enveloped, icosahedral shape arranged in a crystalline manner. The virus mainly infects the connective tissue of hepatopancreas, gills, muscle and develops in the cytoplasm. RT-PCR reconfirmed the presence of reovirus in the hepatopancreas of spontaneously infected mud crab Scylla serrata. The current study shows the importance of monitoring the MCRV prevalence in polyculture farms to minimize its spread and precautionary measures can be taken by screening the brooders from the crab hatchery and stocking of wild crabs without screening should be avoided in order to prevent MCRV outbreak., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Channel catfish (Ictalurus punctatus) are a food fish extensively reared in aquaculture facilities throughout the world and are also among the most abundant wild catfish species in North America, making them a popular target of anglers. Furthermore, channel catfish are important members of aquatic ecosystems; for example, they serve as a glochidial host for the endangered winged mapleleaf mussel (Quadrula fragosa), making them critical for conserving this species through hatchery-based restoration efforts. During a routine health inspection, a novel aquareovirus was isolated from channel catfish used in mussel propagation efforts at a fish hatchery in Wisconsin. This virus was isolated on brown bullhead cells (ATCC CCL-59) and identified through metagenomic sequencing as a novel member of the family Spinareoviridae, genus Aquareovirus. The virus genome consists of 11 segments, as is typical of the aquareoviruses, with phylogenetic relationships based on RNA-dependent RNA polymerase and major outer capsid protein amino acid sequences showing it to be most closely related to golden shiner virus (aquareovirus C) and aquareovirus C/American grass carp reovirus (aquareovirus G) respectively. The potential of the new virus, which we name genictpun virus 1 (GNIPV-1), to cause disease in channel catfish or other species remains unknown., (© 2024 John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Emerging infectious diseases are a major challenge to human and animal health. While predicting the emergence of pathogens is complex, the advent of high-throughput sequencing technologies has allowed the rapid identification of unknown microbiology diversity within organisms. Here, we discuss an example of a metatranscriptomics output to decipher viral evolution., Competing Interests: The authors declare no conflict of interest.

Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21-99.98) and specificity (100%, 95% CI 96.31-100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation., (© 2021. The Author(s).)

We isolated a novel Aquareovirus (hirame aquareovirus: HAqRV) from Japanese flounder Paralichthys olivaceus suffering from reovirus-like infection. In electron microscopy, the spherical virion (75 nm in diameter) was observed with multi-layered capsid structure. The viral genome consisted of 11 segments and regions encoding 7 virion structural proteins and 5 non-structural proteins were predicted. The deduced amino acid sequences of those proteins were highly similar to those of the aquareoviruses. However, the similarity of complete genome sequence between the HAqRV and other aquareoviruses was less than 60%. Phylogenetic analyses based on the deduced amino acid sequences suggested that the HAqRV is not classified into the known species of Aquareovirus. Pathogenicity of HAqRV was clearly demonstrated in accordance with Koch's postulates by experimental infection using Japanese flounder. The results suggest that the HAqRV is a new Aquareovirus species which is highly virulent for the Japanese flounder at early life stages., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The hemorrhagic disease of grass carp (HDGC) caused by grass carp reovirus (GCRV) still poses a great threat to the grass carp industry. Isolation and identification of the GCRV genotype I (GCRV-I) has been rarely reported in the past decade. In this study, a new GCRV was isolated from diseased fish with severe symptoms of enteritis and mild hemorrhages on the body surface. The isolate was further identified by cell culture, transmission electron, indirect immunofluorescence, and SDS-PAGE electrophoretic pattern analysis of genomic RNA. The results were consistent with the new isolate as a GCRV-I member and tentatively named GCRV-GZ1208. Both grass carp and rare minnow infected by the GCRV-GZ1208 have no obvious hemorrhagic symptoms, and the final mortality rate was ≤10%, indicating that it may be a low virulent isolate. GZ1208 possessed highest genomic homology to 873/GCHV (GCRV-I) and golden shiner reovirus (GSRV). Additionally, it was found a 90.7-98.3% nucleotide identity, a 96.4-100% amino acid identity, and <50% identity with GCRV-II and III genotypes. Interestingly, the sequences of some segments of GZ1208 were similar to GCRV-8733/GCHV, whereas the remaining segments were more closely related to GSRV, suggesting that a recombination event had occurred. Bootscan analysis of the complete genomic sequence confirmed this hypothesis, and recombination events between 873/GCHV and other GSRV-like viruses were also accompanied by gene mutations.

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57-96.71% protein sequence identity) but shared only 22.65-45.85% and 23.37-43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus -C ( AqRV -C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 10 4 copies/μL.

Objective: To investigate the influence of oncolytic reovirus on the biological activities of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) as a novel virotherapy strategy., Materials and Methods: The Cell Counting Kit-8 assay was used to detect the viability of hUC-MSCs infected with different multiplicities of infection (MOIs) of reoviruses. The biological activities (proliferation, marker expression, multipotency, and migration) of hUC-MSCs were verified by assaying osteogenic and adipogenic differentiation potential, flow cytometry, and electrical cell-substrate impedance sensing, respectively., Results: The viability of hUC-MSCs slightly decreased by infection with low titers of reoviruses. A MOI of 1 had no effect on the viability of hUC-MSCs within 96 h. The biological activities (proliferation, marker expression, multipotency, and migration) of hUC-MSCs were not affected by reovirus infection at a MOI of 1., Conclusions: Reovirus at a MOI of 1 had no effect on the biological activities of hUC-MSCs.

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of Muscovy duck reovirus (MDRV) RNA in clinical samples is described. The assay is based on TaqMan-MGB technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the sigma B-protein gene of MDRV. This technique also includes an Internal Positive Control (IPC). The real-time RT-PCR assay was able to detect MDRVs, whereas other common waterfowl-origin viral pathogens were not recognised by the established oligonucleotide set, thus showing that the test was specific for MDRV. The sensitivity of the assay was 2.83 × 10 1 copies/μL and was 100 times higher than that of the conventional RT-PCR. The variation coefficients of intra-assay and inter-assay were less than 1.5% which verified sufficient repeatability of this assay. The use of β-actin mRNA as an IPC in order not to reduce the efficiency of the assay was adopted. The detection for 100 clinical samples showed that the positive rate of the established TaqMan-MGB real-time RT-PCR method was 87% (87/100), while the positive rate of the conventional RT-PCR was 83% (83/100), with the coincidence rate was 97.14%. Sensitivity and positive rate for clinical samples of TaqMan fluorescent quantitative RT-PCR were higher than conventional RT-PCR. The high specificity, sensitivity, and rapidity TaqMan-MGB real-time RT-PCR assay with the use of IPC to monitor for false negative results can make this method suitable for the pathogenic surveillance and epidemiological investigation of MDRV infection., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Ltd.)

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important insect vector of Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing, which is the most destructive disease of citrus worldwide. Sequences for putative Diaphorina citri reovirus (DcRV) were identified from some worldwide populations of D. citri. Here, field surveys indicated that the virus was common in D. citri populations from Hawaii and Fuzhou of PR China. Electron microscopy showed that DcRV virions possessed a typical reovirus-like morphology. The U. S. and Chinese DcRV isolates both showed 10 segments of double-stranded RNA sharing >96% nucleotide sequence identity, and encoding 11 deduced proteins. All genome segments contained conserved 5' and 3' terminal nucleotide sequences and inverted repeats that are hallmarks of reovirus sequence. Phylogenetic analysis showed that DcRV may be considered a new species of the genus Fijivirus sharing a most recent common ancestor with the insect-specific fijivirus Nilaparvata lugens reovirus., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A cypovirus was isolated from larvae of the Japanese peppered moth, Biston robustus. The viral genome is 23,954 bp comprising 10 segmented double-stranded RNAs with a new electropherotype among cypoviruses. Each segment encodes one putative protein and has non-coding regions that contain conserved sequences at their 5' and 3' termini, 5'-AGAA(U/A)U-3' and 5'-UGC-3', respectively. Seven proteins encoded in the genome are homologous to those of other cypoviruses at a cut-off E-value of 1 × 10 -5 . The maximal sequence identities of these proteins with cypovirus homologs are 24.30%-39.40%. These results indicate that the virus isolated is a novel cypovirus; herein designated as Biston robustus cypovirus 24 (BrCPV-24). This newly isolated BrCPV-24 infects the larvae of the silkworm Bombyx mori., Competing Interests: Declaration of Competing Interest The author declares no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Grass carp (Ctenopharyngodon idella) hemorrhagic disease, which is characterized by external and internal hemorrhage, is a serious infectious disease affecting grass carp production. Strains of the causative agent, grass carp reovirus (GCRV), are divided into genotypes I, II and III, which are represented by the isolates GCRV-873, GCRV-HZ08 and GCRV-104, respectively. In this study, a real-time reverse-transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed to detect the genotype III grass carp reovirus GCRV-104. The assay was based on the detection of the vp55 gene which encodes the outer fiber protein of the virus. A portable ESE-Quant Tube scanner, with a dimension of 17.4 × 18.8 cm, weighing about 1 kg, and equipped with temperature settings to amplify the DNA isothermally and spectral devices to detect the amplified products using fluorescence, was used to complete the assay. Under the optimal conditions, the assay took approximately 10 min to complete at 37 °C and showed no cross-reactions with other aquatic viruses. Consequently, this rapid real-time RT-RPA assay is a useful method for the simple, rapid and reliable detection of genotype III GCRV strains in resource-limited diagnostic laboratories., Competing Interests: Declaration of Competing Interest We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) is the most important disease for grass carp aquaculture. Its typical clinical symptom is haemorrhaging, although the mechanism was remained unclear. In this study, we investigated the differences in blood parameters and histopathological features between grass carp infected with a virulent and avirulent isolates of genotype II GCRV. Infection with the virulent isolate resulted in increases in 8 routine blood and 2 serum biochemical parameters (P < 0.05); while 9 routine blood and 5 biochemical parameters were significantly decreased (P < 0.05) compared with fish infected with the avirulent isolate. The majority of these alterations were related to hemorrhage, inflammatory reactions and organic damage. The histopathologic changes were primarily vasodilation and hyperaemia in multiple organs, lymphocyte and macrophage infiltration as well as severe vacuolar degeneration in spleen, kidney and liver. The histopathology changes in fish infected with the avirulent isolate were minimal. These results indicated that the pathogenicity of GCRV was primarily reflected in destruction of the blood circulatory system and parenchymatous organs. This study lays the foundation for further research on the pathogenesis of bleeding caused by GCRV infection and the use of blood parameters and histopathology as tools for disease diagnosis., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Turkey arthritis reovirus (TARV) causes tenosynovitis in turkeys, resulting in decreased profits for producers due to the increase in morbidity, mortality, and feed conversion ratio. There is limited information on TARV epidemiology, including the dynamics of diagnostic submissions to veterinary diagnostic laboratories. In this study, we retrospectively analyzed 719 cases of lameness in turkeys submitted to the Minnesota Veterinary Diagnostic Laboratory from March 2010 to May 2018. Almost all submissions were tendon pools, which were tested by virus isolation and/or real-time reverse transcription-polymerase chain reaction. Most of the submissions were from Minnesota. We found 52% of the submitted cases to be positive for TARV. The TARV-positive submissions increased considerably in the last few years. There was no statistical evidence that TARV diagnostic submissions were seasonal, although positive submissions were higher in January, April, July, and December. TARV-positive submissions also increased as flocks aged. In summary, we found that TARV submissions have increased in the last few years, have varied over time, and are correlated with age of the bird. This information is important guidance for conducting more studies to understand TARV infection dynamics.

Retrospective analysis of pigeon necropsy submissions to the California Animal Health and Food Safety Laboratory System from 2000 to 2018 revealed 14 submissions diagnosed with rotavirus A hepatic necrosis or "reoviruslike" viral hepatitis. Nine of the 14 submissions (64%) occurred in 2018. Submissions were racing pigeons and squab breeders from flocks with increased mortality. Juvenile and adult pigeons were submitted with a history of depression, diarrhea, regurgitation, labored breathing, and weakness. Flock morbidity peaked at 80% and mortality at 28%. The most consistent findings on postmortem examination were variably congested, mottled, and enlarged livers and spleens. Microscopically, mild to severe hepatic necrosis was observed with variable bile duct hyperplasia, sinusoidal congestion, hemosiderosis, and portal lymphoplasmacytic inflammation. Rotavirus A was detected in hepatocytes and inflammatory cells by immunohistochemistry. Negative-stain electron microscopy identified viral particles consistent with a member of Reoviridae in all negatively stained liver homogenates. Eleven cases were analyzed by reverse transcriptase-PCR targeting rotavirus A viral protein (VP) 6 and VP7 genes. Subsequent phylogenetic analysis of the VP6 and VP7 sequences compared to published Chinese, Nigerian, and German rotavirus A VP6 and VP7 sequences demonstrated the formation of two and three distinct clades, respectively. To the authors' knowledge, rotavirus A hepatic necrosis in pigeons has not been previously reported in the United States and represents a significant emerging disease for the pigeon industry due to the potential for high flock mortality and lost production.

Southern rice black-streaked dwarf virus (SRBSDV), which causes severe disease symptoms in rice ( Oriza sativa L.) has been emerging in the last decade throughout northern Vietnam, southern Japan and southern, central and eastern China. Here we attempt to quantify the prevalence of SRBSDV in the Honghe Hani rice terraces system (HHRTS)-a Chinese 1300-year-old traditional rice production system. We first confirm that genetically diverse rice varieties are still being cultivated in the HHRTS and categorize these varieties into three main genetic clusters, including the modern hybrid varieties group (MH), the Hongyang improved modern variety group (HY) and the traditional indica landraces group (TIL). We also show over a 2-year period that SRBSDV remains prevalent in the HHRTS (20.1% prevalence) and that both the TIL (17.9% prevalence) and the MH varieties (5.1% prevalence) were less affected by SRBSDV than were the HY varieties (30.2% prevalence). Collectively we suggest that SRBSDV isolates are freely moving within the HHRTS and that TIL, HY and MH rice genetic clusters are not being preferentially infected by particular SRBSDV lineages. Given that SRBSDV can cause 30-50% rice yield losses, our study emphasizes both the need to better monitor the disease in the HHRTS, and the need to start considering ways to reduce its burden on rice production.

Marbled eel reovirus (MERV) is an aquareovirus (AQRV) isolated from diseased marbled eels (Anguilla marmorata) with petechial skin hemorrhage. In this study, we propagated MERV in a cell line derived from the brain of Aequidens rivulatus and purified viral particles by using a discontinuous cesium chloride gradient. Genomic RNA sequences were obtained through next-generation sequencing. MERV, similar to most other AQRVs, showed the presence of 11 double-stranded RNA segments encoding 12 proteins; however, the genome sequence displayed very little similarity to known AQRV sequences. Furthermore, the structural proteins of MERV were most closely related to American grass carp reovirus with sequence identity values of no more than 64.89%. Phylogenetic analysis based on the sequences of structural proteins indicated that MERV shows an evolutionary history between AQRV-B and -G, which belong to the saline and freshwater environment subgroups, respectively. We also observed that MERV showed a closer relationship to orthoreoviruses based on the protein sequences of NS38 and NS73. In summary, MERV is a novel AQRV that could be classified as a member of the new proposed AQRV species "Aquareovirus H". The taxonomic assignments and evolution of AQRVs thus warrant further investigation.

The collapse of iconic, keystone populations of sockeye ( Oncorhynchus nerka ) and Chinook ( Oncorhynchus tshawytscha ) salmon in the Northeast Pacific is of great concern. It is thought that infectious disease may contribute to declines, but little is known about viruses endemic to Pacific salmon. Metatranscriptomic sequencing and surveillance of dead and moribund cultured Chinook salmon revealed a novel arenavirus, reovirus and nidovirus. Sequencing revealed two different arenavirus variants which each infect wild Chinook and sockeye salmon. In situ hybridisation localised arenavirus mostly to blood cells. Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture., Competing Interests: GM, KM, ED, AS, KK, TM, SL, AT, AT, DP, HF, CS No competing interests declared, (© 2019, Mordecai et al.)

Diaphorina citri reovirus (DcRV) was previously identified based on metagenomics surveys for virus discovery. Here, we demonstrated that DcRV induces persistent infection in its psyllid host, Diaphorina citri DcRV was efficiently vertically passed to offspring in a biparental manner. Transmission electron microscopic and immunological analyses showed that the DcRV-encoded nonstructural protein P10 assembled into a virion-packaging tubular structure which is associated with the spread of DcRV throughout the bodies of D. citri insects. P10 tubules containing virions were associated with oocytes of female and sperm of male D. citri insects, suggesting a role in the highly efficient biparental transmission of DcRV. Knocking down P10 by RNA interference for males reduced the percentage of DcRV-infected progeny and for females reduced the viral accumulation in progeny. These results, for the first time, show that a nonstructural protein of a novel insect reovirus provides a safe and pivotal channel for virus spread and biparental transmission to progeny. IMPORTANCE The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important pest in the worldwide citrus industry. It is the vector of " Candidatus Liberibacter asiaticus," the bacterial pathogen of Huanglongbing, which is currently considered the most destructive disease of citrus worldwide. DcRV was previously identified based on metagenomics surveys for virus discovery. Here, we found that this novel and persistent insect reovirus took advantage of a virus-encoded nonstructural protein, P10, for efficient vertical transmission from parents to progeny. P10 assembled into a virion-packaging tubular structure and was associated with oocytes of female D. citri and sperm of males. Consistent with this, knockdown of P10 for either male or female D. citri insects inhibited DcRV transmission to offspring. This tubular strategy for viral spread and biparental transmission might serve as a target for controlling viral vertical transmission and population expansion., (Copyright © 2019 Chen et al.)

Acute infectious gastroenteritis cases in Shanghai, reported over three years, were analyzed. Pathogens were identified in 1031 patients; of these, 725 and 306 were bacterial and viral cases, respectively. Vibrio parahemolyticus and Salmonella were the dominant bacteria, and Caliciviridae and Reoviridae were the dominant viral families in the local area. The acute gastroenteritis epidemic peaks appeared in August and January, which represented the active peak periods of bacteria and viruses, respectively. Logistic regression analyses with sex stratification showed that abdominal pain, fever and ingestion of unsafe food at restaurants were independent factors more frequently associated with bacterial gastroenteritis irrespective of sex; red cell-positive fecal matter was associated with bacterial gastroenteritis with an odds ratio (OR) of 3.28 only in males; and white blood cell count was associated with bacterial gastroenteritis with an OR of 1.02 only in females. Pathogen stratification showed that age, vomiting and red cell-positive fecal matter were associated with males with ORs of 0.99, 0.61 and 1.71, respectively, in bacterial gastroenteritis; and the migrant ratio was higher in males with an OR of 2.29 only in viral gastroenteritis. In conclusion, although bacterial and viral gastroenteritis shared many features, epidemiological and clinical factors differed between sexes and pathogens.

Field-collected US strain C18 of Cryphonectria parasitica, the chestnut blight fungus, was earlier reported to be infected by a double-stranded RNA virus, mycoreovirus 2 (MyRV2). Next-generation sequencing has revealed co-infection of C18 by a positive-strand RNA virus, hypovirus 4 (CHV4). The current molecular and genetic analyses showed interesting commensal interactions between the two viruses. CHV4 facilitated the stable infection and enhanced vertical transmission of MyRV2, which was readily lost during subculturing and showed reduced vertical transmission in single infections. Deletion of a key antiviral RNA silencing gene, dcl2, in isolate C18 increased stability of MyRV2 in single infections. The ability of CHV4 to facilitate stable infection with MyRV2 appears to be associated with the inhibitory effect of CHV4 on RNA silencing via compromising the induction of transcriptional up-regulation of dcl2. These results suggest that natural infection of isolate C18 by MyRV2 in the field was facilitated by CHV4 co-infection., (Copyright © 2019. Published by Elsevier Inc.)

In 2011, ticks were collected from livestock following an outbreak of Crimean Congo hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV), respectively. Traditional reverse transcription-PCR (RT-PCR) identification of known viruses was unsuccessful, but a next-generation sequencing (NGS) approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus, and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identities, KUNDV is proposed to be a new species of Coltivirus Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other orbi- and coltiviruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis. IMPORTANCE Ticks and mosquitoes, as well Culicoides , can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammavanpettai virus (KVPTV), and, with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum , which is a known vector for zoonotic pathogens, such as Crimean Congo hemorrhagic fever virus, Babesia , Theileria , and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock., (Copyright © 2019 American Society for Microbiology.)

The oleander hawk moth, Daphnis nerii, is a serious pest of plants belonging to the family Apocynaceae. Thus far, pathogen infection has not been reported in D. nerii. In this study, a new cytoplasmic polyhedrosis virus (cypovirus; CPV) was isolated from naturally diseased D. nerii larvae and named DnCPV-23. Virions were observed in ultrathin sections of DnCPV polyhedral bodies. Electrophoretic analysis revealed that the DnCPV genome consisted of 10 segments of double-stranded RNA (dsRNA). cDNA copies of these dsRNA segments were amplified using the method of full-length amplification of cDNAs (FLAC), cloned, and sequenced. Sequencing results showed that all segments contained one open reading frame (ORF); They shared the conserved terminal sequences AGUCAAA and AGC at 5' and 3' ends respectively, except segment 4, which is different from previously reported 22 cypoviruses. Phylogenetic analysis based on amino acid sequences of polyhedrin (encoded by segment 10) indicated that this CPV was closely related to CPV type 19. Altogether, DnCPV-23 is a new type of cypovirus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aquareovirus is a genus of viruses in family Reoviridae, subfamily Spinareovirinae, members of which infect fish, shellfish and crustaceans. Grass carp reovirus (GCRV), a genotype 1 reovirus isolated from grass carp, has served as a model strain for investigating aquareovirus-host interactions. Herein, we report a neglected open reading frame (ORF), tentatively named NS12, residing between NS16 and NS31 in segment 7 (S7) of the virus genome. With an additional reading frame, the nucleotide sequence of NS12 partially overlaps with the 3' expressible nucleotide sequence of NS16. NS12 is not a pseudogene during virus replication, as confirmed in fish cells infected with GCRV and based on amino acid sequence analysis and protein expression pattern. Bioinformatics analysis indicated that NS12 is a transmembrane protein, which was confirmed by its exclusive presence in the membrane-associated fraction of the cell lysate. However, unlike fusion protein NS16, NS12 alone could not induce visible syncytium formation in fish cells. Thus, NS12 is functionally distinct from known aquareovirus membrane-associated protein NS16. NS12-like ORFs (with an AUG or non-AUG initiator codon) are also present in the S7 segment of other aquareoviruses, suggesting that NS12 homologues may be widely distributed in the genus Aquareovirus.

Mammalian reovirus (MRV) infects many species. Over the past decades, MRV infections in pigs have been reported, and several highly pathogenic MRV strains have recently been isolated in the United States. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) against the σ1 protein from a serotype 3 reovirus strain (MPC/04) was established to detect antibodies in pigs. The assay did not react with antisera against other pig pathogens and was consistent with the indirect immunofluorescence assay (IFA) and virus neutralization test (VNT). In conclusion, the assay is specific and highly sensitive, providing a method for large-scale monitoring of the serotype 3 MRV infection epidemiology in pigs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Indigenous small cyprinid fish species play an important role in Great Lakes ecosystems and also comprise the backbone of a multimillion-dollar baitfish industry. Due to their widespread use in sport fisheries of the Laurentian Great Lakes, there are increasing concerns that baitfish may introduce or disseminate fish pathogens. In this study, we evaluated whether baitfish purchased from 78 randomly selected retail bait dealers in Michigan harbored fish viruses. Between September 2015 and June 2016, 5,400 baitfish divided into 90 lots of 60 fish were purchased. Fish were tested for the presence of viral hemorrhagic septicemia virus (VHSV), spring viremia of carp virus (SVCV), golden shiner reovirus (GSRV), fathead minnow nidovirus (FHMNV), fathead minnow picornavirus (FHMPV), and white sucker bunyavirus (WSBV). Using the epithelioma papulosum cyprini cell line and molecular confirmation, we demonstrated the presence of viruses in 18 of the 90 fish lots (20.0%) analyzed. The most prevalent virus was FHMNV, being detected in 6 of 30 lots of Fathead Minnow Pimephales promelas and 3 of 42 lots of Emerald Shiners Notropis atherinoides. We also confirmed GSRV in two fish species: the Golden Shiner Notemigonus crysoleucas (5 of 11 lots) and Fathead Minnow (3 of 30 lots). Two VHSV (genotype IVb) isolates were recovered from a single lot of Emerald Shiners. No SVCV, FHMPV, or WSBV was detected in any of the fish examined. Some of the infected fish exhibited clinical signs and histopathological alterations. This study demonstrates that live baitfish are a potential vector for the spread of viral pathogens and underscores the importance of fish health certifications for the Great Lakes baitfish industry., (© 2018 American Fisheries Society.)

Currently, serological assays for grass carp reovirus genotype II (GCRV-II) diagnosis are not available. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against GCRV-II was developed. The structural protein VP38 of GCRV-II was used as the coating antigen. Monoclonal antibodies (mAb) against IgM of grass carp labelled with HRP were used as a secondary antibody. The antigen concentration and serum dilution were optimized using chess board titration. Furthermore, the specificity of indirect ELISA assay was confirmed by cross check with sera positive for other grass carp pathogens. In comparison with results obtained from indirect immunofluorescence assay (IFA) and Western blot by testing of 60 serum samples to evaluate the sensitivity and specificity of the ELISA, agreement between 90% and 96.7% was reached, respectively. A serological survey was performed using the assay with grass carp field serum samples. The seropositive rate of the 242 serum samples was 69.8%. In conclusion, the developed indirect ELISA is a very specific and sensitive test that will be useful for large-scale serological surveys to detect indirectly GCRV II infections as well as to monitor the changes of antibody level after immunization., (© 2018 John Wiley & Sons Ltd.)

The fountain darter Etheostoma fonticola (FOD) is a federally endangered fish listed under the US Endangered Species Act. Here, we identified and characterized a novel aquareovirus isolated from wild fountain darters inhabiting the San Marcos River. This virus was propagated in Chinook salmon embryo (CHSE)-214, rainbow trout gonad-2 and fathead minnow cells at 15°C. The epithelioma papulosum cyprini cell line was refractory at all temperatures evaluated. High throughput sequencing technologies facilitated the complete genome sequencing of this virus utilizing ribosomal RNA-depleted RNA extracted from infected CHSE-214 cells. Conventional PCR primer sets were developed for the detection and confirmation of this virus to assist diagnostic screening methods. Phylogenetic analysis suggests this virus belongs to the Aquareovirus A genus. This research provides requisite initial data critical to support hatchery and refugia biosecurity measures for this endangered species.

The eucalyptus brown looper, Thyrinteina arnobia (Stoll, 1782) (Lepidoptera: Geometridae), is the main lepidopteran defoliator of eucalyptus plantations in Brazil. Outbreaks of this insect pest are common in Brazil and can affect the productivity of planted forests severely. T. arnobia caterpillars from a laboratory colony with viral infection symptoms were analyzed by electron microscopy that revealed polyhedral occlusion bodies (OBs) with several icosahedral virus particles embedded. Analysis of its genetic material showed ten segments of dsRNA, which confirmed this virus as a possible member of the genus Cypovirus. Phylogenetic analysis of the whole genome sequence revealed its close relationship with other isolates of Cypovirus 14 species and according to these results we proposed the name Thyrinteina arnobia cypovirus 14 (TharCPV-14) for this new virus isolate. Further research will be necessary in order to analyze the potential of this virus as a biopesticide., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Understanding the ecology of diseases is important to understanding variability in abundance, and therefore management, of marine animals exploited commercially. The blue crab Callinectes sapidus fills a crucial benthic-pelagic niche in Atlantic estuarine ecosystems and supports large commercial fisheries in both North and South America. In the USA, pre-molt blue crabs are typically held in short-term shedding (ecdysis) facilities to produce soft-shell crabs of increased value. However, mortality rates in these facilities are high and commonly associated with the pathogenic C. sapidus reovirus 1 (CsRV1). To assess whether crab mortalities in these facilities might increase CsRV1 prevalence in wild crab populations, tissue sampled from crabs collected over 2 summers either near to or far from shedding facilities using flow-through water systems were tested by reverse transcription quantitative PCR (RT-qPCR) for the presence of CsRV1 RNA. In support of our hypothesis, PCR data identified the probability of detecting CsRV1 in wild crabs sampled close to shedding facilities to be 78% higher than in crabs sampled from far sites. PCR detections were also 61-72% more probable in male crabs and 21% more likely in male and female crabs over the minimum landing size. As the prevalence at which CsRV1 was detected varied within seasons, among locations and between years, blue crab migration and/or population fluctuations appear to also be involved.

Southern rice black-streaked dwarf virus (SRBSDV) is a devastating newly emerged rice reovirus in Eastern and Southeastern Asia transmitted by a long-distance migratory pest, the white-backed planthopper (WBPH). We previously showed that SRBSDV infection decreased the cold tolerance but improved the heat tolerance of its vector, WBPH. Comparative metabolomic analysis was used to explore the potential mechanisms underlying these changes in temperature stress response. Fourth-generation WBPH nymphs were treated with SRBSDV and/or extreme temperature stress and were analyzed using gas chromatography-time of flight-mass spectrometry. A total of 605 distinguishable peaks were identified and 165, 207, and 202 differentially accumulated metabolites were identified in WBPH after virus infection, cold, or heat stress, respectively. The nucleic acids and fatty acids were the major categories of metabolites regulated by SRBSDV infection, whereas temperature stress regulated tricarboxylic acid cycle compounds, sugars, and polyols. For the WBPH samples infected with SRBSDV and subjected to temperature stress, amino acids, sugars, and polyols were the most significant regulated metabolites. The metabolomics study suggests that SRBSDV may influence the extreme temperature tolerance of WBPH by regulating the accumulation of amino acids, sugars, and polyols in the insect body.

Liao ning virus (LNV) was first isolated in 1996 from mosquitoes in China, and has been shown to replicate in selected mammalian cell lines and to cause lethal haemorrhagic disease in experimentally infected mice. The first detection of LNV in Australia was by deep sequencing of mosquito homogenates. We subsequently isolated LNV from mosquitoes of four genera (Culex, Anopheles, Mansonia and Aedes) in New South Wales, Northern Territory, Queensland and Western Australia; the earliest of these Australian isolates were obtained from mosquitoes collected in 1988, predating the first Chinese isolates. Genetic analysis revealed that the Australian LNV isolates formed two new genotypes: one including isolates from eastern and northern Australia, and the second comprising isolates from the south-western corner of the continent. In contrast to findings reported for the Chinese LNV isolates, the Australian LNV isolates did not replicate in vertebrate cells in vitro or in vivo, or produce signs of disease in wild-type or immunodeficient mice. A panel of human and animal sera collected from regions where the virus was found in high prevalence also showed no evidence of LNV-specific antibodies. Furthermore, high rates of virus detection in progeny reared from infected adult female mosquitoes, coupled with visualization of the virus within the ovarian follicles by immunohistochemistry, suggest that LNV is transmitted transovarially. Thus, despite relatively minor genomic differences between Chinese and Australian LNV strains, the latter display a characteristic insect-specific phenotype.

Grass carp reovirus (GCRV) caused severe hemorrhagic disease with significant losses of fingerling and yearling grass carp, Cyenopharyngodon idellus, in southeast Asian. It was first isolated in 1983 in China, and clade analysis of the different GCRV isolates indicates there are at least three different genotypes I, II, and III. In recent years, GCRV genotype II has been determined as a dominant virus type which cause severe obvious clinical signs in fish but no cytopathic effect onto presently available cell culture. TCID 50 is one of standard method to quantity infectious virus particles. In the present study, an indirect immunofluorescence assay (IFA) was developed using antibody against a protein encoded by segment 10 of GCRV genotype II. Moreover, the specific assay to differentitate GCRV of different genotypes and a sensitive assay for determination of GCRV genotype II were developed respectively. The results showed the IFA only can recognize genotype II virus at the lowest initial concentration of 550 genomic copies/ml. Furthermore, comparison of results obtained from qPCR and the TCID 50 assay combined IFA was conducted. The results indicated that TCID 50 of GCRV isolates JX0901 and HZ08 differs with 2 log steps reduction in the numbers of viruses compared with the number of genome copies detected by qPCR. The immunofluorescence assay developed is sensitive, specific, and the TCID 50 combined with IFA will be a standardizable technique for the quantitation and detection of infectious GCRV in cell culture without cytolysis., (Copyright © 2017. Published by Elsevier Ltd.)

Eleven viral isolates derived mostly in albopictus C6/36 cells from mosquito pools collected in Southeast Asia and the Americas between 1966 and 2014 contained particles with electron microscopy morphology typical of reoviruses. Metagenomics analysis yielded the near complete genomes of three novel reoviruses, Big Cypress orbivirus, Ninarumi virus, and High Island virus and a new tetravirus, Sarawak virus. Strains of previously characterized Sathuvarachi, Yunnan, Banna and Parry's Lagoon viruses (Reoviridae), Bontang virus (Mesoniviridae), and Culex theileri flavivirus (Flaviviridae) were also characterized. The availability of these mosquito virus genomes will facilitate their detection by metagenomics or PCR to better determine their geographic range, extent of host tropism, and possible association with arthropod or vertebrate disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Salmon are paramount to the economy, ecology, and history of the Pacific Northwest. Viruses constitute one of the major threats to salmon health and well-being, with more than twenty known virus species that infect salmon. Here, we describe the isolation and characterization of the fall Chinook aquareovirus, a divergent member of the species Aquareovirus B within the family Reoviridae., Methods: The virus was first found in 2014 as part of a routine adult broodstock screening program in which kidney and spleen tissue samples from healthy-appearing, adult fall Chinook salmon (Oncorhynchus tshawytscha) returning to a hatchery in Washington State produced cytopathic effects when inoculated onto a Chinook salmon embryo cell line (CHSE-214). The virus was not able to be confirmed by an RT-PCR assay using existing aquareovirus pan-species primers, and instead was identified by metagenomic next-generation sequencing. Metagenomic next-generation sequencing was used to recover the full genome and completed using 3' RACE., Results: The genome of the fall Chinook aquareovirus contains 11 segments of double-stranded RNA totaling 23.3 kb, with each segment flanked by the canonical sequence termini found in the aquareoviruses. Sequence comparisons and a phylogenetic analysis revealed a nucleotide identity of 63.2% in the VP7 gene with the Green River Chinook virus, placing the new isolate in the species Aquareovirus B. A qRT-PCR assay was developed targeting the VP2, which showed rapid growth of the isolate during the initial 5 days in culture using CHSE-214 cells., Conclusions: This sequence represents the first complete genome of an Aquareovirus B species. Future studies will be required to understand the potential pathogenicity and epidemiology of the fall Chinook aquareovirus.

Background: The small brown planthopper (SBPH) is an important pest of cereal crops and acts as a transmission vector for multiple RNA viruses. Rapid diagnosis of virus in the vector is crucial for efficient forecast and control of viral disease. Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. The traditional RT-PCR contains a RNA isolation step and is widely used for virus detection in insect. However, using the traditional RT-PCR for detecting RNA virus in individual SBPHs becomes challenging because of the expensive reagents and laborious procedure associated with RNA isolation when processing a large number of samples., Results: We established a simplified RT-PCR method without RNA isolation for RNA virus detection in a single SBPH. This method is achieved by grinding a single SBPH in sterile water and using the crude extract directly as the template for RT-PCR. The crude extract containing the virus RNA can be prepared in approximately two minutes. Rice stripe virus (RSV), rice black streaked dwarf virus (RBSDV) and Himetobi P virus (HiPV) were successfully detected using this simplified method. The detection results were validated by sequencing and dot immunobinding assay, indicating that this simplified method is reliable for detecting different viruses in insects. The evaluation of the sensitivity of this method showed that both RSV and HiPV can be detected when the cDNA from the crude extract was diluted up to 10 3 fold. Compared to the traditional RT-PCR with RNA isolation, the simplified RT-PCR method greatly reduces the sample processing time, decreases the detection cost, and improves the efficiency by avoiding RNA isolation., Conclusions: A simplified RT-PCR method is developed for rapid detection of RNA virus in a single SBPH without the laborious RNA isolation step. It offers a convenient alternative to the traditional RT-PCR method.

Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons. The amplicons were hybridized to specific biotinylated DNA probes and the products were detected colorimetrically using horseradish peroxidase and a microplate reader. The new method is able to detect GCRV at 14 copies/μL within 5h and had a diagnostic sensitivity and a specificity of 100% when GCRV-II and non-target virus were tested. This NASBA-ELISA was evaluated using a panel of clinical samples (n=103) to demonstrate that it is a rapid, effective and sensitive method for GCRV detection in grass carp aquaculture., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Many insects, including mosquitoes, planthoppers, aphids and leafhoppers, are the hosts of bacterial symbionts and the vectors for transmitting viral pathogens 1-3 . In general, symbiotic bacteria can indirectly affect viral transmission by enhancing immunity and resistance to viruses in insects 3-5 . Whether symbiotic bacteria can directly interact with the virus and mediate its transmission has been unknown. Here, we show that an insect symbiotic bacterium directly harbours a viral pathogen and mediates its transovarial transmission to offspring. We observe rice dwarf virus (a plant reovirus) binding to the envelopes of the bacterium Sulcia, a common obligate symbiont of leafhoppers 6-8 , allowing the virus to exploit the ancient oocyte entry path of Sulcia in rice leafhopper vectors. Such virus-bacterium binding is mediated by the specific interaction of the viral capsid protein and the Sulcia outer membrane protein. Treatment with antibiotics or antibodies against Sulcia outer membrane protein interferes with this interaction and strongly prevents viral transmission to insect offspring. This newly discovered virus-bacterium interaction represents the first evidence that a viral pathogen can directly exploit a symbiotic bacterium for its transmission. We believe that such a model of virus-bacterium communication is a common phenomenon in nature.

In an attempt to determine whether or not genetic variants of the Tasmanian strain of Atlantic salmon aquareovirus (TSRV) exist, 14 isolates of TSRV, originating from various locations in Tasmania, covering a 20-year period (1990-2010), obtained from various host species and tissues, and isolated on different cell lines, were selected for this study. Two categories, termed "typical" and "atypical", of variants of TSRV were identified based on preliminary genotypic and phenotypic characterization carried out on these 14 different isolates. In addition, electron microscopic examination indicated the existence of at least three variants based on viral particle size. Finally, this study demonstrated the existence of at least one new variant of TSRV isolates, other than the more commonly isolated typical TSRV isolates, in farmed Tasmanian Atlantic salmon.

Diseases caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) considerably decrease grain yield. Therefore, determining rice cultivars with high resistance to SRBSDV and RRSV is necessary. In this study, rice cultivars with high resistance to SRBSDV and RRSV were evaluated through field trials in Shidian and Mangshi county, Yunnan province, China. SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to quantitatively detect virus gene expression levels in different rice varieties. The following parameters were applied to evaluate rice resistance: acre yield (A.Y.), incidence of infected plants (I.I.P.), virus load (V.L.), disease index (D.I.), and insect quantity (I.Q.) per 100 clusters. Zhongzheyou1 (Z1) and Liangyou2186 (L2186) were considered the most suitable varieties with integrated higher A.Y., lower I.I.P., V.L., D.I. and I.Q., Features: In order to investigate the mechanism of rice resistance, comparative label-free shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomic approaches were applied to comprehensively describe the proteomics of rice varieties' SRBSDV tolerance. Systemic acquired resistance (SAR)-related proteins in Z1 and L2186 may result in the superior resistance of these varieties compared with Fengyouxiangzhan (FYXZ).

Maize rough dwarf disease (MRDD) has long been known as one of the most devastating viral diseases of maize worldwide and is caused by single or complex infection by four fijiviruses: Maize rough dwarf virus (MRDV) in Europe and the Middle East, Mal de Rio Cuarto virus (MRCV) in South America, rice black-streaked dwarf virus (RBSDV), and Southern rice black-streaked dwarf virus (SRBSDV or Rice black-streaked dwarf virus 2, RBSDV-2) in East Asia. These are currently classified as four distinct species in the genus Fijivirus, family Reoviridae, but their taxonomic status has been questioned. To help resolve this, the nucleotide sequences of the ten genomic segments of an Italian isolate of MRDV have been determined, providing the first complete genomic sequence of this virus. Its genome has 29144 nucleotides and is similar in organization to those of RBSDV, SRBSDV, and MRCV. The 13 ORFs always share highest identities (81.3-97.2%) with the corresponding ORFs of RBSDV and phylogenetic analyses of the different genome segments and ORFs all confirm that MRDV clusters most closely with RBSDV and that MRCV and SRBSDV are slightly more distantly related. The results suggest that MRDV and RBSDV should be classified as different geographic strains of the same virus species and we suggest the name cereal black-streaked dwarf fijivirus (CBSDV) for consideration.

To analyze the variation in rice black-streaked dwarf virus (RBSDV) in an area with high incidence of maize rough dwarf disease (MRDD), the RBSDV S1 segment in a collection of 100 maize isolates (sample population A100) from Jining, Shandong Province, was sequenced. An additional 21 maize and rice isolates (subpopulation B21) that were sampled from nine other geographic locations in China in 2012 and 2013 were used as a control. A total of 914 nucleotide mutations, including 239 singleton variable and 675 parsimony-informative sites were detected among the segment 1 (S1) sequences from A100. A total of 614 nucleotide mutation sites including 164 singleton variable and 450 parsimony-informative sites were detected among the S1 sequences from B21, while 97.55 % of the parsimony-informative sites from B21 were also detected in A100. The nucleotide sequence diversities of A100 (π = 0.0479) and B21 (π = 0.0396) were significantly different (P = 0.0002) but showed similar trends. Phylogenetic analysis showed that the 121 RBSDV isolates could be classified into two groups based on their S1 sequences, independent of subpopulation, with a combination of host species and locations. A100 and B21 were under the same level of negative and purifying selection, with Ka/Ks ratios of 0.0337 and 0.0369, respectively. The combined RBSDV population, including 121 isolates, was expanding, with negative values for Tajima's D, Fu and Li's D, and Fu and Li's F in both A100 and B21, except Tajima's D in A100. Based on S1, the RBSDV population in China has long-term phytogeographic stability, and there do not appear to be any newly-emerging strains.

A vertically integrated monitoring program was set up for breeders hatched in 2013 and their offspring to detect differences in performance related to the reovirus vaccination schedule. Within the same organization in Belgium, 17 breeder flocks were vaccinated with one dose of live and one dose of inactivated reovirus vaccine, while 14 flocks received two doses of inactivated vaccine without live priming. The hatchability of the eggs produced by these birds was examined. Further, the daily growth, feed conversion, mortality, slaughterhouse condemnation, production index, and antibiotic use were monitored in 110 broiler flocks derived from the breeders. All gathered data were examined statistically. In eggs obtained from breeders vaccinated twice with inactivated reovirus vaccine, a significant 2.88% higher hatchability rate was observed. The progeny broiler flocks of these breeders showed a significant 18.2% lower mortality during the fattening period. Although not statistically significant, the slaughterhouse condemnation rate was 10.1% lower as well. The results may indicate that-under the epidemiologic conditions of this study-double administration of inactivated reovirus vaccine in broiler breeders can at least contribute to higher hatchability of breeder eggs and lower broiler mortality.

Viruses are extensively studied as pathogens and exploited as molecular tools and therapeutic agents. Existing methods to purify viruses such as gradient ultracentrifugation or chromatography have limitations, for example demand for technical expertise or specialized equipment, high time consumption, and restricted capacity. Our laboratory explores mutations in oncolytic reovirus that could improve oncolytic activity, and makes routine use of numerous virus variants, genome reassortants, and reverse engineered mutants. Our research pace was limited by the lack of high-throughput virus purification methods that efficiently remove confounding cellular contaminants such as cytokines and proteases. To overcome this shortcoming, we evaluated a commercially available resin (Capto Core 700) that captures molecules smaller than 700 kDa. Capto. Core 700 chromatography produced virion purity and infectivity indistinguishable from CsCl density gradient ultracentrifugation as determined by electron microscopy, gel electrophoresis analysis and plaque titration. Capto Core 700 resin was then effectively adapted to a rapid in-slurry pull-out approach for high-throughput purification of reovirus and adenovirus. The in-slurry purification approach offered substantially increased virus purity over crude cell lysates, media, or high-spin preparations and would be especially useful for high-throughput virus screening applications where density gradient ultracentrifugation is not feasible., Competing Interests: K.T. James, M. Hitt, K. Agopsowicz, M.A. Trevors, D. Stoltz and M. Shmulevitz declare no conflicts of interest. B. Cooney is a Field Application Specialist affiliated with GE Healthcare Canada and was involved in conceptual design of approach, provided GE Healthcare reagents at no cost for this project, and may benefit indirectly from increased sales of associated reagents and equipment. B. Cooney remained at arms-length during experimentation and analysis.

The novel Eriocheir sinensis reovirus (EsRV) is a pathogen that causes severe disease and high mortality rates in cultivated crabs. Here, we established a highly sensitive and specific rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that was cheaper and more suitable for field applications in crab aquaculture than those of traditional reverse transcription-polymerase chain reaction (RT-PCR) analysis. The amplification was completed within 45 min under isothermal conditions at 65°C. The RT-LAMP test for EsRV had a detection limit of 15 pg, and sensitivity was 100 times greater than that of conventional RT-PCR. The LAMP primers for EsRV were not amplified by other pathogen strains, indicating good specificity. In addition to detection by electrophoresis, RT-LAMP results were detectable by visual observations of reaction tube turbidity, and calcein was added to visually detect the amplification products. These results indicate that this highly convenient, rapid and sensitive RT-LAMP assay can be used to detect EsRV-infected aquatic organisms., Significance and Impact of the Study: Tremor disease (TD) is one of the most serious diseases of Eriocheir sinensis. A novel E. sinensis reovirus (EsRV) was identified from E. sinensis afflicted with TD and caused high mortality. We developed a reverse transcription loop-mediated isothermal amplification assay with high specificity, sensitivity and rapidity to detect EsRV, which can be used to diagnose aquatic animal diseases, particularly where expensive diagnostic instruments are not available., (© 2016 The Society for Applied Microbiology.)

Reoviruses (family Reoviridae) infect vertebrate and invertebrate hosts with clinical effects ranging from inapparent to lethal. Here, we describe the discovery and characterization of Largemouth bass reovirus (LMBRV), found during investigation of a mortality event in wild largemouth bass (Micropterus salmoides) in 2015 in WI, USA. LMBRV has spherical virions of approximately 80 nm diameter containing 10 segments of linear dsRNA, aligning it with members of the genus Orthoreovirus, which infect mammals and birds, rather than members of the genus Aquareovirus, which contain 11 segments and infect teleost fishes. LMBRV is only between 24 % and 68 % similar at the amino acid level to its closest relative, Piscine reovirus (PRV), the putative cause of heart and skeletal muscle inflammation of farmed salmon. LMBRV expands the known diversity and host range of its lineage, which suggests that an undiscovered diversity of related pathogenic reoviruses may exist in wild fishes.

In order to develop a novel effective biological insecticide for controlling oleander hawk moth, a new pathogen was isolated from naturally diseased Daphnis nerii. Based on scanning electron microscopy, full-length amplification of cDNAs (FLAC), and phylogenetic analysis of genome segments 2and 10,the virus was identified as a new type of cypovirus (Da phnis nerii cypovirus [DnCPV]). Electrophoresis analysis showed that DnCPV had a genome comprising 10double-stranded RNA (dsRNA) segments, ranging from 892 to 4160bp.Using FLAC, the cDNAs from the 10 dsRNA segments of the new CPV were cloned and genome segments 2and 10 were sequenced. Sequencing results showed that segment 2 encoded RNA-dependent-RNA-polymerases (RdRps) and segment 10 encoded polyhedrin. These two segments shared conserved terminal sequences of AGUCAAA and AGC at the 5’and 3’ends,respectively.These conserved terminal sequences were not consistent with any of the known CPV types.Phylogenetic analysis of the RdRp and polyhedrin indicated that this CPV was more closely related to CPV type 19 and type 5than other CPV types. Based on the unique conserved terminal sequences and the electrophoresis pattern of the new virus, we tentatively named it DnCPV Nanchang isolate: DnCPV-NC.

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.

The aim of this study is to determine if enteric viruses are the cause of diarrhea in broiler flocks in Jordan. Intestinal content samples were collected from 101 broiler flocks from several regions of Jordan to detect the presence of astrovirus, coronavirus, reovirus, and rotavirus, by using reverse transcriptase polymerase chain reaction (RT-PCR). Forty-six of these flocks were clinically healthy with no enteric disease, and the other 55 flocks were clinically suffering from diarrhea. The samples were collected between 5 and 16 d of age. The results show that 79% of total 101 flocks tested were infected with one or more of the above enteric viruses. Coronavirus was the most common virus, detected in 56.4% of these flocks, with astrovirus in 29.7% of the flocks, and rotavirus (9.9%) and reovirus (5.6%) being the least common. None of these flocks were found to be infected with all four viruses, but one of the flocks was found to be infected with astrovirus, coronavirus, and rotavirus simultaneously. Individual infection was noted with astrovirus, coronavirus and rotavirus but not with reovirus, whereas all flocks infected with reovirus were also infected with coronavirus. There was no statistical evidence to link these viruses as the main cause of diarrhea in the flocks tested. This is the first study in Jordan to detect all of these viruses and to correlate their presence with diarrhea in chicken flocks., (© 2016 Poultry Science Association Inc.)