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Avian reoviruses continue to cause disease in turkeys with varied pathogenicity and tissue tropism. Turkey enteric reovirus has been identified as a causative agent of enteritis or inapparent infections in turkeys. The new emerging variants of turkey reovirus, tentatively named turkey arthritis reovirus (TARV) and turkey hepatitis reovirus (THRV), are linked to tenosynovitis/arthritis and hepatitis, respectively. Turkey arthritis and hepatitis reoviruses are causing significant economic losses to the turkey industry. These infections can lead to poor weight gain, uneven growth, poor feed conversion, increased morbidity and mortality and reduced marketability of commercial turkeys. To combat these issues, detecting and classifying the types of reoviruses in turkey populations is essential. This research aims to employ clustering methods, specifically K-means and Hierarchical clustering, to differentiate three types of turkey reoviruses and identify novel emerging variants. Additionally, it focuses on classifying variants of turkey reoviruses by leveraging various machine learning algorithms such as Support Vector Machines, Naive Bayes, Random Forest, Decision Tree, and deep learning algorithms, including convolutional neural networks (CNNs). The experiments use real turkey reovirus sequence data, allowing for robust analysis and evaluation of the proposed methods. The results indicate that machine learning methods achieve an average accuracy of 92%, F1-Macro of 93% and F1-Weighted of 92% scores in classifying reovirus types. In contrast, the CNN model demonstrates an average accuracy of 85%, F1-Macro of 71% and F1-Weighted of 84% scores in the same classification task. The superior performance of the machine learning classifiers provides valuable insights into reovirus evolution and mutation, aiding in detecting emerging variants of pathogenic TARVs and THRVs., (© The Author(s) 2024. Published by Oxford University Press.)

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection. However, the role of miRNAs in host response to ARV infection is still not clear. In this study, we show that ARV infection markedly increased gga-miR-30c-5p expression in DF-1 cells and that transfection of cells with gga-miR-30c-5p inhibited ARV replication while knockdown of endogenous gga-miR-30c-5p enhanced viral growth in cells. Importantly, we identified the autophagy related 5 (ATG5), an important proautophagic protein, as a bona fide target of gga-miR-30c-5p. Transfection of DF-1 cells with gga-miR-30c-5p markedly reduced ATG5 expression accompanied with reduced conversion of ARV-induced-microtubule-associated protein 1 light chain 3 II (LC3-II) from LC3-I, an indicator of autophagy in host cell, while knockdown of endogenous gga-miR-30c-5p enhanced ATG5 expression as well as ARV-induced conversion of LC3-II, facilitating viral growth in cells. Furthermore, knockdown of ATG5 by RNA interference (RNAi) or treatment of cells with autophagy inhibitors (3-MA and wortmannin) markedly reduced ARV-induced LC3-II and syncytium formation, suppressing viral growth in cells, while overexpression of ATG5 increased ARV-induced LC3-II and syncytium formation, promoting viral growth in cells. Thus, gga-miR-30c-5p suppressed viral replication by inhibition of ARV-induced autophagy via targeting ATG5. These findings unraveled the mechanism of how host cells combat against ARV infection by self-encoded small RNA and furthered our understanding of the role of microRNAs in host response to pathogenic infection. IMPORTANCE Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, and retarded growth, leading to considerable economic losses to the poultry industry across the globe. Elucidation of the pathogenesis of ARV infection is crucial to guiding the development of novel vaccines or drugs for the effective control of these diseases. Here, we investigated the role of miRNAs in host response to ARV infection. We found that infection of host cells by ARV remarkably upregulated gga-miR-30c-5p expression. Importantly, gga-miR-30c-5p suppressed ARV replication by inhibition of ARV-induced autophagy via targeting autophagy related 5 (ATG5) accompanied by suppression of virus-induced syncytium formation, thus serving as an important antivirus factor in host response against ARV infection. These findings will further our understanding of how host cells combat against ARV infection by self-encoded small RNAs and may be used as a potential target for intervening ARV infection.

In mid-2020, using next-generation sequencing (NGS) technology, we identified a recombinant cluster 2 avian orthoreovirus (ARV) variant named PHC-2020-0545, isolated from tendons of 33-day-old broilers with leg swelling in China. Complete genomic sequencing and analyses demonstrated that the isolate was genetically significantly distinct from known ARV strains in M1 and M3 genes and its σC coding gene had an extremely high variability, compared with the identified ARV strains grouped into other genotyping cluster. Further analysis showed that many base substitutions were silent and non-silent substitutions are most likely to occur in the first positions of codons. Multiple segmental recombination, intra-segmental recombination and accumulation of point mutations might contribute to the emergence of this isolate. The PHC-2020-0545 strain had a strong replication ability in 1-day-old broilers, and mainly affected the movement, digestion and metabolism of broilers. In addition, the infection route of the isolate is related to its pathogenicity to broilers. Therefore, combined with its unique genetic characteristics and potential origin, we determined that the PHC-2020-0545 field strain is a novel recombinant ARV strain, which has certain reference value for the preparation and evaluation of new vaccines., (Copyright © 2021. Published by Elsevier B.V.)

Inclusion body hepatitis (IBH) is, in some cases, a fatal disease affecting fowl by adenovirus strains which are subdivided into 5 species (A-E). In the current study, we investigated sequences from the Loop L1 region of the hexon gene of sequences of adenovirus field stains 1/A and 11/D isolated from a poultry flock co-infected with IBH and avian reoviruses ARVs. In early 2021, an epidemiologic survey highlighted the coinfection adenoviruses with other viruses (orthoreovirus infection) as being particularly deleterious within the poultry industry. Here, we investigated the Loop L1 HVR1-4 region of the hexon gene with relative synonymous codon usage (RSCU) designation and RSCU inclusive of all the mutations. These are the first results that have been presented on fowl adenovirus species A and D with simultaneous reovirus infection in 38-days old broiler chickens in Poland., Competing Interests: The authors have declared that no competing interests exist.

Autophagy plays a momentous role in cellular responses against pathogens. However, the influence of the autophagy machinery on Muscovy duck reovirus (MDRV) infection is not yet confirmed. In this study, it was shown that MDRV infection significantly increased the number of autophagy-like vesicles in DF-1 cells under electron microscope and the LC3-I/LC3-II conversion, which was considered important indicators of autophagy. It was worth noting that the level of autophagy was positively correlated with MDRV replication. Further test results showed that MDRV-induced autophagy can promote virus replication in DF-1 cells, and both the envelope protein sigma A and non-structural protein sigma NS that play an important role in virus replication process can colocalize with the autophagosome marker molecule LC3-II by confocal immunofluorescence analysis. These results indicated that MDRV utilized the autophagosomes for replication. Through transfection of the dual fluorescent plasmid mcherry-EGFP-LC3 and fluorescence microscope observation, it was found that autophagosomes were more likely to fuse with lysosomes in MDRV-infected cells compared with the blank group. The phenomenon of pEGFP-LC3B fluorescent spot and LAMP1 co-localization appeared in MDRV infected cells, indicating that MDRV infection would promote the fusion of autophagosomes and the lysosomes. Conversely, accumulation of p62 was observed by immunoblotting, suggesting that autolysosomes does not exert effective degradation. MDRV infection triggered a incomplete autophagic response. Further studies found that the expression of LAMP1, a marker protein of late endosome/early lysosome, increased significantly in MDRV-infected cells, suggesting an increase in the number of immature lysosomes. In addition, the experiment detected the maturation of the lysosomal acid hydrolase Cathepsin D in the cells, and found that the expression of the 33 kDa mature form of Cathepsin D was significantly reduced after MDRV infection, indicating that MDRV inhibits the maturation of lysosomes. In general, MDRV infection induces autophagy of DF-1 cells, promotes the fusion of autophagosomes and lysosomes, inhibits autophagolysosome degradation, and promotes virus replication., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Since 2017, duck spleen necrosis caused by new variant duck orthoreovirus (N-DRV) infection had been observed in several provinces in China. This disease retards the growth and development of ducks, thereby reducing feed return rate. N-DRV infection causes damage to duck spleen and other immune organs, leading to immunosuppression and susceptibility to other pathogens. In this study, we successfully constructed a breeding duck artificial infection model and found that N-DRV infection can cause pathologic changes, such as ovarian hemorrhage, follicle atrophy, and fallopian tube bleeding in breeding ducks, resulting in significantly reduced fertilization rate and egg hatching rate. Viral RNA was present in egg vitelline membrane, duck embryo, and duckling's spleen samples, as determined through quantitative polymerase chain reaction (qPCR). Autopsy revealed obvious pathologic changes in the spleen and other organs, although there were no obvious early clinical symptoms observed in ducklings. Sequence distance and phylogenetic analysis confirmed that N-DRV-SD19 re-isolated from the spleen samples of ducklings was consistent with the strain N-DRV-XT18 used for infecting breeding ducks. The findings in this study confirmed that N-DRV can be vertically transmitted through eggs, which provide an important reference for the disease prevention and control., (Copyright © 2020. Published by Elsevier B.V.)

Hydropericardium hepatitis syndrome (HHS) is a fatal disease caused by fowl adenovirus serotype 4 (FAdV-4). Avian viral arthritis is an infectious disease characterized by movement disorders caused by avian orthoreovirus (ARV). In the early 2019, our epidemiologic survey on poultry diseases in eight commercial broiler farms in China showed that FAdV-4 and ARV have a high coinfection rate, accounting for 63 % of all ARV-positive samples. We designed chicken embryo and animal models to investigate the synergistic pathogenicity of FAdV-4 and ARV. Weakness and inappetence were observed in all specific-pathogen-free (SPF) chickens of the experimental group. FAdV-4 and ARV coinfection caused severe embryonic body and hepatic hemorrhage in SPF chicken embryos. Compared with the singular ARV-infected group, joint swelling was more severe in all coinfected groups. Compared with single virus infection, the coinfection of the two viruses increased the mortality of SPF chicken embryos and chickens. FAdV-4 and ARV coinfection resulted in significantly severe macroscopic and microscopic lesions of the liver, spleen, and kidney of SPF chickens. The detection results of viral load in allantoic fluid, liver, and cloacal swabs indicated that ARV enhanced FAdV-4 replication in SPF chicken embryos and chickens. Cytokine detection showed a significant change in interleukin-1 (IL-1), IL-6, and interferon-α (IFN-α) levels in coinfected groups compared with those in the single-infected groups. Additionally, FAdV-4 and ARV coinfection caused severe damage to the SPF chicken's immune system. In summary, these findings provide insights into the pathology, prevention, and treatment of FAdV-4 and ARV coinfection., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Duck reovirus (DRV) is a fatal member of the genus Orthoreovirus in the family Reoviridae. The disease caused by DRV leads to huge economic losses to the duck industry. Post-translational modification is an efficient strategy to enhance the immune responses to virus infection. However, the roles of protein phosphorylation in the responses of ducklings to Classic/Novel DRV (C/NDRV) infections are largely unknown. Using a high-resolution LC-MS/MS integrated to highly sensitive immune-affinity antibody method, phosphoproteomes of Cairna moschata spleen tissues under the C/NDRV infections were analyzed, producing a total of 8,504 phosphorylation sites on 2,853 proteins. After normalization with proteomic data, 392 sites on 288 proteins and 484 sites on 342 proteins were significantly changed under the C/NDRV infections, respectively. To characterize the differentially phosphorylated proteins (DPPs), a systematic bioinformatics analyses including Gene Ontology annotation, domain annotation, subcellular localization, and Kyoto Encyclopedia of Genes and Genomes pathway annotation were performed. Two important serine protease system-related proteins, coagulation factor X and fibrinogen α-chain, were identified as phosphorylated proteins, suggesting an involvement of blood coagulation under the C/NDRV infections. Furthermore, 16 proteins involving the intracellular signaling pathways of pattern-recognition receptors were identified as phosphorylated proteins. Changes in the phosphorylation levels of MyD88, NF-κB, RIP1, MDA5 and IRF7 suggested a crucial role of protein phosphorylation in host immune responses of C. moschata. Our study provides new insights into the responses of ducklings to the C/NDRV infections at PTM level.

Duck spleen necrosis disease (DSND) is an emerging infectious disease that causes significant economic loss in the duck industry. In 2018, a duck reovirus (named DRV/GX-Y7) and Salmonella indiana were both isolated from the spleens and livers of diseased ducks with DSND in China. The DRV/GX-Y7 strain could propagate in the Vero, LMH, DF-1 and DEF cells with obvious cytopathic effects. The genome of DRV/GX-Y7 was 23,418 bp in length, contained 10 dsRNA segments, ranging from 3959 nt (L1) to 1191 nt (S4). The phylogenetic analysis showed that the DRV/GX-Y7 strain was in the same branch with the new waterfowl-origin reovirus cluster, but was obviously far distant from the clusters of other previous waterfowl-origin reoviruses Muscovy duck reovirus (MDRV) and goose-origin reovirus (GRV), broiler/layer-origin reovirus (ARV) and turkey-origin reovirus (TRV). The RDP and SimPlot program analysis revealed that there were two potential genetic reassortment events in the M2 and S1 segments of the genome. In order to have a clear insight into the pathogenic mechanism of DRV/GX-Y7 and S. Indiana in clinical DSND, an infection experiment was further conducted by challenging commercial ducklings with the two isolates individually and with both. The results showed that DRV/GX-Y7 produced severe hemorrhagic and/or necrotic lesions in the immune organs (thymus, spleen, and bursae) of experimentally infected ducklings. And, that the co-infection of DRV/GX-Y7 and S. Indiana could greatly enhance the pathogenesis by increasing the morbidity and mortality in ducklings whose clinical symptoms and lesions were similar to the natural clinical DSND cases. In summary, the results suggested that the pathogen causing duck spleen necrosis was an emerging unique genetic reassortment strain of duck Orthoreovirus that was significantly different from any previously reported waterfowl-derived Orthoreovirus and the co-infection with the Salmonella isolate could increase the severity of the disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Muscovy duck reovirus (MDRV) is highly pathogenic to young Muscovy ducklings. Although MDRV infection results in ducklings' acute watery diarrhea, the effect of MDRV infection on the composition of host's intestinal microbiota remains poorly understood. This study was conducted to investigate the impacts of MDRV on the composition of Muscovy ducklings' intestinal bacterial community. Three-day-old Muscovy ducklings were inoculated with either the virulent MDRV strain MW9710 or sterile Hank's solution, respectively. The cecal microbiota was analyzed between control and mock MDRV-infected ducklings using Illumina MiSeq sequencing at 6 dpi and 17 dpi, respectively. The results indicated that MDRV infection damaged the intestinal mucosa. In addition, MDRV infection caused severe perturbations of gut microbiota by decreasing microbial richness, altering the abundance of certain genera of the gut microbiota at 6 dpi. Specifically, the relative abundance of short chain fatty acids-producing bacteria (including Shuttleworthia, Streptococcus, and Ruminococcus) was reduced in MDRV-infected ducklings than those of control group, whereas, with an enrichment of Enterobacteriaceae (including Plesiomonas, Escherichia_Shigella and Proteus). Furthermore, microbiota analysis showed that the gut microbiota dysbiosis caused by MDRV infection was basically recovered at 17 dpi. Collectively, this study demonstrated that the gut microbiota of Muscovy ducklings were altered due to MDRV infection, mainly featuring as a net loss of beneficial bacteria and a compensatory proliferation of pathogenic bacteria, which may lead to severe pathology to the intestinal mucosa, and ultimately acute diarrhea. These results will provide insights into the pathology of MDRV infection.

In recent years, emerging avian reovirus (ARV) strains causing viral arthritis have become a challenge to the worldwide chicken industry, and were responsible for significant economic losses. In this study, we characterized emerging variant ARV strains and examined their genetic relationship and pathogenicity variation with reference strains. A total of 18 emerging variant ARV strains were isolated from tendon and capsular synovial fluid of broiler chickens with clinical cases of arthritis/tenosynovitis at commercial farms in China. Comparative analysis based on σC sequence showed that 4/18 isolates were in the same cluster (Cluster 1) as vaccine strains (S1133), whereas 14 of 18 isolates were in Clusters 2, 3, and 6. The field isolates shared a rather low identity (38.1 to 81.9%) with S1133 in Cluster 1, especially for those from Cluster 6 (38.1 to 67.2%). A higher ARV isolation rate was observed in chicken embryos (47/61) compared to cell culture (37/61) through PCR with a detection primer. A total of 3 isolates were selected to infect specific-pathogen-free (SPF) chickens, showing that the tested isolates, especially that from Cluster 6, displayed greater pathogenicity than S1133 strain, characterized by higher incidence. These findings suggest that the virulence of Chinese ARVs has been increasing rapidly in recent years, and the vaccine need to be updated correspondingly., (© 2019 Poultry Science Association Inc.)

This study describes the molecular characterization of avian reoviruses (ARVs) isolated during an outbreak in commercial chickens between 2015 and 2016. In addition, a pathogenicity study of a selected ARV strain isolated from a field case of viral tenosynovitis in commercial broiler chickens was performed. On the basis of phylogenetic analysis of a 1088-bp fragment of the ARV S1 gene, the investigated sequences were differentiated into five distinct genotypic clusters (GCs), namely GC1, GC2, GC3, GC4, and GC6. Specific-pathogen-free (SPF) and commercial broiler chickens were challenged with the GC1 genetic type MK247011, at 14 days of age via the interdigital toe web. No significant effects in body weight gain and feed conversion were detected in both chicken types. The Δ interdigital web thickness was most severe at 4 days postchallenge (DPC) in both the SPF and broiler subgroups. The inflammation in SPF birds was slightly more severe compared with broilers. Neither mortality nor clinical signs occurred in the infected groups for the duration of the experiment, despite the presence of significant microscopic lesions in challenged birds. Microscopic changes of tenosynovitis became evident at 3 DPC, with the highest incidence and severity detected at 14 and 21 DPC, respectively. Seroconversion against ARV occurred 3 wk postchallenge, and the microscopic lesions detected in tendon and heart sections were highly compatible with those described in the field. Increased severity of tenosynovitis and epicarditis lesions were noted in the ARV-challenged groups compared with the control groups. Although SPF and broiler chickens showed comparable responses to the challenge with an ARV genetic variant, detected lesions were subclinical, denoting the limitations of our challenge approach. The age selected in this experiment possibly influenced the course of the infection. Data from this study highlight the genotypic diversity of isolates in California, and the outcome of the pathogenicity study can be used as a basis to improve protocols for pathogenicity studies to characterize ARV variants causing clinical disease in the field.

Avian orthoreovirus (ARV) infections of broiler flocks cause arthritis/tenosynovitis syndrome and significant economic losses. ARV variants were detected in the USA and Canada. Viral arthritis/tenosynovitis syndrome has occurred frequently in China in recent years. In this study, a variant ARV strain associated with viral arthritis/tenosynovitis syndrome was isolated from broilers and designated as LY383. Genomic sequence and phylogenetic analysis of the σC nucleic acid and amino acid sequences revealed that the isolate was closely related to ARV field strains Reo/PA/Layer/01224B/14, Reo/PA/Broiler/1551/13, GA/14602/2014, GA/13569/2013 and GA/13542/2013, in cluster V, but distinct from most Chinese field strains or commercial vaccine strains. Experimental challenge showed that the isolate could cause arthritis/tenosynovitis syndrome in broilers, which possessed a high level of maternal antibodies induced by commercial ARV vaccines (S1133, 1733 and T98). Furthermore, viral nucleic acid could be detected in cloacal swabs of all challenged birds throughout the entire test from 5 dpi onward. These results suggest that a novel ARV genotype emerges and might become prevalent in broiler flocks in China. RESEARCH HIGHLIGHTS A variant avian orthoreovirus was isolated from a vaccinated broiler flock in North China. The ARV field strain was distinct from previous China-origin ARV isolates and vaccine strains. The current commercial ARV vaccine could not provide effective protection of broilers against the field isolate infection. These findings indicated that variant ARV field strains might become frequent in broiler flocks in China and effective measures should be conducted to prevent and control the disease.

Muscovy duck reovirus (MDRV) causes serious immunodeficiency in the intestinal mucosa, although the underlying histopathological mechanisms remain unclear. Thus, we investigated the impact of MDRV infection on intestinal morphology using hematoxylin and eosin staining. Immune-related cells were also quantified by staining with hematoxylin and eosin, toluidine blue, and periodic acid-Schiff stain, or by immunohistochemistry and cytochemistry for lectin. Similarly, CD4 + and CD8 + cells were quantified by flow cytometry, and the expression of several immune-related molecules was quantified by radioimmunoassay. We found that MDRV clearly damaged the intestinal mucosa, based on tissue morphology, villus length, villus width, intestinal thickness, villus height/crypt depth ratio, and villus surface area. MDRV also altered the density or distribution of lymphocytes, mastocytes, and goblet cells in the small intestinal mucosa, as well as microfold cells in Peyer's patches. In addition, MDRV markedly depleted CD4 + cells from the intestinal mucosa and lowered the CD4 + :CD8 + ratio in peripheral blood. Moreover, MDRV diminished the levels of secretory IgA and mucosal addressin cell adhesion molecule-1 (p < 0.01), but elevated those of histamine and nitric oxide (p < 0.01 or p < 0.05). Finally, MDRV significantly suppressed IL-1β, IL-4, IL-5, and IL-8 levels (p < 0.01 or p < 0.05) mid-infection. Collectively, our data suggest that MDRV severely damages the structure and function of the intestinal mucosa by modulating immune cells and immune-related factors, thus leading to local immunodeficiency. Our findings lay the foundation for further research on the pathogenesis of MDRV., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Adenosine triphosphate (ATP) is an energy source for many types of viruses for facilitating virus replication. This is the first report to demonstrate that the structural protein σA of avian reovirus (ARV) functions as an activator of cellular energy. Three cellular factors, isocitrate dehydrogenase 3 subunit beta (IDH3B), lactate dehydrogenase A (LDHA), and vacuolar-type H+-ATPase (vATPase) co-immunoprecipitated with ARV σA and were identified by 2D-LC/MS/MS. ARV enhances glycolytic flux through upregulation of glycolytic enzymes. Increased ATP levels in both ARV-infected and σA-transfected cells were observed by a fluorescence resonance energy transfer-based genetically encoded indicator, Ateams. Furthermore, σA upregulates IDH3B and glutamate dehydrogenase (GDH) to promote glutaminolysis, activating HIF-1α. Both HIF-1α level and viral yield in IDH3B-depleted and glutamine-deprived cells, and inhibition of glutaminolysis was significantly reduced. The σA R155/273A mutant loses its ability to enter the nucleolus, impairing its ability to regulate glycolysis. In addition, we have identified the conserved untranslated regions (UTR) of the 5'- and 3'-termini of the ARV genome segments that are required for viral protein synthesis in an ATP-dependent manner. Deletion of either the 5'- or 3'-UTR impaired viral protein synthesis. Knockdown of σA reduced the ATP level and significantly decreased virus yield, suggesting that σA enhances ATP formation to promote virus replication. Collectively, this study provides novel insights into σA-modulated suppression of LDHA and activation of IDH3B and GDH to activate the mTORC1/eIF4E/HIF-1α pathways to upregulate glycolysis and the TCA cycle for virus replication., (© 2018 John Wiley & Sons Ltd.)

Duck reovirus (DRV) is an typical aquatic bird pathogen belonging to the Orthoreovirus genus of the Reoviridae family. Reovirus causes huge economic losses to the duck industry. Although DRV has been identified and isolated long ago, the responses of Cairna moschata to classical/novel duck reovirus (CDRV/NDRV) infections are largely unknown. To investigate the relationship of pathogenesis and immune response, proteomes of C. moschata liver cells under the C/NDRV infections were analyzed, respectively. In total, 5571 proteins were identified, among which 5015 proteins were quantified. The differential expressed proteins (DEPs) between the control and infected liver cells displayed diverse biological functions and subcellular localizations. Among the DEPs, most of the metabolism-related proteins were down-regulated, suggesting a decrease in the basal metabolisms under C/NDRV infections. Several important factors in the complement, coagulation and fibrinolytic systems were significantly up-regulated by the C/NDRV infections, indicating that the serine protease-mediated innate immune system might play roles in the responses to the C/NDRV infections. Moreover, a number of molecular chaperones were identified, and no significantly changes in their abundances were observed in the liver cells. Our data may give a comprehensive resource for investigating the regulation mechanism involved in the responses of C. moschata to the C/NDRV infections.

Avian reovirus (ARV) infections characterised by severe arthritis, tenosynovitis, pericarditis, and depressed growth have become increasingly frequent in recent years. In this study, we isolated and identified 11 ARV field strains from chickens with viral arthritis and reduced growth in northern China. Comparative analysis of the σC nucleotide and amino acid sequences demonstrated that all isolates, except LN05 and JS01, were closely related to ARV S1133 and clustered in the first genetic lineage. LN05 and JS01 strains were clustered in the third lineage with the ARV 138 strain. Using S1133 as a reference, five isolates were selected to infect specific-pathogen-free chickens, and we found that the recent isolated Chinese ARV strains had higher replication ability in vivo and caused enhanced mortality than the S1133 strain. These findings suggest that the pathogenicity of Chinese ARVs has been changing in recent years and disease control may become more difficult. This study provides genetic and pathogenic characterisations of ARV strains isolated in northern China and calls for a sustained surveillance of ARV infection in China in order to support a better prevention and control of the disease.

The novel duck reovirus (NDRV) is an emerging, contagious infection. To better realize the pathogenic mechanism of NDRV in ducks, an infection experiment was conducted. The resulting data demonstrated that typical gross lesions were observed in the infected ducks. NDRV was able to replicate in various tissues, leading to these pathological lesions, especially on the liver and spleen. Real-time quantitative PCR showed that the expression of most innate immune-related genes was up-regulated and the antiviral innate immune response could be established in both the liver and spleen. This study indicates that NDRV is a pantropic virus. To resist viral infection, several pathogen recognition receptors can cooperatively recognize NDRV and initiate innate immunity, but the responses are different between different tissues. As far as we know, this is the first systematic investigation of the pathogenicity of NDRV in Cherry Valley ducks based on the host's innate immunity, and these data will provide new insights into the further study of the disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Using next-generation sequencing (NGS) for full genomic characterization studies of the newly emerging avian orthoreovirus (ARV) field strains isolated in Pennsylvania poultry, we identified two co-infection ARV variant strains from one ARV isolate obtained from ARV-affected young layer chickens. The de novo assembly of the ARV reads generated 19 contigs of two different ARV variant strains according to 10 genome segments of each ARV strain. The two variants had the same M2 segment. The complete genomes of each of the two variant strains were 23,493 bp in length, and 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1), encoding 12 viral proteins. Sequence comparison of nucleotide (nt) and amino acid (aa) sequences of all 10 genome segments revealed 58.1-100% and 51.4-100% aa identity between the two variant strains, and 54.3-89.4% and 49.5-98.1% aa identity between the two variants and classic vaccine strains. Phylogenetic analysis revealed a moderate to significant nt sequence divergence between the two variant and ARV reference strains. These findings have demonstrated the first naturally occurring co-infection of two ARV variants in commercial young layer chickens, providing scientific evidence that multiple ARV strains can be simultaneously present in one host species of chickens.

A novel strain of duck reovirus (DRV) associated with a high mortality in Pekin ducklings in China, 2013, was isolated and characterized. This strain (designated as HN5d) grew well in Vero cells and produced marked cytopathic effects. HN5d contains 10 dsRNA genome segments, a typical feature of avian orthoreovirus. Following cloning, sequencing, and sequence analysis of the genome segments, a unique deletion of 18 amino acids was found in the sigma C protein of HN5d when compared with that of the recent Chinese waterfowl reoviruses (e.g., DRV 091). Phylogenetic analysis of cDNA amplicons of segments encoding for the outer capsid proteins revealed that HN5d is a novel genotype 2 waterfowl reovirus isolate. Inoculation of Pekin ducklings with HN5d resulted in splenic necrosis, a typical feature of "Duck spleen necrosis disease" (DSND) discovered in China in 2006. Unlike the typical DSND, HN5d produced severer hemorrhagic and/or necrotic lesions in livers of experimentally infected ducklings. 20-30% of death was observed during the first 7 day in the experimentally exposed birds. These findings suggest that HN5d is a novel duck reovirus isolate with severer pathogenicity in Pekin ducklings., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Particulate material is more efficient in eliciting immune responses. Here we describe the production of microspheres formed by protein muNS-Mi from avian reoviruses, loaded with foreign epitopes by means of IC-Tagging, for their use as vaccines.

Turkey arthritis reovirus (TARV) has been isolated from the gastrocnemius tendons and tibiotarsal joint fluid of lame male turkeys >12 weeks old in the Midwest. Two experiments were conducted to compare the pathogenicity in turkeys of three TARVs (TARV-MN2, TARV-MN4 and TARV-O'Neil), one turkey enteric reovirus (TERV strain MN1) and one chicken arthritis reovirus (CARV strain MN1). Two hundred microlitres of virus were inoculated by the oral, intratracheal, or footpad route into 6-day-old poults placed in isolator units. Poults were necropsied at 1 and 4 weeks post infection in Experiment 1, and at 2 and 4 weeks post infection in Experiment 2. Reovirus was detected by reverse transcription-polymerase chain reaction and virus isolation in tendons of TARV-inoculated poults at 1, 2 and 4 weeks post infection. TARV-O'Neil and TARV-MN2 were detected in tendons of sentinal birds at 1 and 4 weeks and 1 week p.i., respectively. In general, TARVs produced lymphocytic tenosynovitis of the gastrocnemius and digital flexor tendon sheaths without inflammation of the tendons proper. In Experiment 1, poults inoculated with TARV-MN2 and TARV-O'Neil had significantly higher gastrocnemius tendon inflammation scores, as determined by histology, than those inoculated with TERV-MN1 or CARV-MN1. In Experiment 2, poults inoculated with TARV-MN2 and TARV-O'Neil had significantly higher gastrocnemius tendon inflammation scores than those inoculated with TARV-MN4 and virus-free medium (negative control group). Koch's postulates was fulfilled when TARV-MN2 and TARV-O'Neil were re-isolated from tendons of poults that had originally been challenged with either of these viruses. Results of these experiments indicate that TARVs have a unique ability to induce gastrocnemius tenosynovitis in turkeys and that administration of TARV-O'Neil through the oral or intratracheal route is a reproducible model to study pathogenesis of TARV infection.

Avian reovirus (ARV) is an important pathogen in poultry industry and causes great economic losses. As attenuated Salmonella typhimurium is already being used as an effective vehicle for the transfer of DNA vaccines, so in this study we evaluated two DNA vaccines mediated by S. typhimurium on their ability of eliciting antibody production. SPF chickens were respectively immunized with SL7207 (pVAX-σB), SL7207 (pVAX-σC) and SL7027 (pVAX-σB-σC) three times. The results showed that the antibody production was highly dependent on the immunizing times, detectable antibodies of serum antibody IgG and small intestinal mucosal antibody IgA were generated at week 4 and were further improved at week 6 and antibody titers in group SL7207 (pVAX-σC) were higher than that in group SL7207 (pVAX-σB), demonstrating that SL7207 (pVAX-σC) was more powerful than SL7207 (pVAX-σB) in antibody production. The higher antibody titer in SL7027 (pVAX-σB-σC) than that in SL7207 (pVAX-σC) group showed that co-expressing σB and σC could improve antibody production. IFN-γ detection showed that significant higher IFN-γ was generated both in groups SL7027 (pVAX-σB-σC) and SL7207 (pVAX-σC). Subsequent challenge showed that SL7207 (pVAX-σB), SL7207 (pVAX-σC) and SL7027 (pVAX-σB-σC) conferred 50%, 75% and 87.5% respectively., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Avian reovirus (ARV) is an important cause of disease in poultry. Although ARV is known to induce apoptosis in infected cells, the interaction between ARV and its target cells requires further elucidation. In this report, we show that the ARV isolate strain GX/2010/1 induces autophagy in both Vero and primary chicken embryonic fibroblast (CEF) cells based on the appearance of an increased number of double-membrane vesicles, the presence of GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) dot formation, and the elevated production of LC3II. We further demonstrate that the class I phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway contributes to autophagic induction by ARV infection. Moreover, treatment of ARV-infected cells with the autophagy inducer rapamycin increased viral yields, while inhibition of the autophagosomal pathway using chloroquine led to a decrease in virus production. Altogether, our studies strongly suggest that autophagy may play a critical role in determining viral yield during ARV infection.

Birds are most susceptible to infection by avian reovirus, genus Orthoreovirus family Reoviridae, at a young age. Although chicks are protected by antibodies transferred from vaccinated maternal flocks, due to the many variants in the field, the efficiency of the vaccines is limited. The level of antibodies against viruses is generally determined by enzyme-linked immunosorbent assay (ELISA), using the whole virus as the antigen. This has some disadvantages: first, the test measures antibodies against all capsid proteins, most of which are irrelevant for neutralizing the virus, and as such does not reflect the real protection status; second, it is impossible to distinguish between vaccine- and infection-derived antibodies. In the case of a virus that changes frequently, a third disadvantage is the inability to distinguish among serotypes. The aim of this study was to develop a test that would address these concerns. Four prototypes of the avian reovirus protein sigma C were used as antigens on the ELISA plate. Sigma C is the main protein inducing neutralizing antibodies and the most variable among strains and isolates, and it is used for reovirus classification. This differentiating ELISA enabled distinguishing between vaccine and field strains of the virus, identifying the infection source, and in the case of vaccination, exclusively determining the level of protective antibodies. Whereas the whole virus detected antibodies against all strains, differentiating ELISA enabled differentiating between infected and vaccinated animals (DIVA) and in most cases, identifying the sigma C genotype. In a field study, a correlation was found between disease symptoms and antibodies identified against virulent strains in the flock. Thus virulent strains can be identified in the field, enabling adjustment of the relevant vaccines., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Avian orthoreoviruses (ARVs) are responsible for considerable economic losses in broiler chickens; yet, the genetic characterization of most ARV strains is limited to a few genes, and the full coding region has been determined for only S1133 and 138, two ARV strains associated with tenosynovitis. Recently, in parts of the United States, ARVs with novel neutralization antigen type were isolated from chickens afflicted with runting-stunting syndrome. One such strain, AVS-B, was selected for full genome sequencing and phylogenetic analysis. The complete genome was 23,494 bp in size and included 12 open reading frames. The lengths of the coding regions, as well as those of the 5' and 3' ends, were fairly well conserved between AVS-B and other reference strains. In pairwise comparisons to the S1133 and 138 strains, the AVS-B strain shared a wide range of sequence identities along each genome segment, i.e., a range of 54-55% for the σC coding region of S1 genome segment and 91-93% for the S2 genome segment. Phylogenetic analyses of individual genes of AVS-B did not identify any single common ancestor among more completely characterized ARV strains for which sequence data are available. One exception to this lack of identity was strain 138, which shared 90-93% nt identity with AVS-B along seven of ten genome segments; only M2, M3, and S1 segments of these strains shared lower sequence identities. Collectively, our analyses indicated that multiple reassortment events and strong divergence caused by the accumulation of point mutations could have led to the observed assortment and genetic heterogeneity of the AVS-B genome.

The effects of avian reovirus (ARV) p17 protein on cell cycle progression and host cellular protein translation were studied. ARV infection and ARV p17 transfection resulted in the accumulation of infected and/or transfected cells in the G(2)/M phase of the cell cycle. The accumulation of cells in the G(2)/M phase was accompanied by upregulation and phosphorylation of the G(2)/M-phase proteins ATM, p53, p21(cip1/waf1), Cdc2, cyclin B1, Chk1, Chk2, and Cdc25C, suggesting that p17 induces a G(2)/M cell cycle arrest through activation of the ATM/p53/p21(cip1/waf1)/Cdc2/cyclin B1 and ATM/Chk1/Chk2/Cdc25C pathways. The G(2)/M cell cycle arrest resulted in increased virus replication. In the present study, we also provide evidence demonstrating that p17 protein is responsible for ARV-induced host cellular protein translation shutoff. Increased phosphorylation levels of the eukaryotic translation elongation factor 2 (eEF2) and initiation factor eIF2alpha and reduced phosphorylation levels of the eukaryotic translation initiation factors eIF4E, eIF4B, and eIF4G, as well as 4E-BP1 and Mnk-1 in p17-transfected cells, demonstrated that ARV p17 suppresses translation initiation factors and translation elongation factors to induce host cellular protein translation shutoff. Inhibition of mTOR by rapamycin resulted in a decrease in the levels of phosphorylated 4E-BP1, eIF4B, and eIF4G and an increase in the levels eEF2 but did not affect ARV replication, suggesting that ARV replication was not hindered by inhibition of cap-dependent translation. Taken together, our data indicate that ARV p17-induced G(2)/M arrest and host cellular translation shutoff resulted in increased ARV replication.

Avian reovirus (ARV) strain S1133 causes apoptosis in host cells in the middle to late stages of infection. This study investigated the early-stage biological response and intracellular signaling in ARV S1133-infected Vero and chicken cells. Treatment with conditioned medium from ARV S1133-infected cells increased the chemotactic activity of U937 cells. Neutralizing antibodies against IL-1beta and IL-6 showed that both cytokines contribute to viral-induced inflammation but neither affect cell survival. Inhibition of Akt, NF-kappaB, and Stat3 released the chemotactic activity and anti-apoptotic effect elicited by ARV S1133. ARV S1133 activated PI 3-kinase-dependent Akt/NF-kappaB and p70 S6 kinase, as well as Stat3; however, p70 S6 kinase was not involved in ARV S1133-mediated effects. DF1 cells over-expressing constitutively active PI 3-kinase and Stat3 showed association with enhancement of anti-apoptotic activity. In conclusion, in the early stages of ARV S1133 infection, activation of cell survival signals contributes to virus-induced inflammation and anti-apoptotic response., (Copyright 2010 Elsevier Inc. All rights reserved.)

Stimulated by energetic stress, AMP-activated protein kinase (AMPK) controls several cellular functions. It was discovered here that infection of Vero cells with avian reovirus (ARV) upregulated AMPK and mitogen-activated protein kinase (MAPK) p38 phosphorylation in a time- and dose-dependent manner. Being an energy status sensor, AMPK is potentially an upstream regulator of MAPK p38. Treatment with 5-amino-4-imidazolecarboxamide ribose (AICAR), a well-known activator of AMPK, induced phosphorylation of MAPK p38. Unlike AICAR, wortmannin or rapamycin did not induce phosphorylation of MAPK p38, suggesting that mTOR inhibition is not a determining factor in MAPK p38 phosphorylation. Inhibition of AMPK by compound C antagonized the effect of AICAR on MAPK p38 in Vero cells. Specific inhibition of AMPK by small interfering RNA or compound C also suppressed ARV-induced phosphorylation of MAPK kinase (MKK) 3/6 and MAPK p38 in Vero and DF-1 cells, thereby providing a link between AMPK signalling and the MAPK p38 pathway. The mechanism of ARV-enhanced phosphorylation of MKK 3/6 and MAPK p38 in cells was not merely due to glucose deprivation, a probable activator of AMPK. In the current study, direct inhibition of MAPK p38 by SB202190 decreased the level of ARV-induced syncytium formation in Vero and DF-1 cells, and decreased the protein levels of ARV sigma A and sigma C and the progeny titre of ARV, suggesting that activation of MAPK p38 is beneficial for ARV replication. Taken together, these results suggested that AMPK could facilitate MKK 3/6 and MAPK p38 signalling that is beneficial for ARV replication. Although well studied in energy metabolism, this study provides evidence for the first time that AMPK plays a role in modulating ARV and host-cell interaction.

The interplay between avian reovirus (ARV) replication and apoptosis and proteasome pathway was studied in cultured cells. It is shown that inhibition of the proteasome did not affect viral entry and host cell translation but had influence on ARV replication and ARV-induced apoptosis. Evidence is provided to demonstrate that ubiquitin-proteasome blocked ARV replication at an early step in viral life cycle. However, viral transcription and protein translation were also reduced markedly after addition of proteasome inhibitor MG132. Treatment of BHK-21 cells with the MG132 markedly decreased virus titer as well as prevented virus-induced apoptosis. The expression of ARV proteins sigmaC, sigmaA, and sigmaNS was also reduced markedly, suggesting that suppression of virus replication is due to down-regulation of these ARV proteins by ubiquitin-proteasome system. MG132 was also shown to suppress ARV sigmaC-induced phosphrylation of p53 on serine 46, caspase 3 activities, and DNA fragmentation leading to complete inhibition of ARV-induced apoptosis.

The influences of avian reovirus (ARV) infection at 1 day of age on bursa development, antibody responses after vaccinations to avian influenza virus (AIV), Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV), and pathogenecity of virulent IBDV (v-IBDV) were studied in chickens of SPF-origin. The results indicated that LY strain ARV infection in 1-day-old chickens caused atrophy of the Bursa and decreased lymphocyte numbers in the bursa, but it gave no significant negative effects on growth rates and antibody titers to AIV and NDV after vaccination. LY strain ARV infection decreased antibody titers to IBDV in B87 vaccinated birds but all vaccinated birds infected with ARV were still full protected from death or clinical syndromes after v-IBDV challenge. Although all B87-vaccinated birds were full protected from death after v-IBDV challenges, the antibody titers to AIV or NDV after vaccinations with inactivated vaccines were significantly lower in v-IBDV challenged birds than controls. Supprisingly, ARV infection at ages of 1-7 days could compromise the immune suppression induced by v-IBDV in B87-vaccinated birds as HI antibody titers to AIV and NDV in ARV-infected groups were significantly higher than chicknes with no ARV infection. A discussion was made on the interactions between ARV infection and vaccine IBDV or v-IBDV to explain such sophisticated phenomena in the bird experiments.

The present study describes the pathogenesis of infection of chicks with a new avian reovirus strain, belonging to the so-called enteric reovirus strains (ERS) that is capable of causing central nervous system signs in SPF white leghorns. After intramuscular (IM) or oral inoculation birds were either observed for clinical signs or sacrificed for macroscopic, histological and virological examination for 21 days. Virus isolation was performed on the brain, leg muscle, hock joint, liver and spleen. For the detection of viral antigen the immunohistochemistry (IHC) technique was performed on the caudal part of the cerebrum, spinal cord including spinal ganglia and right N. Ischiadicus. High mortality (79% in 7 days) was seen in birds that were inoculated IM. Survivors were depressed and stayed small until the end of the experiment. One bird had tremor and showed torticollis at 9 days after IM inoculation. Birds that were inoculated orally were depressed from day 4 and stayed small until the end of the experiment. One bird showed a torticollis at 10 days after inoculation. After both IM and oral inoculation ERS was isolated from the brain between 3 and 10 days after inoculation. Other examined organs were positive for virus isolation from day 1 or 5 until day 21. IHC revealed viral antigen positive cells in the Plexus chorioideus (plexus epithelial cells or cells within the underlying connective tissue) and in a spinal ganglion. The results indicate that the pathogenesis of ERS infection in chickens bears some resemblance with that of the mammalian reoviruses serotype 1 in mice.

Reoviruses have recently been shown to be associated with disease in young geese and to be involved in epizooties of severe outcome in Hungary. To assess the genetic variability among these pathogenic goose reoviruses (GRVs), we sequenced the S4 genome segment of five GRV strains isolated from different diseased flocks. We found that the GRV S4 genome segment, consisting of two partially overlapping open reading frames (ORFs), shares substantial structural similarity with its counterpart in muscovy duck reoviruses (DRVs). ORF1 is predicted to encode a polypeptide highly similar to the p10 polypeptide of DRV, and ORF2 supposedly encodes the minor outer capsid protein, sigma1/sigmaC. In one of the five GRV strains examined, we identified a single uracil base insertion close to the middle of ORF2. This insertion resulted in a frameshift and in concomitant acquisition of a termination codon (UAA) a few codons downstream, apparently causing truncation of the C-terminal part of the protein. The functional consequences of this assumed mutation, which would result in loss of more than a half of the protein, have yet to be determined. Nonetheless, the sequence and structural similarities between the genome segment encoding sigmal/sigmaC in GRVs and DRVs suggest that these viruses belong to a species distinct from other established species within subgroup 2 of orthoreoviruses.

Previous attempts to extend the host range of the avian sarcoma/leukosis virus (ASLV)-based RCASBP vectors produced two viral vectors, RCASBP M2C (4070A) and RCASBP M2C (797-8), which replicate using the amphotropic murine leukemia virus 4070A Env protein (2). Both viruses were adapted to replicate efficiently in the avian cell line DF-1, but RCASBP M2C (4070A) caused extensive cytopathic effects (CPE) in DF-1 cells whereas RCASBP M2C (797-8) induced low levels of CPE. The two viruses differed only at amino acid 242 of the polyproline-rich region in the surface (SU) subunit of the Env protein. In RCASBP M2C (4070A), an isoleucine replaced the wild-type proline residue, whereas a threonine residue was found in RCASBP M2C (797-8). In the present study, we show that other amino acid substitutions at position 242 strongly influence the CPE and replication rate of the chimeric viruses. There was a correlation between the amount of unintegrated linear retroviral DNA present in infected DF-1 cells and the level of CPE. This suggests that there may be a role for superinfection in the CPE. The treatment of RCASBP M2C (4070A)-infected cells with dantrolene, which inhibits the release of calcium from the endoplasmic reticulum (ER), reduced the amount of CPE seen during infection with the highly cytotoxic virus. Dantrolene treatment did not appear to affect virus production, suggesting that Ca2+ release from the ER had a role in the CPE caused by these viruses.

Avian reoviruses that have been shown to be genetically distinct from chicken origin reoviruses were isolated from commercial turkey flocks in the Southeastern US and Texas that were experiencing enteritis. The pathogenesis of these turkey origin reoviruses (TRVs) was evaluated in commercial and specific pathogen free (SPF) turkey poults and SPF chickens. Mortality, clinical disease, gross lesions, microscopic lesions and body weights were observed. TRVs replicated poorly and did not cause disease in chickens. Clinical disease induced by the TRV isolates, characterized by diarrhoea and depression, was mild in both SPF and commercial origin poults. Several TRV isolates caused moderate to severe bursal atrophy in poults. Additionally, each of the TRV isolates caused significant body weight decreases in SPF and/or commercial poults as compared with sham inoculates. Molecular characterization of the isolates revealed that the TRVs and chicken origin reoviruses had identical electropherotype profiles.

The avian reovirus non-structural protein sigma NS has previously been shown to bind single-stranded (ss) RNA in vitro in a sequence-independent manner. The results of the present study further reveal that sigma NS binds poly(A), poly(U) and ssDNA, but not poly(C), poly(G) or duplex nucleic acids, suggesting that sigma NS has some nucleotide-sequence specificity for ssRNA binding. The current findings also show that sigma NS is present in large ribonucleoprotein complexes in the cytoplasm of avian reovirus-infected cells, indicating that it exists in intimate association with ssRNAs in vivo. Removal of RNA from the complexes generates a sigma NS protein form that sediments between 4.5 and 7 S, suggesting that RNA-free sigma NS associates into small oligomers. Expression and purification of recombinant sigma NS in insect cells allowed us to generate specific antibodies and to perform a variety of assays. The results of these assays revealed that: (i) RNA-free sigma NS exists as homodimers and homotrimers; (ii) the minimum RNA size for sigma NS binding is between 10 and 20 nt; (iii) sigma NS does not have a preference for viral mRNA sequences; and (iv) its RNA-binding activity is conformation-dependent. Baculovirus expression of point and deletion sigma NS mutants in insect cells showed that the five conserved basic amino acids that are important for RNA binding and ribonucleoprotein-complex formation are dispersed throughout the entire sigma NS sequence, suggesting that this protein binds ssRNA through conformational domains. Finally, the properties of the avian reovirus protein sigma NS are compared with those of its mammalian reovirus counterpart.

Avian reoviruses (ARV) are economically important pathogens, especially in the poultry industry, where they cause viral arthritis and tenosynovitis. Mycophenolic acid (MPA) is an inhibitor of inosine monophosphate dehydrogenase (mainly used clinically for immunosuppression) that inhibits the replication of several viruses. We demonstrate in this study that MPA also is capable of inhibiting ARV replication in QM5 quail fibrosarcoma cells. The selectivity index of MPA in QM5 cells was determined as approximately 41. Concentrations of > or =3 microg/ml MPA inhibited infectious ARV progeny production in QM5 cells by more than 100-fold. Inhibition of ARV replication also was seen in other cell lines, including HD-11 and Vero. Addition of exogenous guanosine to MPA-treated ARV-infected QM5 cells restored viral replicative capacity to nearly normal levels.

Members of our laboratory previously generated and described a set of avian reovirus (ARV) temperature-sensitive (ts) mutants and assigned 11 of them to 7 of the 10 expected recombination groups, named A through G (M. Patrick, R. Duncan, and K. M. Coombs, Virology 284:113-122, 2001). This report presents a more detailed analysis of two of these mutants (tsA12 and tsA146), which were previously assigned to recombination group A. The capacities of tsA12 and tsA146 to replicate at a variety of temperatures were determined. Morphological analyses indicated that cells infected with tsA12 at a nonpermissive temperature produced approximately 100-fold fewer particles than cells infected at a permissive temperature and accumulated core particles. Cells infected with tsA146 at a nonpermissive temperature also produced approximately 100-fold fewer particles, a larger proportion of which were intact virions. We crossed tsA12 with ARV strain 176 to generate reassortant clones and used them to map the temperature-sensitive lesion in tsA12 to the S2 gene. S2 encodes the major core protein sigmaA. Sequence analysis of the tsA12 S2 gene showed a single alteration, a cytosine-to-uracil transition, at nucleotide position 488. This alteration leads to a predicted amino acid change from proline to leucine at amino acid position 158 in the sigmaA protein. An analysis of the core crystal structure of the closely related mammalian reovirus suggested that the Leu(158) substitution in ARV sigmaA lies directly under the outer face of the sigmaA protein. This may cause a perturbation in sigmaA such that outer capsid proteins are incapable of condensing onto nascent cores. Thus, the ARV tsA12 mutant represents a novel assembly-defective orthoreovirus clone that may prove useful for delineating virus assembly.

The avian reovirus (ARV) infection is associated with various disease conditions in poultry. However, the pathogenesis mechanisms are poorly characterized. In the present study, we clearly demonstrated that the sigmaC of ARV S1133 strain induced apoptosis in both BHK-21 and Vero cells. Five kinds of assays for apoptosis were used in analyzing ARV-infected BHK-21 and Vero cells: (1) assay for DNA ladders, (2) ELISA detection of cytoplasmic histone-associated DNA fragments, (3) nuclear staining with acridine orange, (4) Western blot, Northern blot, and immunofluorescent assay (IFA), and (5) flow cytometric analysis. The sigmaC protein of ARV could elicit apoptosis occurring in a dose- and time-dependent manner. The current results further our understanding of the function of sigmaC in cultured cells and suggest that sigmaC is a viral-encoded apoptin and possesses apoptosis-inducing ability. Furthermore, deletion analysis of the ARV sigmaC protein suggests that the carboxyl-terminus of sigmaC is important in mediating sigmaC-induced apoptosis because its deletion abolished the induction of apoptosis.

The results of a previous study demonstrated that avian reovirus is highly resistant to the antiviral effects of interferon and suggested that the double-stranded RNA (dsRNA)-binding sigmaA protein might play an important role in that resistance. To gather more evidence on the interferon-inhibitory activity of sigmaA protein, its gene was cloned into the prokaryotic maltose-binding protein (MBP) gene fusion vector pMalC and into the recombinant vaccinia virus WRS2. The two recombinant sigmaA proteins displayed a dsRNA-binding affinity similar to that of sigmaA protein synthesized in avian reovirus-infected cells. Interestingly, MBP-sigmaA, but not MBP, was able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates by blocking the activation of endogenous dsRNA-dependent enzymes. In addition, transient expression of sigmaA protein in HeLa cells rescued gene expression of a vaccinia virus mutant lacking the E3L gene, and insertion of the sigmaA-encoding gene into vaccinia virus conferred protection for the virus against interferon in chicken cells. Further studies demonstrated that expression of recombinant sigmaA in mammalian cells interfered with dsRNA-dependent protein kinase (PKR) function. From these results we conclude that sigmaA is capable of reversing the interferon-induced antiviral state by down-regulating PKR activity in a manner similar to other virus-encoded dsRNA-binding proteins.

The enteropathogenicity of avian reoviruses (ARVs), isolated from chickens affected with malabsorption syndrome (MAS) from The Netherlands and Germany was studied. In the first trial seven different ARVs isolated from MAS cases were inoculated in 1-day-old specific pathogenic free (SPF) white leghorns. The pathogenicity was compared with 2 ARVs isolated from cases of tenosynovitis, namely reference strain S1133 and a Dutch strain. Although a difference in the severity of the clinical disease was observed, all reoviruses could induce vacuolar degeneration and sloughing of the epithelium of the small intestine at day 2 post inoculation (PI) till day 7 PI. Two Dutch and one German ARV derived from MAS causing the most severe intestinal lesions at day 2 PI, were further studied in the second trial using SPF broilers. These reoviruses did not cause weight gain depression in the broilers although lesions in the small intestine were present from day 1 up to day 4 PI and were more severe than in the white leghorn chickens. In one of the inoculated groups apical denuded villi were already present at day 1 PI. At day 7 PI the small intestine of the infected broilers appeared to be normal. Reovirus antigen was detected in the cytoplasm of the enterocytes at the tip and middle section of the affected villi both in layers and in broilers. To study the role of intestinal CD4+ and CD8+ T-cells and macrophages/monocytes in the pathogenesis of ARV, the numbers of these cells of the jejunal villi of one infected and the control broiler groups were compared. CD4+ T-cells were detected in low numbers and only in the infected broiler group at day 14 PI. The numbers of CD8+ T-cells and macrophages/monocytes were significantly higher in the infected broiler group than in the control broiler group at day 7 and 14 PI and at day 7 PI respectively. Our study indicates that the reovirus alone cannot induce intestinal lesions as found in MAS chickens. Moreover, CD8+ T-cells may play a major role in the pathogenesis and or reovirus clearance in the small intestine.

An avian reovirus, ARV-CU98, has recently been isolated from poults experiencing poult enteritis and mortality syndrome (PEMS). To further understand ARV-CU98 and its role in PEMS, the current study investigates interactions of ARV-CU98 with various cell types in vitro. When macrophages, B cells, T cells, and liver cells of chicken or turkey origin were co-incubated with ARV-CU98, only cells of liver origin demonstrated cytopathic effects, the presence of viral antigen, and reduced metabolic activity over time. Furthermore, distinctive pockets of viral particles were evident in electron microscopic examination of a chicken hepatocellular carcinoma (LMH) cell line, but not in a chicken macrophage cell line (MQ-NCSU) co-incubated with virus. Additional evidence of viral replication in LMH, cells but not MQ-NCSU cells was demonstrated by the presence of two viral bands (43 and 145 kD size) in cell lysates from LMH cells exposed to ARV-CU98. Although not capable of being infected by ARV-CU98, MQ-NCSU cells do appear to be activated by the virus since IL-1 mRNA expression is increased in MQ-NCSU cells 2 h after addition of the virus. LMH cells exposed to the virus demonstrate a decrease in IL-1 mRNA expression by 8 to 10 h after addition of the virus, perhaps corresponding to the initiation of infection by the virus. In conclusion, this study demonstrates that ARV-CU98 actively infects and replicates in LMH cells, but not in lymphocytes or macrophages, suggesting that the liver may be a target and site of replication of ARV-CU98 in poults experiencing PEMS.

The cytopathic effect evidenced by cells infected with avian reovirus S1133 suggests that this virus may induce apoptosis in primary cultures of chicken embryo fibroblasts. In this report we present evidence that avian reovirus infection of cultured cells causes activation of the intracellular apoptotic program and that this activation takes place during an early stage of the viral life cycle. The ability of avian reoviruses to induce apoptosis is not restricted to a particular virus strain or to a specific cell type, since different avian reovirus isolates were able to induce apoptosis in several avian and mammalian cell lines. Apoptosis was also provoked in ribavirin-treated avian reovirus-infected cells and in cells infected with UV-irradiated reovirions, indicating that viral mRNA synthesis and subsequent steps in viral replication are not needed for apoptosis induction in avian reovirus-infected cells and that the number of inoculated virus particles, not their infectivity, is the critical factor for apoptosis induction by avian reovirus. Our finding that apoptosis is no longer induced when intracellular viral uncoating is blocked indicates that intraendosomal virion disassembly is required for apoptosis induction and that attachment and uptake of parental reovirions are not sufficient to cause apoptosis. Taken together, our results suggest that apoptosis is triggered from within the infected cell by viral products generated after intraendosomal uncoating of parental reovirions.

Samples of brain, intestine, liver, lung, spleen, and bursa of Fabricius were collected from five common eider (Somateria mollissima) duckling carcasses during a die-off in the western Gulf of Finland (59 degrees 50'N, 23 degrees 15'E) in June 1996. No viral activity was observed in specific-pathogen-free chicken embryos inoculated with tissue suspensions, but samples of bursa of Fabricius from three birds were positive when inoculated into Muscovy duck (Cairina moschata) embryo fibroblasts. The isolates were characterized as nonenveloped RNA viruses and possessed several characteristics of the genus Orthoreovirus. Virus particles were icosahedral with a mean diameter of 72 nm and were stable at pH 3.0; their genome was separated into 10 segments by polyacrylamide gel electrophoresis. Mallard (Anas platyrhynchos) ducklings experimentally infected with the eider reovirus showed elevated serum activities of aspartate aminotransferase, creatine kinase, and lactate dehydrogenase enzymes and focal hemorrhages in the liver, spleen, and bursa of Fabricius. During 1997-99, the prevalence of neutralizing antibodies to the isolated virus ranged from 0 to 86% in 302 serum samples collected from incubating eider hens at three nesting areas along coastal Finland. The highest seroprevalence was found in Hanko in 1999, just weeks before reports of an uninvestigated mortality event resulting in the death of an estimated 98% of ducklings at that location. These findings raise the question of potential involvement of the virus in poor duckling survival and eider population declines observed in several breeding areas along coastal Finland since the mid-1980s.

Poult enteritis and mortality syndrome (PEMS) is an acute, infectious intestinal disease of turkey poults, characterized by high mortality and 100% morbidity, that decimated the turkey industry in the mid-1990s. The etiology of PEMS is not completely understood. This report describes the testing of various filtrates of fecal material from control and PEMS-affected poults by oral inoculation into poults under experimental conditions, the subsequent isolation of a reovirus, ARV-CU98, from one of the PEMS fecal filtrates, and in vivo and in vitro studies conducted to determine the pathogenicity of ARV-CU98 in turkey poults. In order to identify a filtrate fraction of fecal material containing a putative etiologic agent, poults were challenged in two independent experiments with 220- and 100-nm filtrates of fecal material from PEMS-negative and PEMS-positive poults. The 100-nm filtrate was chosen for further evaluation because poults inoculated with this filtrate exhibited mortality and significantly lower (P < or = 0.05) body weight and relative bursa weight, three clinical signs associated with PEMS. These results were confirmed in a third experiment with 100-nm fecal filtrates from a separate batch of PEMS fecal material. In Experiment 3, body weight and relative bursa and thymus weights were significantly lower (P < or = 0.05) in poults inoculated with 100-nm filtrate of PEMS fecal material as compared with poults inoculated with 100-nm filtrate of control fecal material. Subsequently, a virus was isolated from the 100-nm PEMS fecal filtrate and propagated in liver cells. This virus was identified as a reovirus on the basis of cross-reaction with antisera against avian reovirus (FDO strain) as well as by electrophoretic analysis and was designated ARV-CU98. When inoculated orally into poults reared under controlled environmental conditions in isolators, ARV-CU98 was associated with a higher incidence of thymic hemorrhaging and gaseous intestines. In addition, relative bursa and liver weights were significantly lower (P < or = 0.05) in virus-inoculated poults as compared with controls. Virus was successfully reisolated from virus-challenged poults but not from control birds. Furthermore, viral antigen was detected by immunofluorescence in liver sections from virus-challenged poults at 3 and 6 days postinfection and virus was isolated from liver at 6 days postinfection, suggesting that ARV-CU98 replicates in the liver. In addition to a decrease in liver weight, there was a functional degeneration as indicated by altered plasma alanine aminotransferase and aspartate aminotransferase activities in virus poults as compared with controls. Although this reovirus does not induce fulminating PEMS, our results demonstrated that ARV-CU98 does cause some of the clinical signs in PEMS, including intestinal alterations and significantly lower relative bursa and liver weights. ARV-CU98 may contribute directly to PEMS by affecting the intestine, bursa, and liver and may contribute indirectly by increasing susceptibility to opportunistic pathogens that facilitate development of clinical PEMS.

Attempts to reproduce malabsorption syndrome (MAS) by oral inoculation with several different combinations including intestinal homogenate, reovirus, and hemolytic Escherichia coli obtained from MAS-affected chickens and intestinal homogenate from healthy chickens (healthy homogenate) were performed in 1-day-old specific-pathogen-free (SPF) broilers. The MAS homogenate, serving as a positive control, induced weight gain depression and intestinal lesions such as cystic crypts of Lieberkuhn, villus atrophy, and lymphoid and/or granulocytic infiltration. The healthy homogenate, the formalin-treated MAS homogenate, the formalin-treated healthy homogenate, and phosphate-buffered saline caused neither weight gain depression nor intestinal lesions. We were able to reproduce both weight gain depression and intestinal lesions by inoculation of reovirus either combined with the formalin-treated MAS homogenate or combined with healthy homogenate. Surprisingly, when hemolytic E. coli was added to the combination of reovirus with formalin-treated MAS homogenate, this did not cause weight gain depression although this combination caused the described intestinal lesions. Identical results were obtained with the combination of formalin-treated MAS homogenate with hemolytic E coli or the combination of reovirus with hemolytic E. coli. The intestinal lesions were more severe and developed faster by combinations including reovirus and formalin-treated MAS homogenate. This study indicates that a combination of enteropathogenic reovirus with other agents or substances that are present in an intestinal homogenate from MAS-affected and healthy chickens can induce MAS in SPF broilers. Escherichia coli is not essential for induction of weight gain depression but can play a role in development of intestinal lesions. Furthermore, intestinal lesions alone will not always result in weight gain depression.

The replication of two pathotypes of avian reovirus, 1733 and 2177 in transformed chicken lymphoid and myeloid cell lines was examined, showing that only the macrophage cell line, HD11, supports replication. The virulent strain 1733 causes a lytic infection producing 100-1000 fold more virus than the avirulent strain 2177. Cells infected with strain 2177 display delayed viral RNA and protein synthesis as well as a suppressed expression of the major capsid protein muB. These features may contribute to the lower virulence of the strain 2177 in their natural host in vivo.