Avian Reovirus Research Articles

The nonstructural p17 protein of a fusogenic bat-borne reovirus regulates viral replication in virus species- and host-specific manners

Nelson Bay orthoreovirus (NBV), a member of the family Reoviridae, genus Orthoreovirus, is a bat-borne virus that causes respiratory diseases in humans. NBV encodes two unique nonstructural proteins, fusion-associated small transmembrane (FAST) protein and p17 protein, in the S1 gene segment. FAST induces cell–cell fusion between infected cells and neighboring cells and the fusogenic activity is required for efficient viral replication. However, the function of p17 in the virus cycle is not fully understood. Here, various p17 mutant viruses including p17-deficient viruses were generated by a reverse genetics system for NBV. The results demonstrated that p17 is not essential for viral replication and does not play an important role in viral pathogenesis. On the other hand, NBV p17 regulated viral replication in a bat cell line but not in other human and animal cell lines. Nuclear localization of p17 is associated with the regulation of NBV replication in bat cells. We also found that p17 dramatically enhances the cell–cell fusion activity of NBV FAST protein for efficient replication in bat cells. Furthermore, we found that a protein homologue of NBV p17 from another bat-borne orthoreovirus, but not those of avian orthoreovirus or baboon orthoreovirus, also supported efficient viral replication in bat cells using a p17-deficient virus-based complementation approach. These results provide critical insights into the functioning of the unique replication machinery of bat-borne viruses in their natural hosts.

Identification and Functional Analyses of Host Proteins Interacting with the p17 Protein of Avian Reovirus.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome. However, the precise molecular mechanism remains unclear. Here, we report the host cellular proteins that interact with ARV p17 by yeast two-hybrid screening. In this study, the p17 gene was cloned into pGBKT7 to obtain the bait plasmid pGBKT7-p17. After several rounds of screening of a chicken cDNA library, 43 positive clones were identified as possible host factors that interacted with p17. A BLAST search of the sequences was performed on the NCBI website, which ultimately revealed 19 interacting proteins. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genome analyses indicated that the acquired proteins were involved in multicellular organismal processes, metabolic processes, and biological regulation. When the subcellular localization of the host protein and ARV p17 protein was investigated, we observed colocalization of p17-GFP with IGF2BP1-RED and PQBP1-RED in the transfected cells but not with FGF1-RED. The direct interaction of ARV p17 protein with IGF2BP1 and PQBP1 was confirmed by coimmunoprecipitation and GST pulldown assays. We used RT-qPCR to assess the expression variation during ARV infection. The results showed that IGF2BP1, PAPSS2, RPL5, NEDD4L, PRPS2 and IFI16 were significantly upregulated, whereas the expression of FGF1, CDH2 and PQBP1 was markedly decreased in DF-1 cells infected with ARV. Finally, we demonstrated that IGF2BP1 had a positive effect on ARV replication, while PQBP1 had the opposite effect. Our findings provide valuable information for better insights into ARV's pathogenesis and the role of the p17 protein in this process.

Molecular Detection, Characterisation and Serological Survey of Chicken Astrovirus from Broiler Flocks in Malaysia

Astroviruses have been associated with enteric and extra-intestinal disorders in many animal species, including chickens. Here, we describe the detection and characterisation of chicken astrovirus (CAstV) in broilers and its seroprevalence in broiler breeder flocks. Based on PCR protocol, viral confirmation was carried out on clinical tissue samples from broiler chickens suffering from uneven growth and poor performance. The tissues were molecularly detected for CAstV with differential diagnostic testing against the Newcastle disease virus, infectious bronchitis virus, avian nephritis virus, avian rotavirus, fowl adenovirus and avian reovirus. Polymerase gene-based phylogenetic analyses of the twenty samples detected positive for CAstV indicate they belong to Group I and are related to strains from the US, UK, India and Poland. From these 20 samples, CastV could be isolated from 3 samples upon inoculation in 5-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECE); virus-infected embryos showed dwarfing, haemorrhages, oedema and gelatinous lesions at harvest. The enzyme-linked immunosorbent assay (ELISA) results revealed a high prevalence of antibodies against CAstV amongst the broiler breeder flocks tested. It is the first study that describes the detection and prevalence of CAstV in broiler chickens and broiler breeder flocks in Malaysia.

Rapid and visual detection of Mycoplasma synoviae by recombinase-aided amplification assay combined with a lateral flow dipstick

Mycoplasma synoviae (MS) is an important avian pathogen that has brought substantial economic losses to the global poultry industry. Fast and accurate diagnosis is one of the critical factors for the control of MS infection. This study established a simple, rapid and visual detection method for MS using a recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). The reaction temperature and time of the RAA-LFD assay were optimized after selecting the primers and probe, and the specificity and sensitivity rates were analyzed. The results showed that RAA could amplify the target gene in 20 min at a constant temperature of 38°C, and the amplification products could be visualized by LFD within 5 min. There was no cross-reaction with Mycoplasma gallisepticum (MG), Pasteurella multocida (P. multocida), Escherichia coli (E. coli), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian reovirus (ARV). Furthermore, the RAA-LFD assay exhibited high sensitivity with a detection limit of 10 copies/μL. A total of 128 clinical samples with suspected infection of MS were tested by RAA-LFD, PCR, and real-time fluorescence quantitative PCR (RFQ-PCR). The coincidence rate of the detection results was 95.3% between RAA-LFD and PCR, and 98.4% between RAA-LFD and RFQ-PCR. These results suggested that the RAA-LFD method established in the present study was easy to use and was associated with strong specificity and high sensitivity. This method was very suitable for the rapid detection of MS in clinical practice.

Virulence of Emerging Arthrotropic Avian Reoviruses Correlates With Their Ability to Activate and Traffic Interferon-γ Producing Cytotoxic CD8+ T Cells Into Gastrocnemius Tendon.

Newly emerging arthrotropic avian reoviruses (ARVs) are genetically divergent, antigenically heterogeneous, and economically costly. Nevertheless, the mechanism of emerging ARV-induced disease pathogenesis and potential differences in virulence between virus genotypes have not been adequately addressed. In this study, the life cycle of ARV, including the formation of cytoplasmic ARV neo-organelles, paracrystalline structures, and virus release mechanisms, were characterized in the infected host cell by transmission electron microscopy (TEM). In addition, progressive changes in the structure of infected cells were investigated by time-lapse and field emission scanning electron (FE-SE) microscopy. ARVs from the four genotypic cluster groups included in the study caused gross and microscopic lesions in the infected birds. Marked infiltration of γδT cells, CD4+ and CD8+ T lymphocytes were observed in ARV infected tendon tissues starting day 3 post-infection. The ARV variant from genotype cluster-2 triggered significantly high trafficking of IFN-γ producing CD8+ T lymphocytes in tendon tissues and concomitantly showed high morbidity and severe disease manifestations. In contrast, the ARV variant from genotype cluster-4 was less virulent, caused milder disease, and accompanied less infiltration of IFN-γ producing CD8+ T cells. Interestingly, when we blunted antiviral immune responses using clodronate liposomes (which depletes antigen-presenting cells) or cyclosporin (which inhibits cytokine production that regulates T-cell proliferation), significantly lower IFN-γ producing CD8+ T cells infiltrated into tendon tissues, resulting in reduced tendon tissues apoptosis and milder disease manifestations. In summary, these data suggest that the degree of ARV virulence and tenosynovitis/arthritis are potentially directly associated with the ability of the virus to traffic massive infiltration of cytotoxic CD8+ T cells into the infected tissues. Moreover, the ability to traffic cytotoxic CD8+ T cells into infected tendon tissues and the severity of tenosynovitis differ between variants from different ARV genotype cluster groups. However, more than one virus isolate per genotype group needs to be tested to further confirm the association of pathogenicity with genotype. These findings can be used to further examine the interaction of viral and cellular pathways which are essential for the pathogenesis of the disease at the molecular level and to develop effective disease control strategies.

Development of an indirect enzyme-linked immunosorbent assay for the detection of novel chicken orthoreovirus.

A novel avian orthoreovirus (N-ARV) variant characterized with obvious arthritis and synovial inflammation, was isolated from Shandong, China in May 2016. It caused chicken poor growth and enormous economic losses to the poultry industry of China. However, there are few effective methods for detecting the antibody levels of N-ARV. In this study, a viral structural protein σC was expressed using the prokaryotic expression vector pET32a (+). The target protein was obtained by inducing for 6 hours at an IPTG concentration of 0.6mM. The optimal dilution of the coating antigen and serum antibody were determined to be 1000 fold and 10 fold respectively. A specificity test showed that there was no positive reactivity between N-ARV and other pathogens, and when the positive serum was diluted 100 times detection results were still checkable. The repeatability of this method was determined by the inter assay and intra assay tests with variability ranging from 4.85% to 7.93%. In conclusion, this indirect enzyme linked immunosorbent assay (ELISA) will be useful for large-scale serological surveys and monitoring antibody levels in N-ARV infection.

Avian Reoviruses From Wild Birds Exhibit Pathogenicity to Specific Pathogen Free Chickens by Footpad Route.

Avian reoviruses (ARVs) are ubiquitous in domestic poultry with 80% of them being non-pathogenic and they are frequently found in clinically healthy birds. ARVs have also been known to be the etiological agents of viral arthritis (VA), tenosynovitis, myocarditis, runting-stunting syndrome (RSS), and respiratory and enteric disease in chickens. Significant economic losses during the process of poultry husbandry are due, in part, to unmitigated ARV infections throughout the poultry industry. Recently, many isolates shared genetic similarities between those recovered from wild birds and those recovered from poultry. One explanation may be that there is a degree of spillover and spillback of ARVs between the two groups. However, studies on the role of wild birds in the epidemiology and pathogenicity of ARVs are insufficient. Here, we describe the pathogenicity in specific pathogen-free (SPF) chickens of ARV originating from wild birds. The challenge experiment was conducted in six groups including a negative control group, a positive control group (reference strain of S1133), and four groups (A15-157, A18-13, A18-205, A19-106) infected with ARVs from wild birds. The 7-day-old SPF chickens were inoculated with 106TCID50 ARV to evaluate the clinical signs, changes in weight gain, gross lesions, histological changes, virus replication, and serum antibody levels. The peak of clinical signs was from 3 to 5 days post infection (dpi). In addition, the death of one chicken was found in the group infected with the A18-13 isolate. Reduced body weight was also found in chickens infected with ARVs from wild birds compared to the negative control group. All the ARVs infection groups showed noticeable swelling of the footpad. In addition, ARVs were detected in the bursa, tendon, and hock joint by reverse transcription-polymerase chain reaction (RT-PCR) in all infected groups at 5 and 15 dpi. Histopathological observations revealed acute inflammatory responses on the synovium covering the joint surfaces (arthritis) and tendon sheaths (tenosynovitis), as well as bursa atrophy and lymphocyte depletion. The analysis of the humoral response was performed by ELISA assay, and chickens infected with ARVs showed seroconverted. In conclusion, this study described the typical severe disease of acute VA and tenosynovitis in SPF chickens infected with ARVs derived from wild birds. This study confirmed the pathogenicity of ARVs infection in SPF chickens for the first time, and these results enrich our understanding of the pathogenicity of ARVs derived from wild birds.

Analysis of diagnostic cases of turkey viral enteritis in commercial turkey poults in California

Poult enteritis complex is a multifactorial intestinal disease of young turkeys, which results in considerable economic losses for commercial turkey producers. The objectives of this study were to determine the current prevalence of viral enteritis in California turkeys, assess the methods of detection and the gross and histological presentations of laboratory case submissions. The electronic database at the California Animal Health and Food Safety ( CAHFS ) system was searched for necropsy cases in which intestinal content from commercial turkey poults was submitted for transmission electron microscopy ( TEM ) and/or quantitative reverse transcriptase ( qRT )-PCR for enteric viruses for the period February 1, 2020 to February 1, 2021. Based on these search parameters, the study identified and analyzed a total of 66 poult necropsy cases originating from 22 commercial turkey premises. A diagnosis of viral enteritis was made in 43 of the 66 cases (65.1%) screened for enteric viruses by TEM and/or qRT-PCR. The viruses detected in this study included turkey astroviruses, avian rotavirus, avian reovirus, avian enteric picornavirus, and avian parvovirus. Turkey astroviruses species (32/43, 74.4%) were the most frequently detected viruses, with turkey astrovirus-2 (24/43, 55.8%) being the most common species detected amongst all enteric viruses. A single enteric virus was detected in 44.2% (19/43) of cases submissions, while 2 or more enteric viruses were detected in 55.8% (24/43) of submissions. The age at which enteric viruses were detected ranged from one to 7 wk of age, with the highest frequency of detection occurring in the first 3 wk of life (38/43, 88.4%, 95% CI: 1.2–13.3, odds ratio [ OR ] = 4.1). Within the first 3 wk, the majority (24/43, 55.8%) of poults positive for enteric viruses ranged between 1 and 7 d of age. Our assessment of the different diagnostic methods used at CAHFS indicates that the use of TEM in conjunction with qRT-PCR improved our ability to detect viruses in cases where 2 or more enteric viruses were present. The most common macroscopic findings included pale, thin-walled, fluid-filled small intestines (76.7%); thin, dilated, fluid-filled ceca (69.7%), and primarily litter in the gizzard (60.4%). The most common microscopic findings included villi changes (atrophy, fusion, blunting) (72%), infiltration of the lamina propria with mixed inflammatory cells (51%), hyperplasia of crypt epithelial cells (16%), and increase in the number of goblet cells (16%). This study provides an update on the prevalence of turkey viral enteritis in California commercial turkeys operations and discussed production practices which impact the occurrence of the disease complex. It also identified the current presentation of this disease condition in California and discussed the best approach for diagnosing turkey viral enteritis.

P17-Modulated Hsp90/Cdc37 Complex Governs Oncolytic Avian Reovirus Replication by Chaperoning p17, Which Promotes Viral Protein Synthesis and Accumulation of Viral Proteins σC and σA in Viral Factories.

In this work we have determined that heat shock protein 90 (Hsp90) is essential for avian reovirus (ARV) replication by chaperoning the ARV p17 protein. p17 modulates the formation of the Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation. Inhibition of the Hsp90/Cdc37 complex by inhibitors (17-N-allylamino-17-demethoxygeldanamycin 17-AGG, and celastrol) or short hairpin RNAs (shRNAs) significantly reduced expression levels of viral proteins and virus yield, suggesting that the Hsp90/Cdc37 chaperone complex functions in virus replication. The expression levels of p17 were decreased at the examined time points (2 to 7 h and 7 to 16 h) in 17-AAG-treated cells in a dose-dependent manner while the expression levels of viral proteins σA, σC, and σNS were decreased at the examined time point (7 to 16 h). Interestingly, the expression levels of σC, σA, and σNS proteins increased along with coexpression of p17 protein. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability, maturation, and virus production. Virus factories of ARV are composed of nonstructural proteins σNS and μNS. We found that the Hsp90/Cdc37 chaperone complex plays an important role in accumulation of the outer-capsid protein σC, inner core protein σA, and nonstructural protein σNS of ARV in viral factories. Depletion of Hsp90 inhibited σA, σC, and p17 proteins colocalized with σNS in viral factories. This study provides novel insights into p17-modulated formation of the Hsp90/Cdc37 chaperone complex governing virus replication via stabilization and maturation of viral proteins and accumulation of viral proteins in viral factories for virus assembly. IMPORTANCE Molecular mechanisms that control stabilization of ARV proteins and the intermolecular interactions among inclusion components remain largely unknown. Here, we show that the ARV p17 is an Hsp90 client protein. The Hsp90/Cdc37 chaperone complex is essential for ARV replication by protecting p17 chaperone from ubiquitin-proteasome degradation. p17 modulates the formation of Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation, suggesting a feedback loop between p17 and the Hsp90/Cdc37 chaperone complex. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability and virus production. Depletion of Hsp90 prevented viral proteins σA, σC, and p17 from colocalizing with σNS in viral factories. Our findings elucidate that the Hsp90/Cdc37 complex chaperones p17, which, in turn, promotes the synthesis of viral proteins σA, σC, and σNS and facilitates accumulation of the outer-capsid protein σC and inner core protein σA in viral factories for virus assembly.

Genetic variation analysis of the sigma B protein gene of novel duck reovirus in southeastern China from 2011 to 2020

The novel duck reovirus (NDRV) disease first appeared in China in 2011. Infected ducks may be of various ages and breeds. Sigma B protein, which is the key component of NDRV's outer capsid, may trigger group-specific neutralizing antibodies linked to NDRV infection, pathogenicity, and immune defense. The sigma B protein gene of fourteen NDRV field strains was amplified by RT-PCR and cloned into the pMD-18 T vector for sequencing to examine the genetic variation in sigma B proteins of NDRVs in southeastern China between 2011 and 2020. The sigma B protein gene of the fourteen NDRV southeastern strains included in this analysis had 96.3 %–99.8 % nucleotide, and 96.2 %–99.7 % deduced amino acid sequence homology. Phylogenetic analysis revealed that the fourteen southeastern strains belonged to a well-supported lineage that included NDRV and Muscovy duck reovirus (MDRV) strains. However, Avian reovirus (ARV) formed a distinct genetic lineage in the gene tree. The sigma B protein gene sequences of NDRV strains found in southeastern China are substantially conserved, according to these findings. There is no significant geographical difference between NDRV southeastern strains and DRV strains in other regions of China. Our findings will add to the molecular epidemiological picture of NDRV strain spread in southeastern China between 2011 and 2020, laying the groundwork for potential in-depth research on vaccine collection and comprehensive prevention.

Isolation and Genomic Characterization of Avian Reovirus From Wild Birds in South Korea.

Avian reoviruses (ARVs) cause severe arthritis, tenosynovitis, pericarditis, and depressed growth in chickens, and these conditions have become increasingly frequent in recent years. Studies on the role of wild birds in the epidemiology of ARVs are insufficient. This study provides information about currently circulating ARVs in wild birds by gene detection using diagnostic RT-PCR, virus isolation, and genomic characterization. In this study, we isolated and identified 10 ARV isolates from 7,390 wild birds' fecal samples, including migratory bird species (bean goose, Eurasian teal, Indian spot-billed duck, and mallard duck) from 2015 to 2019 in South Korea. On comparing the amino acid sequences of the σC-encoding gene, most isolates, except A18-13, shared higher sequence similarity with the commercial vaccine isolate S1133 and Chinese isolates. However, the A18-13 isolate is similar to live attenuated vaccine av-S1133 and vaccine break isolates (SD09-1, LN09-1, and GX110116). For the p10- and p17-encoding genes, all isolates have identical fusion associated small transmembrane (FAST) protein and nuclear localization signal (SNL) motif to chicken-origin ARVs. Phylogenetic analysis of the amino acid sequences of the σC-encoding gene revealed that all isolates were belonged to genotypic cluster I. For the p10- and p17-encoding genes, the nucleotide sequences of all isolates indicated close relationship with commercial vaccine isolate S1133 and Chinese isolates. For the σNS-encoding gene, the nucleotide sequences of all isolates indicated close relationship with the Californian chicken-origin isolate K1600657 and belonged to chicken-origin ARV cluster. Our data indicates that wild birds ARVs were derived from the chicken farms. This finding suggests that wild birds serve as natural carriers of such viruses for domestic poultry.

Genotypic characterization of two novel avian orthoreoviruses isolated in Iran from broilers with viral arthritis and malabsorption syndrome.

Avian reovirus (ARV) is a major poultry pathogen associated with arthritis, malabsorption, and enteric diseases in chickens. In recent years, emerging ARV strains have become a growing concern causing significant economic losses in broiler chickens around the world. This report focuses on the isolation of ARV from the clinical occurrence of ARV-associated diseases in commercial broiler chickens in Iran and the genotypic characterization of the selected isolates. In 2018, two distinct clinical diseases, suggestive of malabsorption syndrome (MAS) and viral arthritis, were noticed in commercial broiler chickens in the north of Iran. Laboratory investigations were carried out following necropsy, documentation of the gross lesions, and sampling of the affected tissues for histopathology and virology. Molecular diagnosis and characterization of ARV were performed targeting Sigma C (σC) gene sequences of the virus. Two variant ARV strains were isolated from tendon and gizzard of broilers with clinical viral arthritis and MAS, respectively. Phylogenetic analysis of the ARV σC gene sequences revealed that field isolates were clustered in genotypes 2 and 4 (which were distinct from previous Iranian field ARV strains) with relatively low sequence identity (59.2% and 49.1%) to the classical vaccine strains (S1133 and 1733) in genotype 1. This report, for the first time, represents new emerging ARV variants associated with clinical events in Iran, providing insights on the diversity of endemic ARV field isolates, and urges the need for national-wide surveillance of ARV.

A surveillance for avian coronavirus infectious bronchitis virus, infectious laryngotracheitis virus, avian metapneumovirus, and avian reovirus in poultry flocks with respiratory signs in Türkiye

In this study, avian coronavirus infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (AMPV), and avian reovirus (ARV) were evaluated in broiler and layer flocks. For this purpose, tracheal swabs from 48 broiler and 45 layer flocks with respiratory signs were inoculated SPF embryonated chicken eggs for virus isolation. The viruses were identified by real-time PCR. Results showed that the most common virus in both broiler and layer farms was IBV with incidence rates of 58.33% and 46.67%, respectively. ILTV, AMPV, and ARV incidences in the samples were found to be 22.22%, 13.33%, and 4.44% in layer flocks while 2.08%, 8.33%, and 20.83% in broilers, respectively. The numbers of IBV+AMPV and IBV+ARV coinfections were 5 (11.11%) and 1 (2.22%) in layers, whereas, 1 (2.08%) and 5 (10.42%) broilers, respectively. In addition, 2 broiler flocks (4.17%) had triple infection with IBV, AMPV, and ARV. ILTV was detected always alone from the samples of layer and broiler flocks. Sequencing of S1 gene of selected IBV TR/L45 and TR/B42 isolates showed similarities with IS/1494/06 (HM131453) at the rates of 98.98% and 99.69%, respectively, while TR/L37, TR/L38, and TR/L39 isolates were identical to 4/91 (KF377577) vaccine strain at the rates of 99.01%, 99.01%, and 98.76%, respectively. Sequencing analysis of the ICP4 and TK genes of ILTV isolates revealed that they were all field strains with low virulence. The present data represent actual information on the genotypes of IBV and ILTV circulating in poultry flocks in Türkiye.

Isolation and Molecular Characterization of Fowl Adenovirus and Avian Reovirus from Breeder Chickens in Japan in 2019-2021.

Fowl adenoviruses (FAdVs) and avian reoviruses (ARVs) are ubiquitous in poultry farms and most of them are not pathogenic, yet often cause damage to chicks. A total of 104 chicken fecal samples were collected from 7 farms of breeder chickens (layers and broilers) in Japan from 2019 to 2021, and yielded 26 FAdV plus 14 ARV isolates. By sequencing, FAdV isolates were classified as FAdV-1, 5 and 8b. ARV isolates were classified as genotype II, IV and V. These results suggest that FAdVs and ARVs are resident in the breeder chicken farms in Japan.

Isolation and Molecular Characteristics of a Novel Recombinant Avian Orthoreovirus From Chickens in China.

In recent years, the emergence of avian orthoreovirus (ARV) has caused significant losses to the poultry industry worldwide. In this study, a novel ARV isolate, designated as AHZJ19, was isolated and identified from domestic chicken with viral arthritis syndrome in China. AHZJ19 can cause typical syncytial cytopathic effect in the chicken hepatocellular carcinoma cell line, LMH. High-throughput sequencing using Illumina technology revealed that the genome size of AHZJ19 is about 23,230 bp, which codes 12 major proteins. Phylogenetic tree analysis found that AHZJ19 was possibly originated from a recombination among Hungarian strains, North American strains, and Chinese strains based on the sequences of the 12 proteins. Notably, the σC protein of AHZJ19 shared only about 50% homology with that of the vaccine strains S1133 and 1733, which also significantly differed from other reported Chinese ARV strains. The isolation and molecular characteristics of AHZJ19 provided novel insights into the molecular epidemiology of ARV and laid the foundation for developing efficient strategies for control of ARV in China.

WITHDRAWN: Development and evaluation of an indirect ELISA based on recombinant structural protein σC to detect antibodies against avian orthoreovirus

The avian orthoreovirus (ARV) causes large economic losses to poultry industry around the whole world. ARV viral proteins can be classified into three sizes: λ (large), μ (medium) and σ (small). σC, one of the capsid protein of ARV, contains various specific neutralizing epitopes and can induce robust immune responses in infected chickens. An indirect enzyme-linked immunosorbent assay (I-ELISA) was established using the recombinant σC protein. The optimal dilutions of antigen, serum and conjugate goat anti-chicken IgY conjugate were 2.5 μg/ml, 1:80 and 1:4000, respectively. The optimal antigen coating condition was 4℃ for overnight. Hyperimmune serum of high-yielding hens of different age produced stronger reaction against σC. Chicken serum samples were randomly collected and total coincidence rate between commercial kit and σC-ARV-ELISA kit was 97.8%. Importantly, those discrepant serums were further verified using Western blot assay and three of them were determined to be positive. Thus, the method based on σC protein has higher sensitivity compared with the commercial kit and provide a rapid and simple method for serodiagnosis of ARV, contributing to convenient epidemiological monitoring of ARV.

Gga-miR-29a-3p suppresses avian reovirus-induced apoptosis and viral replication via targeting Caspase-3

Avian reovirus (ARV) is an important pathogen causing multiple types of clinical diseases in chickens, including viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome, leading to considerable economic losses to the poultry industry across the globe. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post transcriptionally 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. Here, we show that infection of DF-1 cells (a chicken fibroblast cell line) with ARV markedly altered the expressions of 583 chicken miRNAs(gga-miR), and that transfection of DF-1 cells with gga-miR-29a-3p, an upregulated miRNA in ARV-infected cells, significantly suppressed ARV-induced apoptosis via directly targeting Caspase-3, retarding ARV growth in cells. In contrast, knockdown of endogenous gga-miR-29a-3p in DF-1 cells by specific miRNA inhibitor enhanced ARV-induced apoptosis and increased the content and activity of caspase-3, facilitating viral growth in cells. Consistently, inhibition of Caspase-3 activity by inhibitors decreased viral titers in cell cultures. Thus, gga-miR-29a-3p plays an important antiviral role in host response to ARV infection by suppression of apoptosis via targeting Caspase-3. This information will further our understandings of how host cells combat against ARV infection by self-encoded small RNA and increase our knowledge of the role of microRNAs in host response to pathogenic infection.

Molecular chaperone TRiC governs avian reovirus replication by protecting outer-capsid protein σC and inner core protein σA and non-structural protein σNS from ubiquitin- proteasome degradation

Avian reoviruses (ARVs) are important pathogens that cause considerable economic losses in poultry farming. To date, host factors that control stabilization of ARV proteins remain largely unknown. In this work we determined that the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC) is essential for avian reovirus (ARV) replication by stabilizing outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV. TriC serves as a chaperone of viral proteins and prevent their degradation via the ubiquitin-proteasome pathway. Furthermore, reciprocal co-immunoprecipitation assays confirmed the association of viral proteins (σA, σC, and σNS) with TRiC. Immunofluorescence staining indicated that the TRiC chaperonins (CCT2 and CCT5) are colocalized with viral proteins σC, σA, and σNS of ARV. In this study, inhibition of TRiC chaperonins (CCT2 and CCT5) by the inhibitor HSF1A or shRNAs significantly reduced expression levels of the σC, σA, and σNS proteins of ARV as well as virus yield, suggesting that the TRiC complex functions in stabilization of viral proteins and virus replication. This study provides novel insights into TRiC chaperonin governing virus replication via stabilization of outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV.

Avian Reoviruses of the Same Genotype Induce Different Pathology in Chickens

The emergence of avian reovirus variant strains has caused negative effects in the poultry industry worldwide. Regardless of the efforts in molecular characterization and classification of these variants, information about the pathogenicity, transmissibility, and immunosuppression in chickens is limited. The genomes of two variant strains (A and B) and a classic S1133 strain (C) belonging to the same sigma C genotype 1 were compared. Additionally, these strains were used in a challenge experiment to evaluate inoculated and indirectly exposed specific-pathogen-free chickens. The whole-genome sequence analysis of the three strains revealed nucleotide identity differences in the L3, M2, and S1 genes. Strains A and B also showed homology differences in the S4 gene, despite having high homologies in all other genes. The in vivo challenge experiments showed that, whereas variant A induced high viral loads in tendons, hearts, and duodena of inoculated chickens, variant B induced high viral loads in indirectly exposed chickens. Likewise, histopathology reflected differences in the pathologic effects induced by these strains. For instance, the B and C strains induced more severe microscopic lesions compared with the A strain. Lymphoid depletion was more severe in bursas than in thymi, and inoculated birds were more affected than exposed birds. In conclusion, different pathologic outcomes in chickens were observed depending on the strain and transmission route. This study provides insights onto the relationship between pathogenicity and genomic composition of avian reoviruses.

Avian Reoviruses of the Same Genotype Induce Different Pathology in Chickens.

The emergence of avian reovirus variant strains has caused negative effects in the poultry industry worldwide. Regardless of the efforts in molecular characterization and classification of these variants, information about the pathogenicity, transmissibility, and immunosuppression in chickens is limited. The genomes of two variant strains (A and B) and a classic S1133 strain (C) belonging to the same sigma C genotype 1 were compared. Additionally, these strains were used in a challenge experiment to evaluate inoculated and indirectly exposed specific-pathogen-free chickens. The whole-genome sequence analysis of the three strains revealed nucleotide identity differences in the L3, M2, and S1 genes. Strains A and B also showed homology differences in the S4 gene, despite having high homologies in all other genes. The in vivo challenge experiments showed that, whereas variant A induced high viral loads in tendons, hearts, and duodena of inoculated chickens, variant B induced high viral loads in indirectly exposed chickens. Likewise, histopathology reflected differences in the pathologic effects induced by these strains. For instance, the B and C strains induced more severe microscopic lesions compared with the A strain. Lymphoid depletion was more severe in bursas than in thymi, and inoculated birds were more affected than exposed birds. In conclusion, different pathologic outcomes in chickens were observed depending on the strain and transmission route. This study provides insights onto the relationship between pathogenicity and genomic composition of avian reoviruses.