Isolation, identification and genetic variation analysis of avian orthoreovirus in commercial broilers in China from 2016 to 2021

Characterization of M-class genome segments of muscovy duck reovirus S14

Assignment of avian reovirus temperature-sensitive mutant recombination groups B, C, and D to genome segments

Based on previous reports characterizing the turkey-origin avian reovirus (TRV) sigmaB (sigma2) major outer capsid protein gene, the TRVs may represent a new group within the fusogenic orthoreoviruses. However, no sequence data from other TRV genes or genome segments has been reported. The sigmaC protein encoded by the avian reovirus S1 genome segment is the cell attachment protein and a major antigenic determinant for avian reovirus. The chicken reovirus S1 genome segment is well characterized and is well conserved in viruses from that species. This report details the amplification, cloning and sequencing of the entire S1 genome segment from two and the entire coding sequences of the sigmaC, p10 and p17 genes from an additional five TRVs. Sequence analysis reveals that of the three proteins encoded by the TRV S1 genome segment, sigmaC shares at most 57% amino acid identity with sigmaC from the chicken reovirus reference strain S1133, while the most similar p10 and p17 proteins share 72% and 61% identity, respectively, with the corresponding S1133 proteins. The most closely related mammalian reovirus, the fusogenic Nelson Bay reovirus, encodes a sigmaC protein that shares from 25% to 28% amino acid identity with the TRV sigmaC proteins. This report supports the earlier suggestion that the TRVs are a separate virus species within the Orthoreovirus genus, and may provide some insight into TRV host specificity and pathogenesis.

Heterologous expression, immunogenic evaluation, and subunit vaccine potential of the σC protein from the Xinjiang avian reovirus (ARV) strain xj-1.1.

The sequence of RNA genome segment S4 of the avian reovirus (ARV) strain S1133 was determined. S4 RNA is 1185 base pairs long and contains one open reading frame encoding a protein of 367 amino acid residues (40.6 kDa), the similar size as the known S4 gene product (sigma NS), with a net charge of -1 at neutral pH. The S4 RNA sequence possesses a pentanucleotide sequence UCAUC at the 3'-terminus of its plus strand like in ARV S1 and S3 segments and ten segments of mammalian reovirus (MRV). The predicted amino acid sequence comparison revealed that the homology is 44.02%, 45.71%, and 42.33% for ARV sigma NS and three serotypes of MRV sigma NS, respectively. The relatively high content of alpha-helix structure in the C-terminal portion of ARV sigma NS suggests that this protein may functionally relate to MRV sigma NS. Northern blot hybridization showed that a 32P-labeled cDNA insert S4-49 from ARV S4 RNA cross-hybridized with the corresponding RNA segments of all seven strains of ARV tested.

Live avian reovirus (ARV) vaccine has long been using in poultry industry. Safety, protectivity and antibody response of each individual vaccine is still not clear. Seven commercial live ARV vaccines were each administrated subcutanously twice to 7 groups of 20 SPF chickens at 1 and 5 weeks of age. A control group was also included in this experiment. Blood from all chickens were collected at 3 and 7 weeks of age for ARV enzyme-linked immunosorbent assay (ELISA) antibody test. At 9-week-old, 10 birds per group were challenged via the footpad with the 1733 and 2408 strains of ARV. The increase in the footpad thickness and swelling at 7 and 14 days post challenge (DPC) were calculated. AII vaccinated birds were survived in good health. At 3 and 7 weeks of age, 1 (14.2%) and 6 (85.5%) vaccinated groups of chickens had geometric mean ELlSA titers (GMT) in excess of 1:1000 respectively. At 7 and 14 DPC, 2 and 1 vaccinated group had the increase of footpad swelling in millimeter significantly (P＜0.05) less than the control group. AII the vaccinated groups had footpad swellings and leg hemorrhage scores significantly (P＜0.05) less than the control group. Commercial live ARV vaccines provide some protectivity against virulent ARV challenge, but the protectivity is the remaining virulence of vaccine virus corelated.

Host protein PRPS2 interact with the non-structural protein p17 of Avian Reovirus and promote viral replication

Genomic characteristics of a novel reovirus from Muscovy duckling in China

Avian reovirus (ARV) is a major causative agent of viral arthritis (VA), tenosynovitis, and malabsorption syndrome (MAS) in chickens, with significant economic consequences due to growth retardation, reduced production performance, and immunosuppression. Despite routine vaccination of breeder chickens against ARV, cases of VA and MAS continue to be reported in commercial flocks in recent years. Moreover, there is a lack of recent data on the genetic characteristics of circulating field ARV strains in India. In light of these concerns, a study was conducted to investigate the involvement of ARV in chickens exhibiting clinical signs suggestive of VA or MAS. Samples were collected from 27 commercial broiler and desi-chicken flocks across the mid-western region of Tamil Nadu, South India. Molecular confirmation was performed using reverse transcription polymerase chain reaction (RT-PCR) targeting a partial region of the σCgene within the S1 segment. Of the 27 flocks sampled, only two samples - both from desi-chickens aged two and three weeks - tested positive for ARV. Sequence analysis of these positive samples, compared against available ARV sequences in GenBank (including vaccine strains) revealed that the identified strains clustered within ARV genogroup II. This represents the first report of cluster II ARV in India, indicating the circulation of genetically distinct ARV strains in Indian poultry populations. These findings underscore the need for routine molecular surveillance of ARV genotypes in India and highlight the potential mismatch between circulating field strains and current vaccine strains. Comprehensive genotype monitoring is essential to upgrade vaccine design and implement effective control strategies for ARV-associated diseases in Indian poultry production.

Development of TaqMan real-time RT-PCR for detection of avian reoviruses

Avian viral arthritis (AVA), caused by avian reovirus (ARV), is a viral disease in chickens that has led to significant economic losses in the poultry industry. Recent studies have shown that traditional ARV vaccines based on the S1133 strain fail to protect against emerging ARV variants. In this study, we isolated and characterized three ARV strains (G4, YV, WF) from immunized chicken flocks with respiratory and arthritic symptoms. Genomic analysis revealed that the σC genes of G4, YV, and WF shared only 55.5%, 55.7%, and 58.7% sequence homology, respectively, with the S1133 strain. Phylogenetic analysis placed them in different branches, indicating they are variant strains. YV and WF belong to genotype III, and G4 falls into genotype VI. Whole genome analysis revealed gene segment reassortment among the variants. Pathogenicity testing in three-week-old SPF chickens showed that G4 (genotype VI) caused swelling of footpads, whereas WF (genotype III) did not. G4-infected chickens exhibited significantly higher viral loads in the thymus, lungs, spleen, and bursa of Fabricius than those in the WF-infected chickens, indicating viruses from different genotypes showed various pathogenesis. These results suggested an urgent need for new updates of vaccines against the variant ARVs, especially the genotype VI virus.

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.

Avian reovirus (ARV) can induce many diseases as well as immunosuppression in chickens, severely endangering the poultry industry. Interferons (IFNs) play an antiviral role by inducing the expression of interferon-stimulated genes (ISGs). The effect of ARV infection on the expression of host ISGs is unclear. Specific-pathogen-free (SPF) chickens were infected with ARV strain S1133 in this study, and real time quantitative PCR was used to detect changes in the dynamic expression of IFNs and common ISGs in joints of SPF chickens. The results showed that the transcription levels of IFNA, IFNB, and several ISGs, including myxovirus resistance (MX), interferon-induced transmembrane protein 3 (IFITM3), protein kinase R (PKR), oligoadenylate synthase (OAS), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), interferon-stimulated gene 12 (ISG12), virus inhibitory protein (VIPERIN), interferon-alpha-inducible protein 6 (IFI6), and integrin-associated protein (CD47), were upregulated in joints on days 1–7 of infection (the levels of increase of MX, IFIT5, OAS, VIPERIN, ISG12, and IFI6 were the most significant, at hundreds-fold). In addition, the expression levels of the ISGs encoding zinc finger protein 313 (ZFP313), and DNA damage–inducible transcript 4 (DDIT4) increased suddenly on the 1st or 2nd day, then decreased to control levels. The ARV viral load in chicken joints rapidly increased after 1 day of viral challenge, and the viral load remained high within 6 days of viral challenge. The ARV viral load sharply decreased starting on day 7. These results indicate that in SPF chicken joints, many ISGs have mRNA expression patterns that are basically consistent with the viral load in joints. IFNA, IFNB, and the ISGs MX, IFITM3, PKR, OAS, IFIT5, ISG12, VIPERIN, IFI6, and CD47 play important roles in defending against ARV invasion, inhibiting ARV replication and proliferation, and promoting virus clearance. These results enrich our understanding of the innate immune response mechanisms of hosts against ARV infection and provide a theoretical basis for prevention and control of ARV infection.

Avian reovirus (ARV) is a non-enveloped virus with a segmented double-stranded RNA genome surrounded by a double icosahedral capsid shell. ARVs are associated with viral arthritis, immunosuppression, and enteric diseases in poultry. The sigma C protein was involved in induction of apoptosis and neutralization antibody. In the present study, sigma C-His protein was expressed in Sf9 insect cells and purified by immobilized metal affinity chromatography. Eight monoclonal antibodies (mAbs) against sigma C-His and three mAbs against His were screened from hybridoma cells produced by fusion of splenocytes from immunized mice with NS1 myeloma cells. Among the eight mAbs against sigma C protein, all belonged to the IgG isotype except three for IgM. It was discovered that all anti-His mAbs were mixtures of IgG and IgM isotypes. mAbs reacted with sigma C-His protein in a conformation-independent manner based on dot blot and western blotting assays. The competitive binding assay indicated that all mAbs recognized the same epitope on sigma C protein that was conserved in different isolates. Compared with the commercial anti-ARV S1133 polyclonal antibody, mAb (D15) had universal reactivity to all serotypes or genotypes of ARVs tested. This monoclonal antibody may therefore be useful for the development of an antigen-capture enzyme-linked immunosorbent assay for rapid detection of field isolates.

Avian reovirus (ARV) infection can cause significant losses to the poultry industry. Disease control has traditionally been attempted mainly through vaccination. However, the increase in clinical outbreaks in the last decades demonstrated the poor effectiveness of current vaccination approaches. The present study reconstructs the evolution and molecular epidemiology of different ARV genotypes using a phylodynamic approach, benefiting from a collection of more than one thousand sigma C (σC) sequences sampled over time at a worldwide level. ARVs’ origin was estimated to occur several centuries ago, largely predating the first clinical reports. The origins of all genotypes were inferred at least one century ago, and their emergence and rise reflect the intensification of the poultry industry. The introduction of vaccinations had only limited and transitory effects on viral circulation and further expansion was observed, particularly after the 1990s, likely because of the limited immunity and the suboptimal and patchy vaccination application. In parallel, strong selective pressures acted with different strengths and directionalities among genotypes, leading to the emergence of new variants. While preventing the spread of new variants with different phenotypic features would be pivotal, a phylogeographic analysis revealed an intricate network of viral migrations occurring even over long distances and reflecting well-established socio-economic relationships.