Reovirus Isolates Research Articles

Isolation and Characterization of Avian Reovirus (Egypt/ARV/Giza 2011) Associated with Arthritis in Broiler Breeder Flocks

Isolation and Characterization of Avian Reovirus (Egypt/ARV/Giza 2011) Associated with Arthritis in Broiler Breeder Flocks

First isolation and genomic characterization of avian reovirus from black swans (Cygnus atratus) in China

Identification and analysis of the avian reovirus from black swan. Isolation of the strain through the chorioallantoic membrane route of duck embryos, identified through transmission electron microscopy and RT-PCR based on the ARV S2 gene. The complete genome of the ARV strain was obtained using next-generation sequencing technology. The isolated strain of ARV was named CD200801 and was identified through transmission electron microscopy and RT-PCR based on the ARV S2 gene. Experimental infection with CD200801 resulted in the death of ducklings with serious spleen and liver focal necrosis. BLAST analysis of CD200801 sequences showed a 35.5 to 98.6% similarity to a novel duck reovirus that was isolated in recent years. Phylogenetic analysis revealed that CD200801 was closely related to ARV isolates YL, GX-Y7, and XT-18. We report the first avian reovirus infection in the black swan. This study provides important new insights into the evolutionary relationships among different ARV strains and highlights the need for continued surveillance and monitoring of these viruses in both domestic and wild bird flocks. These findings have significant implications for the development of effective strategies for disease prevention and control in the poultry industry.

Incidence and Economic Impact of Reovirus in the Poultry Industries in the United States.

Avian reoviruses have sole or partial responsibility for a wide range of conditions that are of economic concern to the poultry industries in the United States. It is difficult, however, to determine the exact cost of reoviral-induced disease to this industry for several reasons. Avian reoviruses are ubiquitous in commercial poultry operations, but vary greatly in their ability to cause disease. Many reovirus isolates are not pathogenic, and this contributes to the difficulty in determining their role in production losses or whether they are merely coincidental findings of little or no importance. Results of infection with this virus can be difficult to measure in a commercial setting, aside from morbidity, mortality, or loss of finished product in a processing plant due to condemnation or trim. The fact that there is a relatively high morbidity rate, and at the same time a low mortality rate, makes it extremely difficult to accurately estimate the economic impact of infection with this virus. Variations in the pathogenicity of the virus, the extent of infection within a flock, the blending of progeny from different breeder source flocks into a broiler house, and the lack of separation of infected birds from uninfected birds at processing makes evaluation of economic variables, such as feed conversion and weight gain, very difficult to ascertain. The broiler and turkey industries have been surveyed on the importance of this virus in their respective operations and the results will be consolidated here to consider the economic impact to the poultry industries in the United States.

Evaluation of Pathogenicity and Antigenicity of Avian Reoviruses and Disease Control Through Vaccination.

Avian reoviruses are ubiquitous in poultry production worldwide and can be transmitted vertically or horizontally among chickens. The pathogenicity of reoviruses can range from very pathogenic viruses that affect multiple tissues and organs to apathogenic. Avian reoviruses have been associated with many disease presentations, and two of the most economically significant diseases are viral arthritis/tenosynovitis and viral enteritis. Viral arthritis/tenosynovitis has been recognized since the 1950s and essentially disappeared after development of attenuated live and inactivated vaccines in the 1980s but re-emerged in 2011 due to the emergence of antigenic variants. Viral enteritis was first recognized in the 1970s and became the predominant reovirus-associated disease between 2006 and 2011 due to the emergence of pathogenic enterotropic reoviruses. Pathogenicity of reovirus isolates can be evaluated in several ways, including inoculation of day-old broiler chicks with low maternal reovirus antibody titers via the foot pad route or the oral and intratracheal route. Pathogenic reoviruses induce foot pad inflammation within 3 days of inoculation, and more pathogenic reoviruses are able to disseminate to and damage visceral organs. Only reovirus infections in young chickens result in disease due to age-related resistance to disease development. Reoviruses exist as many serotypes and subtypes with various degrees of interrelatedness. The earliest reovirus strains in the United States were antigenically related to each other and are referred to as S1133-like viruses, but in the 2000s, reoviruses emerged that were antigenically different from the S1133-like viruses. Virus neutralization assay using polyclonal antisera has been used to classify the emerging variant reoviruses into serogroups. The first reovirus vaccines were developed in the 1970s, and by the 1980s breeder vaccination programs were established that protected breeders, prevented vertical transmission of reovirus, and provided maternal immunity to the progeny during the crucial first 3 wk of life. With the emergence of antigenic variant reoviruses in the 2000s, vaccination programs using S1133-like vaccines became ineffective. The poultry industry has relied on vaccination with autogenous inactivated reovirus vaccines to alleviate losses due to viral arthritis/tenosynovitis and viral enteritis. Virus isolates used for autogenous vaccines must be updated regularly and are selected based on pathotype, serotype, or Sigma C (σC) genotype. Live attenuated S1133 vaccines are still used in breeder chickens for the priming effect, followed by one or more injections of the inactivated licensed and/or autogenous vaccines. The route of vaccination and the number of doses received by breeder chickens are very important for a sufficient antibody response. Intramuscular vaccination with inactivated vaccines elicits the highest antibody response, while subcutaneous vaccination with inactivated vaccines elicits a low antibody response. More recently, research has focused on development of alternative vaccines and vaccination strategies. An inactivated variant reovirus vaccine was developed that elicits protection against multiple variant serotypes, and experimental recombinant and subunit vaccines have been described and show potential. More research needs to be done to develop better vaccines, vaccination programs, and other control measures for preventing reovirus infection, transmission, and losses due to disease.

Isolation and characterization of a duck reovirus strain from mature ducks in China

In 2018, a disease characterized by splenic hemorrhage and necrosis killed ducks in a duck farm in Guangxi province, China. A duck reovirus strain was isolated from the tissues of the dead ducks by inoculating duck embryos and BHK-21 cells. Electron microscopy of the cultured the isolate showed that the viral particles were nearly round in shape and approximately 70 nm in diameter, and they were designated DRV-GL18. Sequence analysis showed that the GL18 strain viral genome was 23,419 nt in length and had 10 dsRNA segments. Phylogenetic analysis of cDNA amplicons of segments encoding the protein σC which are outer capsid proteins showed that the isolate belongs to the branch of the epidemic strains of duck reovirus. The Recombination Detection Program (RDP) and SimPlot program analyses suggested potential genetic recombination events in the M2 segments. Pathogenicity experiments revealed that GL18 produced severe hemorrhaging in livers and necrosis in the spleen of infected SPF ducklings. A death rate of 50% in the experimental ducklings was calculated during the first 7 d, and the rest of the ducklings were observed to undergo spleen necrosis. These data suggested that GL18 is a duck reovirus isolate with severer pathogenicity, and it could be a candidate for development of vaccine. This is the first reported isolation of duck reovirus from mature ducks.

Epidemiological investigation of infectious diseases in geese on mainland China during 2018-2021.

Geese play an important role in agricultural economics, with China producing the vast majority of goose meat consumed worldwide annually. The variations in the avian viruses and co-infections result in substantial economic losses to the goose industry in China. To understand the evolutionary characteristics and co-infections of viruses, a broad epidemiological investigation of epizootic viruses of goose was carried out in nine provinces of China during 2018-2021. Here, the results indicated that, among the 1970 clinical samples, 50.81% (1001/1970) were positive for goose astrovirus (GAstV), 18.22% (359/1970) for avian orthoreovirus, 12.74% (251/1970) for goose parvovirus, 11.02% (217/1970) for H9N2 subtype avian influenza virus, 4.01% (79/1970) for Newcastle disease virus, and 2.08% (41/1970) for fowl adenovirus. Among the six viruses, co-infections comprised a large proportion (66.37%) in the field, of which the dual infection was more common. Additionally, phylogenetic analysis of GAstVs indicated that Chinese GAstVs had formed two distinct groups, that is, GAstV-1 and GAstV-2. GAstV-2 sub-genotype II-c had arisen as the dominant genotype in the whole country. Notably, all the H9N2-AIV isolated strains harboured the mammalian adaptation markers I155T, H183N, and Q226L (H3 numbering) in the HA gene, which promotes preferential binding to human-like α2-6-linked sialic acid receptors. And beyond that, we determined that the goose-origin Muscovy Duck Reovirus isolates, showing 51.7%-96% similarities to that of other waterfowl-origin orthoreovirus isolates in sequence homology analysis of the representative part of σC, are a new variant of waterfowl-origin orthoreovirus. These data provide valuable information about the prevalence of infectious diseases in geese on mainland China.

Candidate 'Avian orthoreovirus B': An emerging waterfowl pathogen in Europe and Asia?

A fusogenic virus was isolated from a flock of breeder Pekin ducks in 2019, Hungary. The affected flock experienced a marked decrease in egg production. Histopathological lesions were seen in the oviduct and in the lungs of birds sent for diagnostic investigation. The fusogenic agent was characterized as an orthoreovirus by viral metagenomics. The assembled viral genome was composed of 10 genomic segments and was 23,433 nucleotides (nt) in length. The study strain, designated Reo/HUN/DuckDV/2019, shared low-to-medium gene-wise sequence identity with avian orthoreovirus strains from galliform and anseriform birds (nt, 38.90%-72.33%) as well as with representative strains of neoavian orthoreoviruses (nt, 40.07%-68.23%). On the contrary, the study strain shared 86.48%-95.01% pairwise nt sequence identities with recent German and Chinese reovirus isolates, D2533/6 and Ych, respectively. Phylogenetic analysis clustered all three unusual waterfowl pathogens on a monophyletic branch, indicating a common evolutionary origin of Reo/HUN/DuckDV/2019 with these enigmatic orthoreoviruses described over the past few years. The finding that a candidate new orthoreovirus species, tentatively called Avian orthoreovirus B, was isolated in recent years in Europe and Asia in moribund ducks seems an alarming sign that needs to be better evaluated by extending laboratory diagnosis of viral pathogens in countries where the waterfowl industry is important.

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.

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.

Parallel isolation of calicivirus and reovirus from lethal co-infected mink during a potential epidemic of farmed mink infections

Mink has been identified as an animal with susceptibility to SARS-CoV-2 and also as the only animal with evidence to transmit the virus back to humans. Thus, the surveillance of viruses among high-density farmed minks has a significant meaning for the control of zoonotic emerging diseases in humans. Within anal swabs of minks that died of unknown causes in a mink farm, mink calicivirus (MCV) and mammalian orthoreovirus (MRV) were detected and simultaneously observed within MDCK cell culture from the sample of the same lethal mink. The parallel isolation was successfully performed by utilizing cell lines from different host sources with distinct viral sensitivities, i.e. Mv.1.Lu and Vero-E6 and the two viruses were independently separated. The prevalence of the virus among the minks and its genomic characteristics were investigated through deep sequencing technology. Phylogenetic analysis of the viral genome showed a close relationship of the newly isolated MCV-GCCDC8-2020 with MCV strains belonging to the genus Vesivirus, but with unique mutations derived from the major structural protein (VP1). The reovirus MRV-GCCDC9-2020 isolated from the same mink belongs to serotype 3 mammalian orthoreovirus and genome analysis showed a potential reassortment derived from reoviruses in different species. This study provides a beneficial reference on viral co-infection within disease investigation in farmed minks and raises the concern for the virus surveillance among the high-density fed animal farms.

Isolation, Identification, and Genomic Analysis of a Novel Reovirus from Healthy Grass Carp and Its Dynamic Proliferation In Vitro and In Vivo.

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57–96.71% protein sequence identity) but shared only 22.65–45.85% and 23.37–43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus-C (AqRV-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 104 copies/μL.

Interactions between Human Reovirus and Free-Living Amoebae: Implications for Enteric Virus Disinfection and Aquatic Persistence.

Free-living amoebae (FLA) are ubiquitous protozoa in aquatic/soil habitats and known to resist various disinfection methods commonly used in drinking and wastewater treatment plants. Reoviruses are emerging as useful infectious enteric virus indicators of wastewater treatment efficacy. The possible enhanced protection FLA may provide reoviruses, however, has not been previously described. Using an infectious clinical reovirus isolate in coculture with three FLA, namely, Vermamoeba vermiformis, Acanthamoeba polyphaga, and Willaertia magna, we followed reovirus persistence (by quantitative reverse transcription polymerase chain reaction (RT-qPCR)) and infectivity (TCID50). Virions present in samples persisted over the experimental time period, with most virions remaining infectious. Surprisingly, electron microscopy revealed virions accumulated within the nucleus of amoebae. The current work appears to be the first report of reovirus being internalized within FLA and remaining infectious, providing a previously unreported environmental reservoir and potential mode of dissemination. FLA also appeared to be providing some logs in protection to internalized viruses during UV irradiation, which if not accounted for when determining UV dosage needed for sufficient disinfection may result in unintentional release of pathogens into surrounding water systems.

Polycistronic Genome Segment Evolution and Gain and Loss of FAST Protein Function during Fusogenic Orthoreovirus Speciation.

The Reoviridae family is the only non-enveloped virus family with members that use syncytium formation to promote cell–cell virus transmission. Syncytiogenesis is mediated by a fusion-associated small transmembrane (FAST) protein, a novel family of viral membrane fusion proteins. Previous evidence suggested the fusogenic reoviruses arose from an ancestral non-fusogenic virus, with the preponderance of fusogenic species suggesting positive evolutionary pressure to acquire and maintain the fusion phenotype. New phylogenetic analyses that included the atypical waterfowl subgroup of avian reoviruses and recently identified new orthoreovirus species indicate a more complex relationship between reovirus speciation and fusogenic capacity, with numerous predicted internal indels and 5’-terminal extensions driving the evolution of the orthoreovirus’ polycistronic genome segments and their encoded FAST and fiber proteins. These inferred recombination events generated bi- and tricistronic genome segments with diverse gene constellations, they occurred pre- and post-orthoreovirus speciation, and they directly contributed to the evolution of the four extant orthoreovirus FAST proteins by driving both the gain and loss of fusion capability. We further show that two distinct post-speciation genetic events led to the loss of fusion in the waterfowl isolates of avian reovirus, a recombination event that replaced the p10 FAST protein with a heterologous, non-fusogenic protein and point substitutions in a conserved motif that destroyed the p10 assembly into multimeric fusion platforms.

Isolation and detection of reovirus from arthritis in chickens

Isolation and detection of reovirus from arthritis in chickens

Genotypic Characterization of Emerging Avian Reovirus Genetic Variants in California

This study focuses on virus isolation of avian reoviruses from a tenosynovitis outbreak between September 2015 and June 2018, the molecular characterization of selected isolates based on partial S1 gene sequences, and the full genome characterization of seven isolates. A total of 265 reoviruses were detected and isolated, 83.3% from tendons and joints, 12.3% from the heart and 3.7% from intestines. Eighty five out of the 150 (56.6%) selected viruses for sequencing and characterization were successfully detected, amplified and sequenced. The characterized reoviruses grouped in six distinct genotypic clusters (GC1 to GC6). The most represented clusters were GC1 (51.8%) and GC6 (24.7%), followed by GC2 (12.9%) and GC4 (7.2%), and less frequent GC5 (2.4%) and GC3 (1.2%). A shift on cluster representation throughout time occurred. A reduction of GC1 and an increase of GC6 classified strains was noticed. The highest homologies to S1133 reovirus strain were detected in GC1 (~77%) while GC2 to GC6 homologies ranged between 58.5 and 54.1%. Over time these homologies have been maintained. Seven selected isolates were full genome sequenced. Results indicated that the L3, S1 and M2 genes, coding for proteins located in the virus capsid accounted for most of the variability of these viruses. The information generated in the present study helps the understanding of the epidemiology of reoviruses in California. In addition, provides insights on how other genes that are not commonly studied add variability to the reovirus genome.

Reovirus infection induces stabilization and up-regulation of cellular transcripts that encode regulators of TGF-β signaling.

Reovirus infection induces dramatic changes in host mRNA expression. We utilized oligonucleotide microarrays to measure cellular mRNA decay rates in mock- or reovirus-infected murine L929 cells to determine if changes in host mRNA expression are a consequence of reovirus-induced alterations in cellular mRNA stability. Our analysis detected a subset of cellular transcripts that were coordinately induced and stabilized following infection with the reovirus isolates c87 and c8, strains that led to an inhibition of cellular translation, but not following infection with Dearing, a reovirus isolate that did not negatively impact cellular translation. The induced and stabilized transcripts encode multiple regulators of TGF- β signaling, including components of the Smad signaling network and apoptosis/survival pathways. The coordinate induction, through mRNA stabilization, of multiple genes that encode components of TGF-β signaling pathways represents a novel mechanism by which the host cell responds to reovirus infection.

Genetic and pathogenic characterisation of 11 avian reovirus isolates from northern China suggests continued evolution of virulence.

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.

Reverse Genetics for Fusogenic Bat-Borne Orthoreovirus Associated with Acute Respiratory Tract Infections in Humans: Role of Outer Capsid Protein σC in Viral Replication and Pathogenesis.

Nelson Bay orthoreoviruses (NBVs) are members of the fusogenic orthoreoviruses and possess 10-segmented double-stranded RNA genomes. NBV was first isolated from a fruit bat in Australia more than 40 years ago, but it was not associated with any disease. However, several NBV strains have been recently identified as causative agents for respiratory tract infections in humans. Isolation of these pathogenic bat reoviruses from patients suggests that NBVs have evolved to propagate in humans in the form of zoonosis. To date, no strategy has been developed to rescue infectious viruses from cloned cDNA for any member of the fusogenic orthoreoviruses. In this study, we report the development of a plasmid-based reverse genetics system free of helper viruses and independent of any selection for NBV isolated from humans with acute respiratory infection. cDNAs corresponding to each of the 10 full-length RNA gene segments of NBV were cotransfected into culture cells expressing T7 RNA polymerase, and viable NBV was isolated using a plaque assay. The growth kinetics and cell-to-cell fusion activity of recombinant strains, rescued using the reverse genetics system, were indistinguishable from those of native strains. We used the reverse genetics system to generate viruses deficient in the cell attachment protein σC to define the biological function of this protein in the viral life cycle. Our results with σC-deficient viruses demonstrated that σC is dispensable for cell attachment in several cell lines, including murine fibroblast L929 cells but not in human lung epithelial A549 cells, and plays a critical role in viral pathogenesis. We also used the system to rescue a virus that expresses a yellow fluorescent protein. The reverse genetics system developed in this study can be applied to study the propagation and pathogenesis of pathogenic NBVs and in the generation of recombinant NBVs for future vaccines and therapeutics.

A duck reovirus variant with a unique deletion in the sigma C gene exhibiting high pathogenicity in Pekin ducklings

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.

Isolation and identification of a new reovirus associated with mortalities in farmed oriental river prawn, Macrobrachium nipponense (de Haan, 1849), in China.

Isolation and identification of a new reovirus associated with mortalities in farmed oriental river prawn, Macrobrachium nipponense (de Haan, 1849), in China.