Analyses and Insights into Genetic Reassortment and Natural Selection as Key Drivers of Piscine orthoreovirus Evolution.

Piscine orthoreovirus (PRV) is a pathogen that causes heart and skeletal muscle inflammation in Salmo salar and has also been linked to circulatory disorders in other farmed salmonids, such as Oncorhynchus kisutch and Oncorhynchus mykiss. The virus has a segmented, double-stranded RNA genome, which makes it possible to undergo genetic reassortment and increase its genomic diversity through point mutations. In this study, genetic reassortment in PRV was assessed using the full genome sequences available in public databases. This study used full genome sequences that were concatenated and genome-wide reassortment events, and phylogenetic analyses were performed using the recombination/reassortment detection program version 5 (RDP5 V 5.5) software. Additionally, each segment was aligned codon by codon, and overall mean distance and selection was tested using the Molecular Evolutionary Genetics Analysis X software, version 10.2 (MEGA X version 10.2). The results showed that there were 17 significant reassortment events in 12 reassortant sequences, involving genome exchange between low and highly virulent genotypes. PRV sequences from different salmonid host species did not appear to limit the reassortment. This study found that PRV frequently undergoes reassortment events to increase the diversity of its segmented genome, leading to antigenic variation and increased virulence. This study also noted that to date, no reassortment events have been described between PRV-1 and PRV-3 genotypes. However, the number of complete genomic sequences within each genotype is uneven. This is important because PRV-3 induces cross-protection against PRV-1, making it a potential vaccine candidate.

Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories.

The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.

Antiviral effects of micafungin against pteropine orthoreovirus, an emerging zoonotic virus carried by bats.

Bat-borne emerging zoonotic viruses cause major outbreaks, such as the Ebola virus, Nipah virus, and/or beta coronavirus. Pteropine orthoreovirus (PRV), whose spillover event occurred from fruits bats to humans, causes respiratory syndrome in humans widely in South East Asia. Repurposing approved drugs against PRV is an effective tool to confront future PRV pandemics. We screened 2,943 compounds in an FDA-approved drug library and identified eight hit compounds that reduce viral cytopathic effects on cultured Vero cells. Real-time quantitative PCR analysis revealed that six of eight hit compounds significantly inhibited PRV replication. Among them, micafungin used clinically as an antifungal drug, displayed a prominent antiviral effect on PRV. Secondly, the antiviral effects of micafungin on PRV infected human cell lines (HEK293T and A549), and their transcriptome changes by PRV infection were investigated, compared to four different bat-derived cell lines (FBKT1 (Ryukyu flying fox), DEMKT1 (Leschenault's rousette), BKT1 (Greater horseshoe bat), YUBFKT1 (Eastern bent-wing bats)). In two human cell lines, unlike bat cells that induce an IFN-γ response pathway, an endoplasmic reticulum stress response pathway was commonly activated. Additionally, micafungin inhibits viral release rather than suppressing PRV genome replication in human cells, although it was disturbed in Vero cells. The target of micafungin's action may vary depending on the animal species, but it must be useful for human purposes as a first choice of medical care.

Occurrence of salmonid alphavirus and piscine orthoreovirus-1 infections in migrating salmon (Salmo salar L.) post-smolt in western Norway.

Viral diseases are a serious problem in Atlantic salmon (Salmo salar L.) farming in Norway, often leading to reduced fish welfare and increased mortality. Disease outbreaks in salmon farms may lead to spread of viruses to the surrounding environment. There is a public concern that viral diseases may negatively affect the wild salmon populations. Pancreas disease (PD) caused by salmonid alphavirus (SAV) and heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus-1 (PRV-1) are common viral diseases in salmon farms in western Norway. In the current study, we investigated the occurrence of SAV and PRV-1 infections in 651 migrating salmon post-smolt collected from three fjord systems (Sognefjorden, Osterfjorden and Hardangerfjorden) located in western Norway in 2013 and 2014 by real-time RT-PCR. Of the collected post-smolts, 303 were of wild origin and 348 were hatchery-released. SAV was not detected in any of the tested post-smolt, but PRV-1 was detected in 4.6% of them. The Ct values of PRV-1 positive fish were usually high (mean 32.0; range: 20.1-36.8). PRV-1 prevalence in post-smolts from the three fjords was 6.1% in Sognefjorden followed by 4.8% in Osterfjorden and 2.3% in Hardangerfjorden. The prevalence PRV-1 was significantly higher in wild (6.9%) compared to hatchery-released post-smolt (2.6%). The occurrence of PRV-1 infection in the fish was lowest in the Hardangerfjorden which has the highest fish farming intensity. Our results suggest that SAV infection are uncommon in migrating smolt while PRV-1 infection can be detected at low level. These findings suggest that migrating smolts were at low risk from SAV or PRV-1 released from salmon farms located in their migration routes in 2013 and 2014.

Nelson Bay Reovirus Isolated from Bats and Blood-Sucking Arthropods Collected in Yunnan Province, China.

Nelson Bay reovirus (NBV) is an emerging zoonotic virus that can cause acute respiratory disease in humans. These viruses are mainly discovered in Oceania, Africa, and Asia, and bats have been identified as their main animal reservoir. However, despite recent expansion of diversity for NBVs, the transmission dynamics and evolutionary history of NBVs are still unclear. This study successfully isolated two NBV strains (MLBC1302 and MLBC1313) from blood-sucking bat fly specimens (Eucampsipoda sundaica) and one (WDBP1716) from the spleen specimen of a fruit bat (Rousettus leschenaultii), which were collected at the China-Myanmar border area of Yunnan Province. Syncytia cytopathic effects (CPE) were observed in BHK-21 and Vero E6 cells infected with the three strains at 48 h postinfection. Electron micrographs of ultrathin sections showed numerous spherical virions with a diameter of approximately 70 nm in the cytoplasm of infected cells. The complete genome nucleotide sequence of the viruses was determined by metatranscriptomic sequencing of infected cells. Phylogenetic analysis demonstrated that the novel strains were closely related to Cangyuan orthoreovirus, Melaka orthoreovirus, and human-infecting Pteropine orthoreovirus HK23629/07. Simplot analysis revealed the strains originated from complex genomic reassortment among different NBVs, suggesting the viruses experienced a high reassortment rate. In addition, strains successfully isolated from bat flies also implied that blood-sucking arthropods might serve as potential transmission vectors. IMPORTANCE Bats are the reservoir of many viral pathogens with strong pathogenicity, including NBVs. Nevertheless, it is unclear whether arthropod vectors are involved in transmitting NBVs. In this study, we successfully isolated two NBV strains from bat flies collected from the body surface of bats, which implies that they may be vectors for virus transmission between bats. While the potential threat to humans remains to be determined, evolutionary analyses involving different segments revealed that the novel strains had complex reassortment histories, with S1, S2, and M1 segments highly similar to human pathogens. Further experiments are required to determine whether more NBVs are vectored by bat flies, their potential threat to humans, and transmission dynamics.

Pteropine orthoreoviruses use cell surface heparan sulphate as an attachment receptor.

Pteropine orthoreoviruses (PRVs) are an emerging group of fusogenic, bat-borne viruses from the Orthoreovirus genus. Since the isolation of PRV from a patient with acute respiratory tract infections in 2006, the zoonotic potential of PRV has been further highlighted following subsequent isolation of PRV species from patients in Malaysia, Hong Kong and Indonesia. However, the entry mechanism of PRV is currently unknown. In this study, we investigated the role of previously identified mammalian orthoreovirus (MRV) receptors, sialic acid and junctional adhesion molecule-1 for PRV infection. However, none of these receptors played a significant role in PRV infection, suggesting PRV uses a distinct entry receptor from MRV. Given its broad tissue tropism, we hypothesized that PRV may use a receptor that is widely expressed in all cell types, heparan sulphate (HS). Enzymatic removal of cell surface HS by heparinase treatment and genetic ablation of HS biosynthesis genes, SLC35B2, exostosin-1, N-deacetylase/N-sulfotransferase I and beta-1,3-glucuronyltransferase 3, significantly reduced infection with multiple genetically distinct PRV species. Replication kinetic of PRV3M in HS knockout cells revealed that HS plays a crucial role in the early phase of PRV infection. Mechanistic studies demonstrated that HS is an essential host-factor for PRV attachment and internalization into cells. To our knowledge, this is the first report on the use of HS as an attachment receptor by PRVs.

Isolation and genetic characterization of a mammalian orthoreovirus from Vespertilio sinensis in Japan.

Throughout East Asia, Europe, and North America, mammalian orthoreovirus (MRV), for which bats have been proposed to be natural reservoirs, has been detected in a variety of domestic and wild mammals, as well as in humans. Here, we isolated a novel MRV strain (designated as Kj22-33) from a fecal sample from Vespertilio sinensis bats in Japan. Strain Kj22-33 has a 10-segmented genome with a total length of 23,580 base pairs. Phylogenetic analysis indicated that Kj22-33 is a serotype 2 strain, the segmented genome of which has undergone reassortment with that of other MRV strains.

Oncolytic Reoviruses: Can These Emerging Zoonotic Reoviruses Be Tamed and Utilized?

Orthoreovirus is a nonenveloped double-stranded RNA virus under the Reoviridae family. This group of viruses, especially mammalian orthoreovirus (MRV), are reported with great therapeutic values due to their oncolytic effects. In this review, the life cycle and oncolytic effect of MRV and a few emerging reoviruses were summarized. This article also highlights the challenges and strategies of utilizing MRV and the emerging reoviruses, avian orthoreovirus (ARV) and pteropine orthoreovirus (PRV), as oncolytic viruses (OVs). Besides, the emergence of potential ARV and PRV as OVs were discussed in comparison to MRV. Finally, the risk of reovirus as zoonosis or reverse zoonosis (zooanthroponosis) were debated, and concerns were raised in this article, which warrant continue surveillance of reovirus (MRV, ARV, and PRV) in animals, humans, and the environment.

Surveillance, isolation and genomic characterization of Pteropine orthoreovirus of probable bat origin among patients with acute respiratory infection in Malaysia.

Pteropine orthoreovirus (PRV), an emerging bat-borne virus, has been linked to cases of acute respiratory infections (ARI) in humans. The prevalence, epidemiology and genomic diversity of PRV among ARI of unknown origin were studied. Among 632 urban outpatients tested negative for all known respiratory viruses, 2.2% were PRV-positive. Patients mainly presented with moderate to severe forms of cough, sore throat and muscle ache, but rarely with fever. Phylogenetic analysis revealed that over 90% of patients infected with the Melaka virus (MelV)-like PRV, while one patient infected with the Pulau virus previously found only in fruit bats. Human oral keratinocytes and nasopharyngeal epithelial cells were susceptible to clinical isolates of PRV, including the newly isolated MelV-like 12MYKLU1034. Whole genome sequence of 12MYKLU1034 using Nanopore technique revealed a novel reassortant strain. Evolutionary analysis of the global PRV strains suggests the continuous evolution of PRV through genetic reassortment among PRV strains circulating in human, bats and non-human primate hosts, creating a spectrum of reassortant lineages with complex evolutionary characteristics. In summary, the role of PRV as a common etiologic agent of ARI is evident. Continuous monitoring of PRV prevalence, pathogenicity and diversity among human and animal hosts is important to trace the emergence of novel reassortants.

In-depth health surveillance and clinical nutrition in farmed Atlantic salmon: a strategic attempt to detect and mitigate an HSMI outbreak.

Fish health personnel have limited tools in combatting viral diseases such as heart and skeletal muscle inflammation (HSMI) in open net-pen farmed Atlantic salmon. In this study, we aimed to predict HSMI by intensified health monitoring and apply clinical nutrition to mitigate the condition. We followed a commercial cohort (G1) of Atlantic salmon that was PRV-1 naïve when transferred to a sea cage at a location where HSMI outbreaks commonly occur. The fish in the other cages (G2-G6) at the location had a different origin than G1 and were PRV-1 positive prior to sea transfer. By continuous analysis of production data and sequentially (approximately every fourth week) performing autopsy, RT-qPCR (for PRV-1 and selected immune genes), blood and histological analysis of 10 fish from G1 and G2, we identified the time of PRV-1 infection in G1 and predicted the onset of HSMI prior to any clinical signs of disease. Identical sequences across partial genomes of PRV-1 isolates from G1 and G2 suggest the likely transfer from infected cages to G1. The isolates were grouped into a genogroup known to be of high virulence. A commercial health diet was applied during the HSMI outbreak, and the fish had low mortality and an unaffected appetite. In conclusion, we show that fish health and welfare can benefit from in-depth health monitoring. We also discuss the potential health value of clinical nutrition as a mean to mitigate HSMI.

Recombinant characteristics, pathogenicity, and transmissibility of a variant goose orthoreovirus derived from inter-lineage recombination.

Since March 2021, an infectious characterized by white necrotic foci throughout the goose body has appeared in the major goose-producing regions in China. This disease has caused economic hardship for goose farms in many regions of China with approximately 50 % mortality. A novel goose-origin orthoreovirus was isolated from the spleen of diseased geese and designated as N-GRV/HN/Goose/2021/China (N-GRV-HN21) strain. Next-generation sequencing and phylogenetic analysis revealed that the isolate was a reassortant virus containing viral gene segments from three ARV serotypes that infect duck, muscovy duck, and goose. Geese infection test showed that both N-GRV-HN21-infected and contacted geese displayed whole-body white necrotic foci. N-GRV RNA was detected in different organs of both infected and contacted geese, indicating that the N-GRV isolate is pathogenic and transmissible in geese. Seroconversion was also observed in experimentally infected and contacted geese. A prevalence study of 323 goose serum samples collected from different goose breeding areas showed that 86 % of the geese were positive for N-GRV. In conclusion, all results warrant the necessity to monitor orthoreovirus epidemiology and reassortment as the orthoreovirus could be an important pathogen for the waterfowl industry and a novel orthoreovirus might emerge to threaten animal and public health.

The µ2 and λ1 Proteins of Mammalian Reovirus Modulate Early Events Leading to Induction of the Interferon Signaling Network.

It has been previously shown that amino acid polymorphisms in reovirus proteins µ2 and λ1 are associated with differing levels of interferon induction. In the present study, viruses carrying these polymorphisms in either or both proteins, were further studied. The two viral determinants exert a synergistic effect on the control of ß-interferon induction at the protein and mRNA level, with a concomitant increase in RIG-I. In contrast, levels of phospho-Stat1 and interferon-stimulated genes are increased in singly substituted viruses but with no further increase when both substitutions were present. This suggests that the viral determinants are acting during initial events of viral recognition. Accordingly, difference between viruses was reduced when infection was performed with partially uncoated virions (ISVPs) and transfection of RNA recovered from early-infected cells recapitulates the differences between viruses harboring the different polymorphisms. Altogether, the data are consistent with a redundant or complementary role of µ2 and λ1, affecting either early disassembly or the nature of the viral RNA in the incoming viral particle. Proteins involved in viral RNA synthesis are thus involved in this likely critical aspect of the ability of different reovirus variants to infect various cell types, and to discriminate between parental and transformed/cancer cells.

Characterization of a Novel Pathogenic Reovirus in Grasshoppers.

Grasshoppers can swarm in the millions and destroy crops over wide areas, posing a major economic threat to agriculture. A wide range of insect-related viruses has recently been reported in the metagenomics of grasshoppers. Here, we identified and isolated a novel reovirus from grasshoppers, named Acrididae reovirus (ARV). The complete genome of ARV was composed of nine dsRNA segments. Phylogenetic analysis revealed that ARV formed a monophyletic lineage with unclassified insect-associated reoviruses and was sufficiently distinct from known genera of Reoviridae. ARV could replicate in its host Locusta migratoria and result in host death. Lower-dose ARV infection affected ovary development and resulted in a significant reduction in fecundity. The identification and characterization of a novel pathogenic reovirus could potentially promote the development of new biological control agents.

Detection and Characterization of a Reassortant Mammalian Orthoreovirus Isolated from Bats in Xinjiang, China.

Mammalian orthoreoviruses (MRVs) are increasingly reported to cause various diseases in humans and other animals, with many possibly originating from bats, highlighting the urgent need to investigate the diversity of bat-borne MRVs (BtMRVs). Here, we report the detection and characterization of a reassortant MRV that was isolated from a bat colony in Xinjiang, China. The BtMRV showed a wide host and organ tropism and can efficiently propagate the cell lines of different animals. It caused mild damage in the lungs of the experimentally inoculated suckling mice and was able to replicate in multiple organs for up to three weeks post-inoculation. Complete genome analyses showed that the virus was closely related to MRVs in a wide range of animals. An intricate reassortment network was revealed between the BtMRV and MRVs of human, deer, cattle, civet and other bat species. Specifically, we found a bat-specific clade of segment M1 that provides a gene source for the reassortment of human MRVs. These data provide important insights to understand the diversity of MRVs and their natural circulation between bats, humans, and other animals. Further investigation and surveillance of MRV in bats and other animals are needed to control and prevent potential MRV-related diseases.

Isolation and characterization of an orthoreovirus from Indonesian fruit bats.

Nelson Bay orthoreovirus (NBV) is an emerging bat-borne virus and causes respiratory tract infections in humans sporadically. Over the last two decades, several strains genetically related to NBV were isolated from humans and various bat species, predominantly in Southeast Asia (SEA), suggesting a high prevalence of the NBV species in this region. In this study, an orthoreovirus (ORV) belonging to the NBV species was isolated from Indonesian fruit bats' feces, tentatively named Paguyaman orthoreovirus (PgORV). Serological studies revealed that 81.2% (108/133) of Indonesian fruit bats sera had neutralizing antibodies against PgORV. Whole-genome sequencing and phylogenetic analysis of PgORV suggested the occurrence of past reassortments with other NBV strains isolated in SEA, indicating the dispersal and circulation of NBV species among bats in this region. Intranasal PgORV inoculation of laboratory mice caused severe pneumonia. Our study characterized PgORV's unique genetic background and highlighted the potential risk of PgORV-related diseases in Indonesia.

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.

Characterization of replication and variations in genome segments of a bat reovirus, BatMRV/B19-02, by RNA-seq in infected Vero-E6 cells.

Mammalian orthoreoviruses (MEVs) that can cause enteric, respiratory, and encephalitic infections have been identified in a wide variety of mammalian species. Here, we report a novel MRV type 1 strain detected in Miniopterus schreibersii that may have resulted from reassortment events. Using next-generation RNA sequencing (RNA-seq), we found that the ratios of the RNA levels of the 10 reovirus segments in infected cells were constant during the late stages of infection. We also discovered that the relative abundance of each segment differed. Notably, the relative abundance of M2 (encoding the µ1 protein) and S4 (encoding the σ3 protein) RNAs was higher than that of the others throughout the infection. Additionally, massive junctions were identified. These results support the hypothesis that defective genome segments are generated and that cross-family recombination occurs. These data may further the study of gene function, viral replication, and virus evolution.

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.