Detection, sequencing, and tissue distribution of piscine orthoreovirus 2-like virus in diseased coho salmon in Alaska.

We performed a diagnostic disease investigation on a cohort of coho salmon (Oncorhynchus kisutch) fingerlings in Alaska exhibiting anorexia, gaping mouths, anemia, and increased mortality. Histologic examination revealed mild-to-severe myocardial degeneration and lymphohistiocytic and neutrophilic myocarditis, moderate splenic histiocytosis, and mild renal histiocytosis. Piscine orthoreoviruses 1 and 3 were not detected by molecular methods, and no other viruses could be cultured on 3 common diagnostic fish cell lines. De novo assembly produced a viral genome of 10 linear segments with >80% homology to piscine orthoreovirus 2 (PRV2) encoding all 11 PRV2 proteins. An in situ hybridization probe using RNAscope was developed against 697 viral nucleotides identified by sequencing, which revealed viral genome in heart, spleen, gill, kidney, liver, blood, and the lamina propria of the intestines. Our findings are supportive of a novel piscine orthoreovirus most closely related to PRV2 associated with morbidity and mortality of coho salmon in the northeastern Pacific.

Attenuated infection by a Pteropine orthoreovirus isolated from an Egyptian fruit bat in Zambia.

BACKGROUND: Pteropine orthoreovirus (PRV) is an emerging bat-borne zoonotic virus that causes severe respiratory illness in humans. Although PRVs have been identified in fruit bats and humans in Australia and Asia, little is known about the prevalence of PRV infection in Africa. Therefore, this study performed an PRV surveillance in fruit bats in Zambia. METHODS: Egyptian fruit bats (Rousettus aegyptiacus, n = 47) and straw-colored fruit bats (Eidolon helvum, n = 33) captured in Zambia in 2017-2018 were screened for PRV infection using RT-PCR and serum neutralization tests. The complete genome sequence of an isolated PRV strain was determined by next generation sequencing and subjected to BLAST and phylogenetic analyses. Replication capacity and pathogenicity of the strain were investigated using Vero E6 cell cultures and BALB/c mice, respectively. RESULTS: An PRV strain, tentatively named Nachunsulwe-57, was isolated from one Egyptian fruit bat. Serological assays demonstrated that 98% of sera (69/70) collected from Egyptian fruit bats (n = 37) and straw-colored fruit bats (n = 33) had neutralizing antibodies against PRV. Genetic analyses revealed that all 10 genome segments of Nachunsulwe-57 were closely related to a bat-derived Kasama strain found in Uganda. Nachunsulwe-57 showed less efficiency in viral growth and lower pathogenicity in mice than another PRV strain, Miyazaki-Bali/2007, isolated from a patient. CONCLUSIONS: A high proportion of Egyptian fruit bats and straw-colored fruit bats were found to be seropositive to PRV in Zambia. Importantly, a new PRV strain (Nachunsulwe-57) was isolated from an Egyptian fruit bat in Zambia, which had relatively weak pathogenicity in mice. Taken together, our findings provide new epidemiological insights about PRV infection in bats and indicate the first isolation of an PRV strain that may have low pathogenicity to humans.

Characterization of taro reovirus and its status in taro (Colocasia esculenta) germplasm from the Pacific.

Taro reovirus (TaRV) has been reported infecting taro (Colocasia esculenta) in the South Pacific, but information on the virus is limited. Here, we report the genome sequence of a reovirus infecting taro in Papua New Guinea that had 10 genomic segments ranging from 1.1 to 3.9 kilobase pairs (kbp) in length with a total genome length of 26.3 kbp. TaRV was most closely related to rice ragged stunt virus (RRSV) but did not cross-react with RRSV polyclonal antisera. TaRV was not detected in 82 germplasm accessions of taro in Hawaii, or samples collected in American Samoa, Fiji, Guam, Palau, or Vanuatu.

Isolation and characterization of mammalian orthoreovirus type 3 from a fecal sample from a wild boar in Japan.

Mammalian orthoreoviruses (MRVs) have been identified in various mammalian species, including humans, bats, and pigs. However, isolation and complete genome sequences of MRVs from wild boars have not yet been reported. In this study, we isolated, sequenced, and analyzed an MRV from a free-living wild boar in Japan using the porcine-sapelovirus-resistant cell line N1380. Complete and empty virus particles were obtained from the N1380 cell culture supernatants, and complete genome sequences were obtained from complete virus particles. Sequence analysis revealed that the isolated MRV, named TY-14, could be classified as MRV3 and had a close genetic relationship to an MRV2 isolate from a lion in a Japanese zoo (L2, L3, and M3 genes) and a human MRV2 isolate from Japan (S2 gene). Phylogenetic analysis showed that TY-14 clustered only with bat MRVs in the M1 phylogenetic tree but formed a cluster with several animal MRVs in the M2 and S3 phylogenetic trees and branched independently in the L1, S1, and S4 phylogenetic trees, suggesting a genetic relationship to viruses of unknown origin. Recombination events were identified in the M2 gene. These results suggest that TY-14 was generated by reassortment and recombination events involving MRVs circulating in Japan, viruses from bats, and other viruses of unknown origin.

Inactivation of Piscine orthoreovirus.

Piscine orthoreovirus infects various salmonid fish species, and the infection is associated with diseases such as heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). There are no vaccines available or genetically selected resistant hosts that can efficiently control piscine orthoreovirus (PRV) infection. Currently, the only prophylactic measure against PRV is general biosecurity measures aiming to break the transmission cycle. Methods to eradicate infectious virus from contaminated facilities are desirable, but the knowledge on how to inactivate PRV is lacking. A major bottleneck for inactivation studies is the lack of ability to propagate PRV in cell culture. Therefore, in this study we developed an in vivo model for detection of infectious PRV particles after treatment of the virus with inactivation tools such as heat, pH, iodine, UV and commercially available disinfectants. The results show that standard iodine treatment is efficient in inactivation of the virus, and similarly are high and low pH extremes and treatment with Virocid, a commercially available disinfectant. A UV dose of at least 50 mJ/cm2 is required for inactivation, and the virus has high resistance against heat treatment.

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.

Cross-sectional study of histopathology and piscine orthoreovirus during a marine production cycle of farmed Atlantic salmon (Salmo salar L.) in British Columbia, Canada.

Two cohorts of farmed Atlantic salmon, Salmo salar L., in British Columbia, Canada, were sampled for histopathology (nine organs) and piscine orthoreovirus (PRV-1) PCR after seawater entry at 2, 4, 6, 8, 10, 13, 16 and 19 months (20 fish per cohort per date). One cohort-from a PRV+ hatchery-remained PRV+ throughout the study (sample prevalence 80%-100%). In an adjacent pen, the other cohort-from a PRV- hatchery-was 0% PRV+ at 78 days, 30% PRV+ at 128 days and ≥95% PRV+ thereafter. Among sample cohorts that were ≥80% PRV+, median Ct values were nominally less among fish sourced from the PRV- hatchery (28.7-33.3) than the PRV+ hatchery (30.8-35.2). No microscopic lesions were associated with PRV Ct value (minimum = 25.6). About 3% of fish in both cohorts had moderate inflammatory heart lesions; among these fish, only one had skeletal muscle inflammation (mild), and PRV Ct values were similar to unaffected cohorts sampled the same day. Also, among 16 moribund or freshly dead fish sampled opportunistically during the study, 14 were PRV+, and none had significant inflammatory heart lesions. These data support the hypothesis that British Columbia PRV-1 does not contribute to mortality.

Piscine orthoreovirus sequences in escaped farmed Atlantic salmon in Washington and British Columbia.

BACKGROUND: Piscine orthoreovirus (PRV) is an emergent virus in salmon aquaculture belonging to the family Reoviridae. PRV is associated with a growing list of pathological conditions including heart and skeletal inflammation (HSMI) of farmed Atlantic salmon. Despite widespread PRV infection in commercially farmed Atlantic salmon, information on PRV prevalence and on the genetic sequence variation of PRV in Atlantic salmon on the north Pacific Coast is limited. METHODS: Feral Atlantic salmon caught in Washington State and British Columbia following a large containment failure at a farm in northern Puget Sound were sampled. Fish tissues were tested for PRV by RT-qPCR assay for segment L1 and conventional RT-PCR for PRV segment S1. The PCR products were sequenced and their relationship to PRV strains in GenBank was determined using phylogenetic analysis and nucleotide and amino acid homology comparisons. RESULTS: Following the escape of 253,000 Atlantic salmon from a salmon farm in Washington State, USA, 72/73 tissue samples from 27 Atlantic salmon captured shortly after the escape tested PRV-positive. We estimate PRV-prevalence in the source farm population at 95% or greater. The PRV found in the fish was identified as PRV sub-genotype Ia and very similar to PRV from farmed Atlantic salmon in Iceland. This correlates with the source of the fish in the farm. Eggs of infected fish were positive for PRV indicating the possibility of vertical transfer and spread with fish egg transports. CONCLUSIONS: PRV prevalence was close to 100% in farmed Atlantic salmon that were caught in Washington State and British Columbia following a large containment failure at a farm in northern Puget Sound. The PRV strains present in the escaped Atlantic salmon were very similar to the PRV strain reported in farmed Atlantic salmon from the source hatchery in Iceland that was used to stock commercial aquaculture sites in Washington State. This study emphasizes the need to screen Atlantic salmon broodstock for PRV, particularly where used to supply eggs to the global Atlantic salmon farming industry thereby improving our understanding of PRV epidemiology.

Piscine orthoreovirus-3 is prevalent in wild seatrout (Salmo trutta L.) in Norway.

In 2017, a PCR-based survey for Piscine orthoreovirus-3 (PRV-3) was conducted in wild anadromous and non-anadromous salmonids in Norway. In seatrout (anadromous Salmo trutta L.), the virus was present in 16.6% of the fish and in 15 of 21 investigated rivers. Four of 221 (1.8%) Atlantic salmon (Salmo salar L.) from three of 15 rivers were also PCR-positive, with Ct-values indicating low amounts of viral RNA. All anadromous Arctic char (Salvelinus alpinus L.) were PCR-negative. Neither non-anadromous trout (brown trout) nor landlocked salmon were PRV-3 positive. Altogether, these findings suggest that in Norway PRV-3 is more prevalent in the marine environment. In contrast, PRV-3 is present in areas with intensive inland farming in continental Europe. PRV-3 genome sequences from Norwegian seatrout grouped together with sequences from rainbow trout (Oncorhynchus mykiss Walbaum) in Norway and Coho salmon (Oncorhynchus kisutch Walbaum) in Chile. At present, the origin of the virus remains unknown. Nevertheless, the study highlights the value of safeguarding native fish by upholding natural and artificial barriers that hinder introduction and spread, on a local or national scale, of alien fish species and their pathogens. Accordingly, further investigations of freshwater reservoirs and interactions with farmed salmonids are warranted.

Detection of piscine orthoreoviruses (PRV-1 and PRV-3) in Atlantic salmon and rainbow trout farmed in Germany.

Piscine orthoreoviruses (PRVs) are emerging pathogens causing circulatory disorders in salmonids. PRV-1 is the etiological cause of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar), characterized by epicarditis, inflammation and necrosis of the myocardium, myositis and necrosis of red skeletal muscle. In 2017, two German breeding farms for Atlantic salmon and rainbow trout (Oncorhynchus mykiss) experienced disease outbreaks with mortalities of 10% and 20% respectively. The main clinical signs were exhaustion and lethargic behaviour. During examinations, PRV-1 in salmon and PRV-3 in trout were detected for the first time in Germany. Further analyses also indicated the presence of Aeromonas salmonicida in internal tissues of both species. While PRV-1 could be putatively linked with the disease in Atlantic salmon, most of the rainbow trout suffered from an infection with A. salmonicida and not with PRV-3. Interestingly, the sequence analysis suggests that the German PRV-3 isolate is more similar to a Chilean PRV-3 isolate from Coho salmon (Oncorhynchus kisutch) than to PRV-3 from rainbow trout from Norway. This indicates a wide geographic distribution of this virus or dispersal by global trade. These findings indicate that infections with PRVs should be considered when investigating disease outbreaks in salmonids.

Mass spectrometry-based identification and whole-genome characterisation of the first pteropine orthoreovirus isolated from monkey faeces in Thailand.

BACKGROUND: The pteropine orthoreovirus (PRV) was isolated from monkey (Macaca fascicularis) faecal samples collected from human-inhabited areas in Lopburi Province, Thailand. These samples were initially obtained to survey for the presence of hepatitis E virus (HEV). RESULTS: Two virus isolates were retrieved by virus culture of 55 monkey faecal samples. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was successfully used to identify the viruses as the segmented dsRNA orthoreovirus. Phylogenetic analysis of the Lopburi orthoreovirus whole-genomes revealed relationships with the well-characterised PRVs Pulau (segment L1), Cangyuan (segments L2, M3 and S3), Melaka (segments L3 and M2), Kampar (segments M1 and S2) and Sikamat (segments S1 and S4) of Southeast Asia and China with nucleotide sequence identities of 93.5-98.9%. RT-PCR showed that PRV was detected in 10.9% (6/55) and HEV was detected in 25.5% (14/55) of the monkey faecal samples. CONCLUSIONS: PRV was isolated from monkey faeces for the first time in Thailand via viral culture and LC-MS/MS. The genetic diversity of the virus genome segments suggested a re-assortment within the PRV species group. The overall findings emphasise that monkey faeces can be sources of zoonotic viruses, including PRV and HEV, and suggest the need for active virus surveillance in areas of human and monkey co-habitation to prevent and control emerging zoonotic diseases in the future.

Identification of a piscine reovirus-related pathogen in proliferative darkening syndrome (PDS) infected brown trout (Salmo trutta fario) using a next-generation technology detection pipeline.

The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) with a mortality rate of near 100% in pre-alpine rivers of central Europe. So far the etiology and causation of this disease is still unclear. The objective of this study was to identify the cause of PDS using a next-generation technology detection pipeline. Following the hypothesis that PDS is caused by an infectious agent, brown trout specimens were exposed to water from a heavily affected pre-alpine river with annual occurrence of the disease. Specimens were sampled over the entire time period from potential infection through death. Transcriptomic analysis (microarray) and RT-qPCR of brown trout liver tissue evidenced strong gene expression response of immune-associated genes. Messenger RNA of specimens with synchronous immune expression profiles were ultra-deep sequenced using next-generation sequencing technology (NGS). Bioinformatic processing of generated reads and gap-filling Sanger re-sequencing of the identified pathogen genome revealed strong evidence that a piscine-related reovirus is the causative organism of PDS. The identified pathogen is phylogenetically closely related to the family of piscine reoviruses (PRV) which are considered as the causation of different fish diseases in Atlantic and Pacific salmonid species such as Salmo salar and Onchorhynchus kisutch. This study also highlights that the approach of first screening immune responses along a timeline in order to identify synchronously affected stages in different specimens which subsequently were ultra-deep sequenced is an effective approach in pathogen detection. In particular, the identification of specimens with synchronous molecular immune response patterns combined with NGS sequencing and gap-filling re-sequencing resulted in the successful pathogen detection of PDS.

Isolation of Pteropine orthoreovirus from Pteropus vampyrus in Garut, Indonesia.

Flying foxes belonging to the genus Pteropus are known to be reservoirs of zoonotic viruses. In this study, we describe the isolation of Pteropine orthoreovirus (PRV) from rectal swab samples of Pteropus vampyrus in Indonesia. PRV is an emerging zoonotic respiratory virus that can be transmitted from bats to humans. Rectal swabs (n = 91) were screened by PCR for PRV and 10 (11%) were positive. Phylogenetic analysis based on nucleotide sequences indicated that the S2, S3, S4, M3, L2, and L3 segments of one isolate (Garut-69) were closely related to previously isolated strains in Indonesia. The remaining gene segments showed both similarity and genetic divergence with other PRV strains, suggesting that re-assortment events had occurred. This is the first report of PRV infection to P. vampyrus in West Java, Indonesia.

Molecular and Antigenic Characterization of Piscine orthoreovirus (PRV) from Rainbow Trout (Oncorhynchus mykiss).

Piscine orthoreovirus (PRV-1) causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). Recently, a novel PRV (formerly PRV-Om, here called PRV-3), was found in rainbow trout (Oncorhynchus mykiss) with HSMI-like disease. PRV is considered to be an emerging pathogen in farmed salmonids. In this study, molecular and antigenic characterization of PRV-3 was performed. Erythrocytes are the main target cells for PRV, and blood samples that were collected from experimentally challenged fish were used as source of virus. Virus particles were purified by gradient ultracentrifugation and the complete coding sequences of PRV-3 were obtained by Illumina sequencing. When compared to PRV-1, the nucleotide identity of the coding regions was 80.1%, and the amino acid identities of the predicted PRV-3 proteins varied from 96.7% (λ1) to 79.1% (σ3). Phylogenetic analysis showed that PRV-3 belongs to a separate cluster. The region encoding σ3 were sequenced from PRV-3 isolates collected from rainbow trout in Europe. These sequences clustered together, but were distant from PRV-3 that was isolated from rainbow trout in Norway. Bioinformatic analyses of PRV-3 proteins revealed that predicted secondary structures and functional domains were conserved between PRV-3 and PRV-1. Rabbit antisera raised against purified virus or various recombinant virus proteins from PRV-1 all cross-reacted with PRV-3. Our findings indicate that despite different species preferences of the PRV subtypes, several genetic, antigenic, and structural properties are conserved between PRV-1 and-3.

Development of a serum neutralization assay to detect Pteropine Orthoreovirus Indonesia/2010 neutralizing antibodies.

Pteropine Orthoreoviruses (PRVs) are fusogenic bat-borne orthoreoviruses that cause flu-like upper respiratory tract infections in humans. The presence of this group of viruses in bats and humans has been well documented in areas where their biological reservoirs - fruit bats (family Pteropodidae) - live densely. In the present study, a serum neutralization (SN) assay to detect neutralizing antibodies against PRV Indonesia/2010 isolate was set up and used to assess the seroprevalence of this virus in Italian domestic animals. The new developed assay was able of detecting PRV neutralizing antibodies in the hyper-immune polyclonal serum produced in rabbits (titer of 1:160). The negative serum was negative at all tested dilutions. No cross-reactions have been evidenced neither against reference MRVs nor against their respective hyper-immune sera. Eight hundred and fifty-three serum samples collected from 524 bovines, 271 small ruminants, and 58 horses (all used as sentinel animals in the Bluetongue and West Nile disease National surveillance program) were also tested with the new developed SN assay. According to the results of this survey, neither PRV nor PRV cross- reacting viruses antibodies have been demonstrated in Italian domestic animals. However, the new developed SN assay could be a very valuable diagnostic tool to detect infection in animals and humans.

Prevalence of piscine orthoreovirus and salmonid alphavirus in sea-caught returning adult Atlantic salmon (Salmo salar L.) in northern Norway.

Heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus (PRV) and pancreas disease (PD) caused by salmonid alphavirus (SAV) are among the most prevalent viral diseases of Atlantic salmon farmed in Norway. There are limited data about the impact of disease in farmed salmon on wild salmon populations. Therefore, the prevalence of PRV and SAV in returning salmon caught in six sea sites was determined using real-time RT-PCR analyses. Of 419 salmon tested, 15.8% tested positive for PRV, while none were positive for SAV. However, scale reading revealed that 10% of the salmon had escaped from farms. The prevalence of PRV in wild salmon (8%) was significantly lower than in farm escapees (86%), and increased with fish length (proxy for age). Sequencing of the S1 gene of PRV from 39 infected fish revealed a mix of genotypes. The observed increase in PRV prevalence with fish age and the lack of phylogeographic structure of the virus could be explained by virus transmission in the feeding areas. Our results highlight the need for studies about the prevalence of PRV and other pathogens in Atlantic salmon in its oceanic phase.

Molecular testing of adult Pacific salmon and trout (Oncorhynchus spp.) for several RNA viruses demonstrates widespread distribution of piscine orthoreovirus in Alaska and Washington.

This research was initiated in conjunction with a systematic, multiagency surveillance effort in the United States (U.S.) in response to reported findings of infectious salmon anaemia virus (ISAV) RNA in British Columbia, Canada. In the systematic surveillance study reported in a companion paper, tissues from various salmonids taken from Washington and Alaska were surveyed for ISAV RNA using the U.S.-approved diagnostic method, and samples were released for use in this present study only after testing negative. Here, we tested a subset of these samples for ISAV RNA with three additional published molecular assays, as well as for RNA from salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV). All samples (n = 2,252; 121 stock cohorts) tested negative for RNA from ISAV, PMCV, and SAV. In contrast, there were 25 stock cohorts from Washington and Alaska that had one or more individuals test positive for PRV RNA; prevalence within stocks varied and ranged from 2% to 73%. The overall prevalence of PRV RNA-positive individuals across the study was 3.4% (77 of 2,252 fish tested). Findings of PRV RNA were most common in coho (Oncorhynchus kisutch Walbaum) and Chinook (O. tshawytscha Walbaum) salmon.

Antiviral Responses and Biological Concequences of Piscine orthoreovirus Infection in Salmonid Erythrocytes.

Salmonid red blood cells are the main target cells for Piscine orthoreovirus (PRV). Three genotypes of PRV (PRV-1,2,3) infect Atlantic salmon (Salmo salar), Chinook salmon (Onchorhynchus tshawytscha), Coho salmon (Oncorhynchus kisutch), rainbow trout (Onchorhynchus mykiss) and brown trout (Salmo trutta), and can cause diseases like heart and skeletal muscle inflammation (HSMI), jaundice syndrome, erythrocyte inclusion body syndrome (EIBS) and proliferative darkening syndrome (PDS). Purified PRV administrated to fish has proven the causality for HSMI and EIBS. During the early peak phase of infection, salmonid erythrocytes are the main virus-replicating cells. In this initial phase, cytoplasmic inclusions called "virus factories" can be observed in the erythrocytes, and are the primary sites for the formation of new virus particles. The PRV-infected erythrocytes in Atlantic salmon mount a strong long-lasting innate antiviral response lasting for many weeks after the onset of infection. The antiviral response of Atlantic salmon erythrocytes involves upregulation of potential inhibitors of translation. In accordance with this, PRV-1 protein production in erythrocytes halts while virus RNA can persist for months. Furthermore, PRV infection in Coho salmon and rainbow trout are associated with anemia, and in Atlantic salmon lower hemoglobin levels are observed. Here we summarize and discuss the recently published findings on PRV infection, replication and effects on salmonid erythrocytes, and discuss how PRV can be a useful tool for the study of innate immune responses in erythrocytes, and help reveal novel immune functions of the red blood cells in fish.

Virulence, pathology, and pathogenesis of Pteropine orthoreovirus (PRV) in BALB/c mice: Development of an animal infection model for PRV.

BACKGROUND: Cases of acute respiratory tract infection caused by Pteropine orthoreovirus (PRV) of the genus Orthoreovirus (family: Reoviridae) have been reported in Southeast Asia, where it was isolated from humans and bats. It is possible that PRV-associated respiratory infections might be prevalent in Southeast Asia. The clinical course of PRV is not fully elucidated. METHODS: The virulence, pathology, and pathogenesis of two PRV strains, a human-borne PRV strain (isolated from a patient, who returned to Japan from Bali, Indonesia in 2007) and a bat-borne PRV (isolated from a bat [Eonycteris spelaea] in the Philippines in 2013) were investigated in BALB/c mice using virological, pathological, and immunological study methods. RESULTS: The intranasal inoculation of BALB/c mice with human-borne PRV caused respiratory infection. In addition, all mice with immunity induced by pre-inoculation with a non-lethal dose of PRV were completely protected against lethal PRV infection. Mice treated with antiserum with neutralizing antibody activity after inoculation with a lethal dose of PRV showed a reduced fatality rate. In this mouse model, bat-borne PRV caused respiratory infection similar to human-borne PRV. PRV caused lethal respiratory disease in an animal model of PRV infection, in which BALB/c mice were used. CONCLUSIONS: The BALB/c mouse model might help to accelerate research on the virulence of PRV and be useful for evaluating the efficacy of therapeutic agents and vaccines for the treatment and prevention of PRV infection. PRV was shown for the first time to be a causative virus of respiratory disease on the basis of Koch's postulations by the additional demonstration that PRV caused respiratory disease in mice through their intranasal inoculation with PRV.