Piscine orthoreovirus (PRV) belongs to the Reoviridae family and is the only known fish virus related to the Orthoreovirus genus. The virus is the causative agent of heart and skeletal muscle inflammation (HSMI), an emerging disease in farmed Atlantic salmon (Salmo salar L.). PRV is ubiquitous in farmed Atlantic salmon and high loads of PRV in the heart are consistent findings in HSMI. The mechanism by which PRV infection causes disease remains largely unknown. In this study we investigated the presence of PRV in blood and erythrocytes using an experimental cohabitation challenge model. We found that in the early phases of infection, the PRV loads in blood were significantly higher than in any other organ. Most virus was found in the erythrocyte fraction, and in individual fish more than 50% of erythrocytes were PRV-positive, as determined by flow cytometry. PRV was condensed into large cytoplasmic inclusions resembling viral factories, as demonstrated by immunofluorescence and confocal microscopy. By electron microscopy we showed that these inclusions contained reovirus-like particles. The PRV particles and inclusions also had a striking resemblance to previously reported viral inclusions described as Erythrocytic inclusion body syndrome (EIBS). We conclude that the erythrocyte is a major target cell for PRV infection. These findings provide new information about HSMI pathogenesis, and show that PRV is an important factor of viral erythrocytic inclusions.

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.

Viral diseases are among the main challenges in farming of Atlantic salmon (Salmo salar). The most prevalent viral diseases in Norwegian salmon aquaculture are heart and skeletal muscle inflammation (HSMI) caused by Piscine orthoreovirus (PRV), and pancreas disease (PD) caused by Salmonid alphavirus (SAV). Both PRV and SAV target heart and skeletal muscles, but SAV additionally targets exocrine pancreas. PRV and SAV are often present in the same locations and co-infections occur, but the effect of this crosstalk on disease development has not been investigated. In the present experiment, the effect of a primary PRV infection on subsequent SAV infection was studied. Atlantic salmon were infected with PRV by cohabitation, followed by addition of SAV shedder fish 4 or 10 weeks after the initial PRV infection. Histopathological evaluation, monitoring of viral RNA levels and host gene expression analysis were used to assess disease development. Significant reduction of SAV RNA levels and of PD specific histopathological changes were observed in the co-infected groups compared to fish infected by SAV only. A strong correlation was found between histopathological development and expression of disease related genes in heart. In conclusion, experimentally PRV infected salmon are less susceptible to secondary SAV infection and development of PD.

Piscine orthoreovirus 1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus is widespread in Atlantic salmon and was present in Norway long before the first description of HSMI in 1999. Furthermore, in Canada the virus is prevalent in farmed Atlantic salmon but HSMI is not and Canadian isolates have failed to reproduce HSMI experimentally. This has led to the hypothesis that there are virulence differences between PRV-1 isolates. In this study we performed a dose standardized challenge trial, comparing six PRV-1 isolates, including two Norwegian field isolates from 2018, three historical Norwegian isolates predating the first report of HSMI and one Canadian isolate. The Norwegian 2018 isolates induced lower viral protein load in blood cells but higher plasma viremia. Following peak replication in blood, the two Norwegian 2018 isolates induced histopathological lesions in the heart consistent with HSMI, whereas all three historical Norwegian and the Canadian isolates induced only mild cardiac lesions. This is the first demonstration of virulence differences between PRV-1 isolates and the phenotypic differences are linked to viral proteins encoded by segment S1, M2, L1, L2 and S4.

Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1–2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection.

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation in farmed Atlantic salmon. The virus is ubiquitous and found in both farmed and wild salmonid fish. It belongs to the family Reoviridae, closely related to the genus Orthoreovirus. The PRV genome comprises ten double-stranded RNA segments encoding at least eight structural and two non-structural proteins. Erythrocytes are the major target cells for PRV. Infected erythrocytes contain globular inclusions resembling viral factories; the putative site of viral replication. For the mammalian reovirus (MRV), the non-structural protein μNS is the primary organizer in factory formation. The analogous PRV protein was the focus of the present study. The subcellular location of PRV μNS and its co-localization with the PRV σNS, µ2 and λ1 proteins was investigated. We demonstrated that PRV μNS forms dense globular cytoplasmic inclusions in transfected fish cells, resembling the viral factories of MRV. In co-transfection experiments with μNS, the σNS, μ2 and λ1 proteins were recruited to the globular structures. The ability of μNS to recruit other PRV proteins into globular inclusions indicates that it is the main viral protein involved in viral factory formation and pivotal in early steps of viral assembly.