Atlantic salmon (Salmo salar L.) mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens and fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999[1], HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom[2]. Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful. Here we provide evidence that HSMI is associated with infection with piscine reovirus (PRV). PRV is a novel reovirus identified by unbiased high throughput DNA sequencing and a bioinformatics program focused on nucleotide frequency as well as sequence alignment and motif analyses. Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Koch's postulates, or prevention or modification of disease through use of specific drugs or vaccines. Nonetheless, as our data indicate that a causal relationship is plausible, measures must be taken to control PRV not only because it threatens domestic salmon production but also due to the potential for transmission to wild salmon populations.
Viral diseases pose a significant threat to the productivity in aquaculture. Heart- and skeletal muscle inflammation (HSMI) is an emerging disease in Atlantic salmon (Salmo salar) farming. HSMI is associated with Piscine orthoreovirus (PRV) infection, but PRV is ubiquitous in farmed Atlantic salmon and thus present also in apparently healthy individuals. This has brought speculations if additional etiological factors are required, and experiments focusing on the causal relationship between PRV and HSMI are highly warranted. A major bottleneck in PRV research has been the lack of cell lines that allow propagation of the virus. To bypass this, we propagated PRV in salmon, bled the fish at the peak of the infection, and purified virus particles from blood cells. Electron microscopy, western blot and high-throughput sequencing all verified the purity of the viral particles. Purified PRV particles were inoculated into naïve Atlantic salmon. The purified virus replicated in inoculated fish, spread to naïve cohabitants, and induced histopathological changes consistent with HSMI. PRV specific staining was demonstrated in the pathological lesions. A dose-dependent response was observed; a high dose of virus gave earlier peak of the viral load and development of histopathological changes compared to a lower dose, but no difference in the severity of the disease. The experiment demonstrated that PRV can be purified from blood cells, and that PRV is the etiological agent of HSMI in Atlantic salmon.
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.
Infectious salmon anemia (ISA) virus is the cause of infectious salmon anemia in farmed Atlantic salmon. The virus has been shown to contain RNA with structural characteristics similar to those of accepted members of the Orthomyxoviridae. Further biochemical, physiochemical, and morphological characterization of ISA virus was undertaken to clarify its taxonomic position. The virus was found to be sensitive to chloroform, heat, and low pH and agglutinated erythrocytes from fish. Erythrocytes from mammals or birds were not agglutinated. Receptor-destroying enzyme activity was detected, and the nature of this enzyme was suggested to be an acetylesterase. The buoyant density of the virus was 1.18 g/ml in sucrose and CsCl gradients. The maximum rate of virus replication was observed at 15°C, while no virus was produced at 25°C. Actinomycin D inhibited viral replication, and viral antigen was detected in nuclei by immunofluorescence. The addition of trypsin to the culture medium during virus replication had a beneficial effect on virus replication. ISA virus contains four major polypeptides with estimated molecular sizes of 71, 53, 43, and 24 kDa. Electron microscopy revealed structures closely resembling the nucleocapsids of influenza virus. Mushroom-shaped surface projections were a distinctive morphological feature, which differed from the rod-shaped hemagglutinin projections of the influenza viruses. The data reported here support the relationship of ISA virus to the Orthomyxoviridae, although ISA virus differs from influenza viruses in some morphological characteristics and in showing restricted hemagglutination, in different specificity of the receptor-destroying enzyme, in different polypeptide profile, in being unable to replicate at temperatures above 25°C, and in host range.
BackgroundCardiomyopathy syndrome (CMS) is a severe disease affecting large farmed Atlantic salmon. Mortality often appears without prior clinical signs, typically shortly prior to slaughter. We recently reported the finding and the complete genomic sequence of a novel piscine reovirus (PRV), which is associated with another cardiac disease in Atlantic salmon; heart and skeletal muscle inflammation (HSMI). In the present work we have studied whether PRV or other infectious agents may be involved in the etiology of CMS.ResultsUsing high throughput sequencing on heart samples from natural outbreaks of CMS and from fish experimentally challenged with material from fish diagnosed with CMS a high number of sequence reads identical to the PRV genome were identified. In addition, a sequence contig from a novel totivirus could also be constructed. Using RT-qPCR, levels of PRV in tissue samples were quantified and the totivirus was detected in all samples tested from CMS fish but not in controls. In situ hybridization supported this pattern indicating a possible association between CMS and the novel piscine totivirus.ConclusionsAlthough causality for CMS in Atlantic salmon could not be proven for either of the two viruses, our results are compatible with a hypothesis where, in the experimental challenge studied, PRV behaves as an opportunist whereas the totivirus might be more directly linked with the development of CMS.
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