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.

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.

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.

The newly described piscine reovirus (PRV) appears to be associated with the development of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar L. PRV seems to be ubiquitous among fish in Norwegian salmon farms, but high viral loads and tissue distribution support a causal relationship between virus and disease. In order to improve understanding of the distribution of PRV in the salmon production line, we quantified PRV by using realtime PCR on heart samples collected at different points in the life cycle from pre-smolts to fish ready for slaughter. PRV positive pre-smolts were found in about 36% of the freshwater cohorts and a general increase in viral load was observed after their transfer to seawater. A reduction in viral loads was recorded when fish approached slaughter (18 mo in sea cages). Sequencing of positive samples did not support the hypothesis that outbreaks are caused by the spreading of a particular (virulent) strain of PRV. KEY WORDS: Piscine reovirus · PCR · Salmon · Aquaculture · HSMI Resale or republication not permitted without written consent of the publisherDis Aquat Org 99: [7][8][9][10][11][12] 2012 After initial success with experimental transmission of the disease (Kongtorp et al. 2004a), HSMI was recently linked to a novel reovirus, piscine reovirus (PRV) (Palacios et al. 2010). Although the virus appears to be ubiquitous in Norwegian salmon farms, elevated viral loads are associated with outbreaks of HSMI (Palacios et al. 2010). A causal relationship is also supported by in situ hybridisation using formalin-fixed heart tissue where a correlation between the presence of viral nucleic acids and histopathological lesions has been found (Palacios et al. 2010). PRV has been detected in healthy, wild salmon, albeit with lower prevalence and virus load. The virus has also been found in archive samples taken from salmon in the late 1980s and also in rainbow trout Oncorhynchus mykiss Walbaum (Palacios et al. 2010). Farmed Atlantic salmon with HSMI-like lesions have also been reported from Scotland (Ferguson et al. 2005).In the present study we quantified PRV loads at various stages in the Atlantic salmon production line, from pre-smolts in fresh water to fish ready for slaughter. Samples were collected from farms where no heart lesions were observed and from farms with clinical outbreaks of HSMI. We also sequenced parts of the PRV genome detected in different samples in order to investigate the molecular epidemiology of the virus and potentially virulent PRV strains. Our results indicate that PRV is a ubiquitous virus in Norwegian fish farms and that careful virus quantification and/or study of tissue distribution of viral particles/genomes need to be complemented with his topathology for correct diagnoses. The results should be useful when interpreting PRV loads both in the context of HSMI/PRV epidemiology and diagnosis. MATERIALS AND METHODSViral loads were established for 2 sets of samples; one set of 171 field samples was collected from 23 mar...

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in μ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-β signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins μ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in μ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.

Aquaculture is the fastest growing food production sector in the world. However, the increased production has been accompanied by the emergence of infectious diseases. Heart and skeletal muscle inflammation (HSMI) is one example of an emerging disease in farmed Atlantic salmon (Salmo salar L). Since the first recognition as a disease entity in 1999 it has become a widespread and economically important disease in Norway. The disease was recently found to be associated with infection with a novel reovirus, piscine reovirus (PRV). The load of PRV, examined by RT-qPCR, correlated with severity of HSMI in naturally and experimentally infected salmon. The disease is characterized by epi-, endo- and myocarditis, myocardial necrosis, myositis and necrosis of the red skeletal muscle. The aim of this study was to investigate the presence of PRV antigens in heart tissue of Atlantic salmon and monitor the virus distribution in the heart during the disease development. This included target cell specificity, viral load and tissue location during an HSMI outbreak. Rabbit polyclonal antisera were raised against putative PRV capsid proteins μ1C and σ1 and used in immunohistochemical analysis of archived salmon heart tissue from an experimental infection. The results are consistent with the histopathological changes of HSMI and showed a sequential staining pattern with PRV antigens initially present in leukocyte-like cells and subsequently in cardiomyocytes in the heart ventricle. Our results confirm the association between PRV and HSMI, and strengthen the hypothesis of PRV being the causative agent of HSMI. Immunohistochemical detection of PRV antigens will be beneficial for the understanding of the pathogenesis of HSMI as well as for diagnostic purposes.

Defense mechanisms in developing fish are poorly known but before maturation of lymphoid organs and immunocompetence, innate mechanisms are essential. The complement system represents a major part of innate immunity. Our main objective was to map the presence of complement components early in fish development. Rainbow trout eggs, embryos, and hatchlings were assayed for the onset and duration of C3-1, C3-3, C3-4, C4, C5, C7, factor B, and factor D transcription using real-time reverse transcription-polymerase chain reaction. In general, complement transcript levels increased steadily from day 28 postfertilization to hatch, followed by a decrease during yolk-sac resorption. All the complement proteins studied were found in unfertilized eggs. There was no correlation between the transcript and protein levels throughout the study period. Complement proteins appeared in the liver, kidney, and intestine between day 7 and 35 but not until day 77 in the heart. This study is the first to address the ontogeny of several complement components and represents the first evidence that maternal transfer of complement components, other than C3, occurs in teleost fish.

No abstract