Øystein Wessel

Transcriptome analyses of Atlantic salmon (Salmo salar L.) erythrocytes infected with piscine orthoreovirus (PRV).

Heart and skeletal muscle inflammation (HSMI) is a widespread disease of farmed Atlantic salmon (Salmo salar L.) and is associated with piscine orthoreovirus (PRV) infection. PRV is detectable in blood long before development of pathology in cardiac- and skeletal muscle appear, and erythrocytes have been identified as important target cells for the virus. The effects of PRV infection on cellular processes of erythrocytes are not known, but haemolytic anemia or systemic lysis of erythrocytes does not seem to occur, even with high virus loads in erythrocytes. In this study, gene expression profiling performed with high-density oligonucleotide microarray showed that PRV infection of erythrocytes induced a large panel of virus responsive genes. These involved interferon-regulated antiviral genes, as well as genes involved in antigen presentation via MHC class I. PRV infection also stimulated negative immune regulators. In contrast, a large number of immune genes expressed prior to infection were down-regulated. Moderate reduction of expression was also found for many genes encoding components of cytoskeleton and myofiber, proteins involved in metabolism, ion exchange, cell-cell interactions as well as growth factors and regulators of differentiation. PRV did not affect expression of genes involved in heme biosynthesis, gas exchange or erythrocyte-specific markers, but some regulators of erythropoiesis showed decreased transcription levels. These results indicate that PRV infection activates innate antiviral immunity in salmon erythrocytes, but suppresses other gene expression programs. Gene expression profiles suggest major phenotypic changes in PRV infected erythrocytes, but the functional consequences remain to be explored.

Piscine orthoreovirus (PRV) replicates in Atlantic salmon (Salmo salar L.) erythrocytes ex vivo

Piscine orthoreovirus (PRV) is a reovirus that has predominantly been detected in Atlantic salmon (Salmo salar L.). PRV is associated with heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon, and recently erythrocytes were identified as major target cells. The study of PRV replication and pathogenesis of the infection has been impeded by the inability to propagate PRV in vitro. In this study we developed an ex vivo cultivation system for PRV in Atlantic salmon erythrocytes. PRV was successfully passaged to naïve erythrocytes using lysates of blood cells from infected salmon. During cultivation a significant increase in viral load was observed by RT-qPCR and flow cytometry, which coincided with the formation of cytoplasmic inclusions. The inclusions resembled viral factories and contained both PRV protein and dsRNA. In addition, the erythrocytes generated an antiviral immune gene activation after PRV infection, with significant up-regulation of IFN-α, RIG-I, Mx and PKR transcripts. Supernatants from the first passage successfully transmitted virus to naïve erythrocytes. This study demonstrates that PRV replicates in Atlantic salmon erythrocytes ex vivo. The ex vivo infection model closely reflects the situation in vivo and can be used to study the infection and replication mechanisms of PRV, as well as the antiviral immune responses of salmonid erythrocytes.

Heart and skeletal muscle inflammation (HSMI) disease diagnosed on a British Columbia salmon farm through a longitudinal farm study

Heart and skeletal muscle inflammation (HSMI) is an emerging disease of marine-farmed Atlantic Salmon (Salmo salar), first recognized in 1999 in Norway, and later also reported in Scotland and Chile. We undertook a longitudinal study involving health evaluation over an entire marine production cycle on one salmon farm in British Columbia (Canada). In previous production cycles at this farm site and others in the vicinity, cardiac lesions not linked to a specific infectious agent or disease were identified. Histologic assessments of both live and moribund fish samples collected at the farm during the longitudinal study documented at the population level the development, peak, and recovery phases of HSMI. The fish underwent histopathological evaluation of all tissues, Twort’s Gram staining, immunohistochemistry, and molecular quantification in heart tissue of 44 agents known or suspected to cause disease in salmon. Our analysis showed evidence of HSMI histopathological lesions over an 11-month timespan, with the prevalence of lesions peaking at 80–100% in sampled fish, despite mild clinical signs with no associated elevation in mortalities reported at the farm level. Diffuse mononuclear inflammation and myodegeneration, consistent with HSMI, was the predominant histologic observation in affected heart and skeletal muscle. Infective agent monitoring identified three agents at high prevalence in salmon heart tissue, including Piscine orthoreovirus (PRV), and parasites Paranucleospora theridion and Kudoa thyrsites. However, PRV alone was statistically correlated with the occurrence and severity of histopathological lesions in the heart. Immunohistochemical staining further localized PRV throughout HSMI development, with the virus found mainly within red blood cells in early cases, moving into the cardiomyocytes within or, more often, on the periphery of the inflammatory reaction during the peak disease, and reducing to low or undetectable levels later in the production cycle. This study represents the first longitudinal assessment of HSMI in a salmon farm in British Columbia, providing new insights on the pathogenesis of the disease.

Differences in gene expression in Atlantic salmon parr and smolt after challenge with Piscine orthoreovirus (PRV).

Heart and skeletal muscle inflammation (HSMI) are a disease of farmed Atlantic salmon (Salmo salar) associated with Piscine orthoreovirus (PRV). The disease appears mainly during the marine production phase. This study examined if smoltification and transfer to seawater could compromise immune responses to PRV. Parr and smolts of the same origin were challenged by cohabitation with intraperitoneally injected salmon. Peak levels of PRV in spleen of cohabitants were reached after 8 weeks, but at a lower level in parr compared to smolts. Thereafter the virus levels declined, but remained significantly lower in parr than in smolts. Both groups developed typical HSMI histopathological heart lesions, which were most prominent after 10 weeks. Microarray and qPCR analyses revealed slightly lower expression of immune genes in spleen and head kidney of smolts before challenge. Infected parr showed earlier induction of genes involved in innate antiviral immunity, as well as for genes related to B and T cell responses. Gene expression profiles also indicated stimulation of heme and iron metabolism and erythropoiesis in smolts, which may indicate replacement of PRV-infected erythrocytes.

Infection with purified Piscine orthoreovirus demonstrates a causal relationship with heart and skeletal muscle inflammation in Atlantic salmon

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) in red and melanised foci in white muscle of Atlantic salmon (Salmo salar).

Melanised focal changes (black spots) are common findings in the white skeletal muscle of seawater-farmed Atlantic salmon (Salmo salar). Fillets with melanised focal changes are considered as lower quality and cause large economic losses. It has been suggested that red focal changes (red spots) precede the melanised focal changes. In the present work, we examined different populations of captive and wild salmon for the occurrence of both types of changes, which were investigated for the presence of different viruses by immunohistochemistry and RT-qPCR. The occurrence of red or melanised foci varied significantly between the populations, from none in wild fish control group, low prevalence of small foci in fish kept in in-house tanks, to high prevalence of large foci in farm-raised salmon. Large amounts of Piscine orthoreovirus (PRV) antigen were detected in all foci. No other viruses were detected. Red focal changes contained significantly higher levels of PRV RNA than apparently non-affected areas in white muscle of the same individuals. Some changes displayed a transient form between a red and melanised pathotype, indicating a progression from an acute to a chronic manifestation. We conclude that PRV is associated with the focal pathological changes in the white muscle of farmed Atlantic salmon and is a premise for the development of focal melanised changes.

The non-structural protein μNS of piscine orthoreovirus (PRV) forms viral factory-like structures

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.

Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells

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.

Infection experiments with novel Piscine orthoreovirus from rainbow trout (Oncorhynchus mykiss) in salmonids

A new disease in farmed rainbow trout (Onchorhyncus mykiss) was described in Norway in 2013. The disease mainly affected the heart and resembled heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar L.). HSMI is associated with Piscine orthoreovirus (PRV), and a search for a similar virus in the diseased rainbow trout led to detection of a sequence with 85% similarity to PRV. This finding called for a targeted effort to assess the risk the new PRV-variant pose on farmed rainbow trout and Atlantic salmon by studying infection and disease pathogenesis, aiming to provide more diagnostic knowledge. Based on the genetic relationship to PRV, the novel virus is referred to as PRV-Oncorhynchus mykiss (PRV-Om) in contrast to PRV-Salmo salar (PRV-Ss). In experimental trials, intraperitoneally injected PRV-Om was shown to replicate in blood in both salmonid species, but more effectively in rainbow trout. In rainbow trout, the virus levels peaked in blood and heart of cohabitants 6 weeks post challenge, along with increased expression of antiviral genes (Mx and viperin) in the spleen, with 80–100% of the cohabitants infected. Heart inflammation was diagnosed in all cohabitants examined 8 weeks post challenge. In contrast, less than 50% of the Atlantic salmon cohabitants were infected between 8 and 16 weeks post challenge and the antiviral response in these fish was very low. From 12 weeks post challenge and onwards, mild focal myocarditis was demonstrated in a few virus-positive salmon. In conclusion, PRV-Om infects both salmonid species, but faster transmission, more notable antiviral response and more prominent heart pathology were observed in rainbow trout.

Piscine orthoreovirus (PRV) ơ3 protein binds dsRNA.

Piscine orthoreovirus (PRV) has a double-stranded, segmented RNA genome and belongs to the family Reoviridae. PRV is associated with heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar L.) and cause intraerythrocytic inclusions. The virus is widespread in both wild and farmed salmonid fish in Europe, North- and South America. In mammalian orthoreovirus (MRV), the outer capsid protein ơ3 has dsRNA binding properties, which serve to inhibit the early innate immune response of the host. Important structural motifs and key amino acid residues are conserved between MRV ơ3 and the homologous PRV protein, and we hypothesized that PRV ơ3 binds dsRNA. Gene regions and amino acid residues predicted to be important for dsRNA binding were determined through bioinformatic analysis and investigated functionally following site-directed mutagenesis and the generation of truncated ơ3 variants. Our results provide evidence that the PRV protein ơ3 binds dsRNA in a sequence independent manner, thus sharing this function with MRV ơ3. Although no specific domain solely responsible for dsRNA binding was determined, the results point to residues within a predominantly basic region to be important for this functional property. We conclude that multiple sites are involved in the dsRNA binding activity of PRV ơ3.