The same strain of Piscine orthoreovirus (PRV-1) is involved in the development of different, but related, diseases in Atlantic and Pacific Salmon in British Columbia

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.

Heart and skeletal muscle inflammation (HSMI) is a significant and often fatal disease of cultured Atlantic salmon in Norway. The consistent presence of Piscine orthoreovirus (PRV) in HSMI diseased fish along with the correlation of viral load and antigen with development of lesions has supported the supposition that PRV is the etiologic agent of this condition; yet the absence of an in vitro culture system to demonstrate disease causation and the widespread prevalence of this virus in the absence of disease continues to obfuscate the etiological role of PRV with regard to HSMI. In this study, we explore the infectivity and disease causing potential of PRV from western North America—a region now considered endemic for PRV but without manifestation of HSMI—in challenge experiments modeled upon previous reports associating PRV with HSMI. We identified that western North American PRV is highly infective by intraperitoneal injection in Atlantic salmon as well as through cohabitation of both Atlantic and Sockeye salmon. High prevalence of viral RNA in peripheral blood of infected fish persisted for as long as 59 weeks post-challenge. Nevertheless, no microscopic lesions, disease, or mortality could be attributed to the presence of PRV, and only a minor transcriptional induction of the antiviral Mx gene occurred in blood and kidney samples during log-linear replication of viral RNA. Comparative analysis of the S1 segment of PRV identified high similarity between this North American sequence and previous sequences associated with HSMI, suggesting that factors such as viral co-infection, alternate PRV strains, host condition, or specific environmental circumstances may be required to cause this disease.

Piscine orthoreovirus (PRV) mediated diseases have emerged throughout salmonid aquaculture. Three PRV subtypes are currently reported as causative agents of or in association with diseases in different salmonid species. PRV-1 causes heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) and is associated with jaundice syndrome in farmed chinook salmon (Oncorhynchus tshawytscha). PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon in Japan. PRV-3 has recently been associated with a disease in rainbow trout (Oncorhynchus mykiss) characterized by anaemia, heart and red muscle pathology; to jaundice syndrome in coho salmon (Oncorhynchus kisutch). In this study, we conducted a 10-week long experimental infection trial in rainbow trout with purified PRV-3 particles to assess the causal relationship between the virus and development of heart inflammation. The monitoring the PRV-3 load in heart and spleen by RT-qPCR shows a progressive increase of viral RNA to a peak, followed by clearance without a measurable change in haematocrit. The development of characteristic cardiac histopathological findings occurred in the late phase of the trial and was associated with increased expression of CD8+, indicating cytotoxic T cell proliferation. The findings indicate that, under these experimental conditions, PRV-3 infection in rainbow trout act similarly to PRV-1 infection in Atlantic salmon with regards to immunological responses and development of heart pathology, but not in the ability to establish a persistent infection.

Heart- and skeletal muscle inflammation (HSMI) caused by infection with Piscine orthoreovirus (PRV) is one of the most common viral diseases in farmed Atlantic salmon (Salmo salar) in Norway, and disease outbreaks have been reported in most countries with large-scale Atlantic salmon aquaculture. Currently there is no vaccine available for protection against HSMI, partly due to the lack of a cell line for efficient virus propagation. Erythrocytes are the primary target cells for PRV invivo and a potential source for isolation of PRV particles. In this study, PRV was purified from infected erythrocytes, inactivated and used in a vaccination trial against HSMI. A single immunization with adjuvanted, inactivated PRV induced protection against HSMI in Atlantic salmon infected by virus injection 6weeks later, while a moderate protection was obtained in fish infected through natural transmission, i.e. cohabitation. The PRV vaccine significantly reduced PRV loads and histopathological lesions typical for HSMI compared to the unvaccinated control group. This is the first demonstration of protective vaccination against PRV, and promising for future control of HSMI in Atlantic salmon aquaculture.

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.

Viral diseases represent a serious problem in Atlantic salmon (Salmo salar L.) farming in Norway. Pancreas disease (PD) caused by salmonid alphavirus (SAV) and heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus (PRV) are among the most frequently diagnosed viral diseases in recent years. The possible spread of viruses from salmon farms to wild fish is a major public concern. Sea trout S. trutta collected from the major farming areas along the Norwegian coast are likely to have been exposed to SAV and PRV from farms with disease outbreaks. We examined 843 sea trout from 4 counties in Norway for SAV and PRV infections. We did not detect SAV in any of the tested fish, although significant numbers of the trout were caught in areas with frequent PD outbreaks. Low levels of PRV were detected in 1.3% of the sea trout. PRV-infected sea trout were caught in both salmon farming and non-farming areas, so the occurrence of infections was not associated with farming intensity or HSMI cases. Our results suggest that SAV and PRV infections are uncommon in wild sea trout. Hence, we found no evidence that sea trout are at risk from SAV or PRV released from salmon farms.

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) infection is common in aquaculture of salmonids. The three known PRV genotypes (PRV-1-3) have host species specificity and cause different diseases, but all infect and replicate in red blood cells (RBCs) in early infection phase. PRV-1 is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar), PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon (Oncorhynchus kisutch), while PRV-3 induces HSMI-like disease in farmed rainbow trout (Oncorhynchus mykiss). PRV-3 can also infect A. salmon without causing clinical disease and has been shown to cross-protect against PRV-1 infection and HSMI, while PRV-2 or inactivated adjuvanted PRV-1 vaccine only partially reduced HSMI pathologic changes. In the present work, we studied the transcriptional responses in blood cells of A. salmon two- and five-weeks post infection with PRV-1, PRV-2, PRV-3, or post injection with inactivated PRV-1 vaccine. PRV-1 and PRV-3 replicated well in A. salmon blood cells, and both induced the typical innate antiviral responses triggered by dsRNA viruses. Two weeks post infection, PRV-3 triggered stronger antiviral responses than PRV-1, despite their similar viral RNA replication levels, but after five weeks the induced responses were close to equal. PRV-2 and the InPRV-1 vaccine did not trigger the same typical antiviral responses as the replicating PRV-1 and PRV-3 genotypes, but induced genes involved in membrane trafficking and signaling pathways that may regulate physiological functions. These findings propose that the protection mediated by PRV-3 against a secondary infection by PRV-1 occur due to a potent and early activation of the same type of innate immune responses. The difference in the timing of antiviral responses may give PRV-1 an evolutionary edge, facilitating its dissemination to A. salmon heart, a critical step for HSMI development.

During the last decade, Piscine orthoreovirus was identified as the main causative agent of heart and skeletal muscle inflammation (HSMI) in Atlantic Salmon, Norway. A recent study showed that PRV-1 sequences from salmonid collected in North Atlantic Pacific Coast (NAPC) grouped separately from the Norwegian sequences found in Atlantic Salmon diagnosed with HSMI. Currently, the routine assay used to screen for PRV-1 in NAPC water and worldwide cannot differentiate between the two groups of PRV-1. Therefore, this study aimed at developing a real-time polymerase chain reaction (RT-qPCR) assay to target the PRV-1 genome segments specific for variants associated with HSMI. The assay was optimized and tested against 71 tissue samples collected from different regions including Norway, Chile and both coast of Canada and different hosts farmed Atlantic Salmon, wild Coho Salmon and escaped Atlantic Salmon collected in British Columbia, West Coast of Canada. This assay has the potential to be used for screening salmonids and non-salmonids that may carry PRV-1 potentially causing HSMI.

The first cases of heart and skeletal muscle inflammation (HSMI), in Atlantic salmon Salmo salar were registered in 1999 in the Hitra/Frøya area of Norway. The disease has since spread south to Rogaland, i.e. the southernmost county with salmon farming in Norway. The disease outbreaks usually start 5 to 9 mo after release into seawater but may occur as early as 2 wk after sea release. The present study focuses on possible pathogens associated with HSMI. It was not possible to find any parasites or bacteria that could explain HSMI, and none of the well-known viruses (infectious salmon anaemia virus, Norwegian salmonid alphavirus, infectious pancreatic necrosis virus, Atlantic salmonid paramyxovirus) were consistently present. Use of transmission electron microscopy showed the presence of epitheliocystis agent in 3 of 4 farms included in this study, and several virus-like particles. Type I and Type II virus particles, previously described for salmon suffering from haemorrhagic smolt syndrome (HSS), and erythrocytic inclusion body syndrome (EIBS) virus were consistently present in salmon suffering from HSMI in all 4 farms included in this study. The 2 HSS viruses (Type I and Type II) were also cultured in Atlantic salmon kidney (ASK) cells from salmon suffering from HSMI. However, a causal relationship between the observed virus particles and HSMI remains to be demonstrated.

This formal comment is in response to Siah et al. [1], Piscine Reovirus: Genomic and Molecular Phylogenetic Analysis from Farmed and Wild Salmonids Collected on the Canada/US Pacific Coast, with a subsequent correction (Siah et al. 2016 [2]). Although a correction for this paper was published on Oct. 12, 2016, (Siah et al. 2016 [2]), there continues to be inadequate supporting evidence for the primary conclusion that PRV genetic sequences are temporally and spatially homogeneous in salmonid species across the northeastern Pacific region. The evidence in this paper warrants thorough consideration. Piscine orthoreovirus (PRV) causes acute infection of the red blood cells in salmon (Finstad et al. [3]; Haatveit et al. 2017 [4]). It is the causative agent of the emerging farm salmon disease Heart and Skeletal Muscle Inflammation (HSMI) (Wessel et al. 2017 [5]) with clinical symptoms which can include lethargy, anemia, anorexia and mortality (Kongtorp et al. 2004 [6]). Palacios et al. [7] expressed concerns about the transfer of PRV from farmed to wild fish due to its contagious nature. PRV is now considered ubiquitous in farmed Atlantic salmon (Haatveit et al. 2017 [4]) and has an estimated 80% prevalence rate among BC farmed salmon (Kibenge et al. 2013 [8]). HSMI has recently been diagnosed in British Columbia (BC), Canada (Di Cicco et al. 2017 [9]). Hence, release of PRV from salmon farms into Pacific salmon habitat is a significant management concern in the eastern Pacific Ocean. In the correction, Siah et al. [2] acknowledge that the conclusion that PRV has not been recently introduced to BC was overstated. However their supporting evidence that … salmonids from western North America Pacific waters carried PRV RNA sequences for at least 13 years with little genetic differentiation among sequence types in selected samples spanning 2001 to 2014 remains insufficient. Their conclusion appears to be highly dependent on six unique sequences of PRV segment S1, detected by Siah et al. [1]: {type:entrez-nucleotide,attrs:{text:KR478642,term_id:931323276,term_text:KR478642}}KR478642: collected in May 2001 {type:entrez-nucleotide,attrs:{text:KR478643,term_id:931323280,term_text:KR478643}}KR478643: collected in Aug. 2001 {type:entrez-nucleotide,attrs:{text:KR478644,term_id:931323283,term_text:KR478644}}KR478644: collected in Aug. 2001 {type:entrez-nucleotide,attrs:{text:KR347078,term_id:931316047,term_text:KR347078}}KR347078: collected in Aug. 2001 {type:entrez-nucleotide,attrs:{text:KR347079,term_id:931316050,term_text:KR347079}}KR347079: collected in Aug. 2001 {type:entrez-nucleotide,attrs:{text:KR347080,term_id:931316053,term_text:KR347080}}KR347080: collected in Mar. 2005 These six Siah et al. [1] sequences collected in 2001 and 2005 (submitted to GenBank April—May 2015) predate those collected by Kibenge et al. [10,11] by seven years and are nearly identical to the isolates Siah et al. [1] collected in 2013 and 2014. Thus, these six PRV isolates appear to be highly resistant to mutation over a 13-year interval 2001–2014, which is atypical for RNA viruses, generally known to exhibit a high mutation rate (Chao et al. [12]). Drake and Holland [13] estimate the genomic mutation rate (U) to be between 1 and 0.1 for most RNA viruses, where U is G x u, G is the genome size in nucleotides, and u is the per-nucleotide mutation rate. Weight of evidence for longer-term PRV presence in BC Siah et al. [1] cite detection of PRV in a wild Steelhead trout (O. mykiss) collected in 1977 in support of longer-term PRV presence in BC. This result is cited from Marty et al. [14], who provided no S1 segment sequence information to verify the PRV strain identity as per the sequence groupings reported by both Kibenge et al. [8] and Garseth et al. [15]. The recent discovery of the widespread occurrence of PRV-2 across the North Pacific (Takano et al. 2016 [16]) raises the question: Was the 1977 steelhead infected with PRV-2 or PRV? In absence of S1 sequencing this uncertainty cannot be resolved. Furthermore, this result could not be replicated by a second laboratory (Purcell and Thompson [17]) and therefore warrants qualification as a non-repeatable result and a suspect positive lacking sufficient robustness to provide evidence critical to the temporal presence of PRV in BC.

Heart and skeletal muscle inflammation (HSMI) is associated with Piscine orthoreovirus (PRV) infection and is an important disease in Atlantic salmon (Salmo salar) aquaculture. Since PRV infects erythrocytes and farmed salmon frequently experience environmental hypoxia, the current study examined mutual effects of PRV infection and hypoxia on pathogenesis and fish performance. Furthermore, effects of HSMI on hypoxia tolerance, cardiorespiratory performance and blood oxygen transport were studied. A cohabitation trial including PRV-infected post-smolts exposed to periodic hypoxic stress (4 h of 40% O2; PRV-H) at 4, 7 and 10 weeks post-infection (WPI) and infected fish reared under normoxic conditions (PRV) was conducted. Periodic hypoxic stress did not influence infection levels or histopathological changes in the heart. Individual incipient lethal oxygen saturation (ILOS) was examined using a standardized hypoxia challenge test (HCT). At 7 WPI, i.e. peak level of infection, both PRV and PRV-H groups exhibited reduced hypoxia tolerance compared to non-infected fish. Three weeks later (10 WPI), during peak levels of pathological changes, reduced hypoxia tolerance was still observed for the PRV group while PRV-H performed equal to non-infected fish, implying a positive effect of the repeated exposure to hypoxic stress. This was in line with maximum heart rate (fHmax) measurements, showing equal performance of PRV-H and non-infected groups, but lower fHmax above 19°C as well as lower temperature optimum (Topt) for aerobic scope for PRV, suggesting reduced cardiac performance and thermal tolerance. In contrast, the PRV-H group had reduced hemoglobin-oxygen affinity compared to non-infected fish. In conclusion, Atlantic salmon suffering from HSMI have reduced hypoxia tolerance and cardiac performance, which can be improved by preconditioning fish to transient hypoxic stress episodes.

The disease Heart and Skeletal Muscle Inflammation (HSMI) is causing substantial economic losses to the Norwegian salmon farming industry where the causative agent, piscine orthoreovirus (PRV), is reportedly spreading from farmed to wild Atlantic salmon (Salmo salar) with as yet undetermined impacts. To assess if PRV infection is epidemiologically linked between wild and farmed salmon in the eastern Pacific, wild Pacific salmon (Oncorhynchus sp.) from regions designated as high or low exposure to salmon farms and farmed Atlantic salmon reared in British Columbia (BC) were tested for PRV. The proportion of PRV infection in wild fish was related to exposure to salmon farms (p = 0.0097). PRV was detected in: 95% of farmed Atlantic salmon, 37–45% of wild salmon from regions highly exposed to salmon farms and 5% of wild salmon from the regions furthest from salmon farms. The proportion of PRV infection was also significantly lower (p = 0.0008) where wild salmon had been challenged by an arduous return migration into high-elevation spawning habitat. Inter-annual PRV infection declined in both wild and farmed salmon from 2012–2013 (p ≤ 0.002). These results suggest that PRV transfer is occurring from farmed Atlantic salmon to wild Pacific salmon, that infection in farmed salmon may be influencing infection rates in wild salmon, and that this may pose a risk of reduced fitness in wild salmon impacting their survival and reproduction.

Chapter 9 - Heart and skeletal muscle inflammation

Piscine orthoreovirus (PRV) causes heart- and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). Erythrocytes are the main target cells for PRV. HSMI causes significant economic losses to the salmon aquaculture industry, and there is currently no vaccine available. PRV replicates and assembles within cytoplasmic structures called viral factories, mainly organized by the non-structural viral protein µNS. In two experimental vaccination trials in Atlantic salmon, using DNA vaccines expressing different combinations of PRV proteins, we found that expression of the non-structural proteins µNS combined with the cell attachment protein σ1 was associated with an increasing trend in lymphocyte marker gene expression in spleen, and induced moderate protective effect against HSMI.