Piscine Myocarditis Virus Research Articles

Monitoring infection with Piscine myocarditis virus and development of cardiomyopathy syndrome in farmed Atlantic salmon (Salmo salar L.) in Norway.

An epidemiological study was carried out in Norway in 2015–2018, investigating the development of infection with Piscine myocarditis virus (PMCV) and development of cardiomyopathy syndrome (CMS) in farmed Atlantic salmon. Cohorts from 12 sites were followed and sampled every month or every other month from sea transfer to slaughter. PMCV was detected at all sites and in all sampled cages, and fish in six sites developed clinical CMS. The initial infection happened between 1 and 7 months post‐sea transfer, and the median time from infection with PMCV until outbreak of CMS was 6.5 months. Generally, fish from sites with CMS had higher viral titre and a higher prevalence of PMCV, compared to sites that did not develop clinical CMS. The virus persisted until the point of slaughter at most (11 out of 12) of the sites. The detection of PMCV in all sites suggests that PMCV is more widespread than previously known. Screening for PMCV as a tool to monitor impending outbreaks of CMS must be supported by observations of the health status of the fish and risk factors for development of disease.

Antiviral defense in salmonids – Mission made possible?

Viral diseases represent one of the major threats for salmonid aquaculture. Survival from viral infections are highly dependent on host innate antiviral immune defense, where interferons are of crucial importance. Neutralizing antibodies and T cell effector mechanisms mediate long-term antiviral protection. Despite an immune cell repertoire comparable to higher vertebrates, farmed fish often fail to mount optimal antiviral protection. In the quest to multiply and spread, viruses utilize a variety of strategies to evade or escape the host immune system. Understanding the specific interplay between viruses and host immunity at depth is crucial for developing successful vaccination and treatment strategies in mammals. However, this knowledge base is still limited for pathogenic fish viruses. Here, we have focused on five RNA viruses with major impact on salmonid aquaculture: Salmonid alphavirus, Infectious salmon anemia virus, Infectious pancreatic necrosis virus, Piscine orthoreovirus and Piscine myocarditis virus. This review explore the protective immune responses that salmonids mount to these viruses and the existing knowledge on how the viruses counteract and/or bypass the immune response, including their IFN antagonizing effects and their mechanisms to establish persisting infections.

Molecular testing of adult Pacific salmon and trout (Oncorhynchus spp.) for several RNA viruses demonstrates widespread distribution of piscine orthoreovirus in Alaska and Washington.

This research was initiated in conjunction with a systematic, multiagency surveillance effort in the United States (U.S.) in response to reported findings of infectious salmon anaemia virus (ISAV) RNA in British Columbia, Canada. In the systematic surveillance study reported in a companion paper, tissues from various salmonids taken from Washington and Alaska were surveyed for ISAV RNA using the U.S.-approved diagnostic method, and samples were released for use in this present study only after testing negative. Here, we tested a subset of these samples for ISAV RNA with three additional published molecular assays, as well as for RNA from salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV). All samples (n=2,252; 121 stock cohorts) tested negative for RNA from ISAV, PMCV, and SAV. In contrast, there were 25 stock cohorts from Washington and Alaska that had one or more individuals test positive for PRV RNA; prevalence within stocks varied and ranged from 2% to 73%. The overall prevalence of PRV RNA-positive individuals across the study was 3.4% (77 of 2,252 fish tested). Findings of PRV RNA were most common in coho (Oncorhynchus kisutch Walbaum) and Chinook (O.tshawytscha Walbaum) salmon.

Cardiomyopathy syndrome in Atlantic salmon Salmo salar L.: A review of the current state of knowledge.

Cardiomyopathy syndrome (CMS) is a severe cardiac disease affecting Atlantic salmon Salmo salar L. The disease was first recognized in farmed Atlantic salmon in Norway in 1985 and subsequently in farmed salmon in the Faroe Islands, Scotland and Ireland. CMS has also been described in wild Atlantic salmon in Norway. The demonstration of CMS as a transmissible disease in 2009, and the subsequent detection and initial characterization of piscine myocarditis virus (PMCV) in 2010 and 2011 were significant discoveries that gave new impetus to the CMS research. In Norway, CMS usually causes mortality in large salmon in ongrowing and broodfish farms, resulting in reduced fish welfare, significant management-related challenges and substantial economic losses. The disease thus has a significant impact on the Atlantic salmon farming industry. There is a need to gain further basic knowledge about the virus, the disease and its epidemiology, but also applied knowledge from the industry to enable the generation and implementation of effective prevention and control measures. This review summarizes the currently available, scientific information on CMS and PMCV with special focus on epidemiology and factors influencing the development of CMS.

Detection and molecular characterization of a novel piscine-myocarditis-like virus from baitfish in the USA

During a survey of apparently healthy baitfish from the state of Minnesota, a novel piscine-myocarditis-like virus (PMCLV) was detected in golden shiners (Notemigonus crysoleucas). The nearly complete genome sequence is 5819nt long, including a partial 5' untranslated region (UTR) of 100nt. The sequence is divided into three ORFs: the complete ORF1 and ORF2, encoding proteins of 818 and 831 amino acids, respectively, and a partial ORF3 encoding 248 amino acids of the corresponding protein. This novel virus sequence was most closely related to piscine myocarditis virus (PMCV), with a 49.0% and 58.2% amino acid identity in the ORF1 (YP005481249)- and ORF2 (YP004581250)-encoded proteins, respectively. Six of 56 retail outlets (e.g., bait shops) were positive during the 2014-2015 survey, indicating a 10.7% prevalence of the novel virus in this population of golden shiners. Currently, there is no disease that is known to be associated with this virus.

Health monitoring in wild anadromous salmonids -Evidence of absence or absence of evidence?

Health monitoring in wild anadromous salmonids -Evidence of absence or absence of evidence?

Effects of functional feeds on the lipid composition, transcriptomic responses and pathology in heart of Atlantic salmon (Salmo salar L.) before and after experimental challenge with Piscine Myocarditis Virus (PMCV).

BackgroundCardiomyopathy syndrome (CMS) is a severe cardiac disease of Atlantic salmon (Salmo salar) recently associated with a double-stranded RNA virus, Piscine Myocarditis Virus (PMCV). The disease has been diagnosed in 75-85 farms in Norway each year over the last decade resulting in annual economic losses estimated at up to €9 million. Recently, we demonstrated that functional feeds led to a milder inflammatory response and reduced severity of heart lesions in salmon experimentally infected with Atlantic salmon reovirus, the causal agent of heart and skeletal muscle inflammation (HSMI). In the present study we employed a similar strategy to investigate the effects of functional feeds, with reduced lipid content and increased eicosapentaenoic acid levels, in controlling CMS in salmon after experimental infection with PMCV.ResultsHepatic steatosis associated with CMS was significantly reduced over the time course of the infection in fish fed the functional feeds. Significant differences in immune and inflammatory responses and pathology in heart tissue were found in fish fed the different dietary treatments over the course of the infection. Specifically, fish fed the functional feeds showed a milder and delayed inflammatory response and, consequently, less severity of heart lesions at earlier and later stages after infection with PMCV. Decreasing levels of phosphatidylinositol in cell membranes combined with the increased expression of genes related with T-cell signalling pathways revealed new interactions between dietary lipid composition and the immune response in fish during viral infection. Dietary histidine supplementation did not significantly affect immune responses or levels of heart lesions.ConclusionsCombined with the previous findings on HSMI, the results of the present study highlight the potential role of clinical nutrition in controlling inflammatory diseases in Atlantic salmon. In particular, dietary lipid content and fatty acid composition may have important immune-modulatory effects in Atlantic salmon that could be potentially beneficial in fish balancing the immune and tissue responses to viral infections.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-462) contains supplementary material, which is available to authorized users.

Clinical cardiomyopathy syndrome in Atlantic salmon, Salmo salar L.

Cardiomyopathy syndrome (CMS) was first described in farmed Atlantic salmon, Salmo salar, in Norway (Ferguson, Poppe & Speare 1990) and then subsequently in the Faroe Islands (Poppe & Seierstad 2003) and Scotland (Rodger & Turnbull 2000). The infectious nature of the disease has been demonstrated by Fritsvold et al. (2009), and a totivirus, the piscine myocarditis virus (PMCV), has been identified as the infectious agent involved (Lovoll et al. 2010; Haugland et al. 2011). The disease primarily affects farmed Atlantic salmon in their second year at sea where it has a significant economic impact due to mortality associated with large-size fish (Brun et al. 2003). CMS has been one of the major diseases in Norwegian aquaculture for the last decade, and viral RNA can be detected in farmed fish for months without any signs of clinical disease (Wiik-Nielsen et al. 2012). Although the disease has only been described in farmed Atlantic salmon, histopathology consistent with CMS was described in wild Atlantic salmon (Poppe & Seierstad 2003), and in a recent study, PCMV was detected and sequenced from wild Atlantic salmon in Norway (Garseth, Biering & Tengs 2012). Little is known about the prevalence of the virus in other marine species, although recent reports suggest that it is not common, having been detected in only one species, the Atlantic argentine, Argentina silus (Ascanius), and sequencing studies suggest that the argentine isolate may be different to those found in Atlantic salmon (B€ockerman et al. 2011; Tengs & B€ockerman 2012). This report describes the occurrence and findings of a case of clinical CMS in Atlantic salmon in Ireland. One marine farm site in the north-west of Ireland reported slightly elevated mortalities in some marine cages in June 2012. The affected fish were in a population of approximately 480 000 reared in 16 cages and were on average 3.5 kg in weight. Although mortalities were less than 10 fish per cage per day, the appearance of the moribund fish was dramatic. Superficial signs included congestion and oedema in the skin (Fig. 1a) and occasional exophthalmia. Internally, blood-tinged ascites, purple to grey livers with diphtheritic fibrinous membranes and petechiae in the caecal fat were observed (Fig. 1b). Swollen, blood-engorged atria of the heart, haemopericardium and/or pale patches of tissue were also commonly seen on the cardiac ventricles (Fig. 1c). Six moribund fish were sampled for histopathology (gill, kidney, spleen, pyloric caeca, pancreas, liver, heart, skin and muscle in 10% buffered formalin) and bacteriology [kidney swabs taken and plated onto tryptone soya agar (TSA), thiosulfate-citrate-bile salts-sucrose agar (TCBS) and TSA plus 1.5% NaCl and incubated at 20–22 °C, plus examination of Gramstained kidney smears]. The histopathology revealed diffuse severe cardiomyopathy of the spongy layers of the heart ventricles and multifocal liver necrosis in all fish sampled (Fig. 1d). There Correspondence H Rodger, Vet-Aqua International, Oranmore Business Park, Oranmore, Co. Galway, Ireland (e-mail: hamishrodger@eircom.net)

Piscine myocarditis virus (PMCV) in wild Atlantic salmon Salmo salar

Cardiomyopathy syndrome (CMS) is a severe cardiac disease of sea-farmed Atlantic salmon Salmo salar L., but CMS-like lesions have also been found in wild Atlantic salmon. In 2010 a double-stranded RNA virus of the Totiviridae family, provisionally named piscine myocarditis virus (PMCV), was described as the causative agent of CMS. In the present paper we report the first detection of PMCV in wild Atlantic salmon. The study is based on screening of 797 wild Atlantic salmon by real-time RT-PCR. The samples were collected from 35 different rivers along the coast of Norway, and all individuals included in the study were classified as wild, based on visual appearance and scale reading. Two samples tested positive during PCR analysis, and the results were confirmed by sequencing.

Genetic variation in Norwegian piscine myocarditis virus in Atlantic salmon, Salmo salar L.

Cardiomyopathy syndrome (CMS) in Atlantic salmon, Salmo salar L., is a severe cardiac disease characterized by a necrotizing myocarditis involving the atrium and the spongious part of the ventricle. The disease is caused by piscine myocarditis virus (PMCV), a double-stranded RNA virus likely belonging to the family Totiviridae. The objective of this study was to evaluate the genetic variation in Norwegian PMCV isolates focusing on the putative structural proteins encoded by open reading frames (ORFs) 1 and 3. The virus isolates were sampled from a total of 36 farms along the Norwegian coastline. This study represents the first investigation of PMCV genome variation and shows that Norwegian isolates are highly similar, with the most divergent isolates sharing 98.6% nucleotide identity. Interestingly, amino acid sequence diversity within ORF3 is approximately threefold higher than for ORF1. While phylogenetic analysis based on concatenated nucleotide data covering ORF1 and ORF3 revealed four main clusters, the maximum sequence variation of 1.4% at the nucleotide level suggests that all Norwegian isolates belong to a single genogroup. Substantial sequence variation within farms was also observed, which may complicate future molecular epidemiological investigations. The genetic homogeneity among the Norwegian isolates might facilitate development of both diagnostic tools and an efficient vaccine against CMS in the future.

Characterization of myocardial lesions associated with cardiomyopathy syndrome in Atlantic salmon, Salmo salar L., using laser capture microdissection

Cardiomyopathy syndrome (CMS) in Atlantic salmon, Salmo salar L., is characterized by focal infiltration in the spongy myocardium and endocardium of the heart. The origin of the mononuclear infiltrate is unknown. Using experimentally infected fish, we investigated localization of the causative agent, piscine myocarditis virus (PMCV), within the heart and characterized the cell population associated with myocardial lesions. Cellular and transcriptional characteristics in the lesions were compared with adjacent non-infiltrated tissues using laser capture microdissection, RT-qPCR and immunohistochemistry. Our results reveal that PMCV is almost exclusively present in myocardial lesions. The inflammatory infiltrate comprises a variety of leucocyte populations, including T cells, B cells, MHC class II(+) and CD83(+) cells, most likely of the macrophage line. Correlation analyses demonstrated co-ordinated leucocyte activity at the site of the virus infection. Cellular proliferation and/or DNA repair was demonstrated within the myocardial lesions. Different cell populations, mainly myocytes, stained positive for proliferating cell nuclear antigen (PCNA). Densities of endothelial cells and fibroblasts were not significantly increased. The simultaneous presence of PMCV and various inflammatory cells in all myocardial lesions analysed may indicate that both viral lytic and immunopathological effects may contribute to the pathogenesis of CMS.

Comparison of Atlantic salmon individuals with different outcomes of cardiomyopathy syndrome (CMS)

BackgroundCardiomyopathy syndrome (CMS) is a severe disease of Atlantic salmon (Salmo salar L.) associated with significant economic losses in the aquaculture industry. CMS is diagnosed with a severe inflammation and degradation of myocardial tissue caused by a double-stranded RNA virus named piscine myocarditis virus (PMCV), with structural similarities to the Totiviridae family. In the present study we characterized individual host responses and genomic determinants of different disease outcomes.ResultsFrom time course studies of experimentally infected Atlantic salmon post-smolts, fish exhibited different outcomes of infection and disease. High responder (HR) fish were characterized with sustained and increased viral load and pathology in heart tissue. Low responder (LR) fish showed declining viral load from 6–10 weeks post infection (wpi) and absence of pathology. Global gene expression (SIQ2.0 oligonucleotide microarray) in HR and LR hearts during infection was compared, in order to characterize differences in the host response and to identify genes with expression patterns that could explain or predict the different outcomes of disease. Virus-responsive genes involved in early antiviral and innate immune responses were upregulated equally in LR and HR at the first stage (2–4 wpi), reflecting the initial increase in virus replication. Repression of heart muscle development was identified by gene ontology enrichment analyses, indicating the early onset of pathology. By six weeks both responder groups had comparable viral load, while increased pathology was observed in HR fish. This was reflected by induced expression of genes implicated in apoptosis and cell death mechanisms, presumably related to lymphocyte regulation and survival. In contrast, LR fish showed earlier activation of NK cell-mediated cytotoxicity and NOD-like receptor signaling pathways. At the late stage of infection, increased pathology and viral load in HR was accompanied by a broad activation of genes involved in adaptive immunity and particularly T cell responses, probably reflecting the increased infiltration and homing of virus-specific T cells to the infected heart. This was in sharp contrast to LR fish, where recovery and reduced viral load was associated with a significantly reduced transcription of adaptive immunity genes and activation of genes involved in energy metabolism.ConclusionsIn contrast to LR, a stronger and sustained expression of genes involved in adaptive immune responses in heart tissue of HR at the late stage of disease probably reflected the increased lymphocyte infiltration and pathological outcome. In addition to controlled adaptive immunity and activation of genes involved in cardiac energy metabolism in LR at the late stage, recovery of this group could also be related to an earlier activation of NOD-like receptor signaling and NK cell-mediated cytotoxicity pathways.

Prevalence of viral RNA from piscine reovirus and piscine myocarditis virus in Atlantic salmon, Salmo salar L., broodfish and progeny

Prevalence of viral RNA from piscine reovirus and piscine myocarditis virus in Atlantic salmon, <i>Salmo salar</i> L., broodfish and progeny

Prevalence of piscine myocarditis virus (PMCV) in marine fish species

Cardiomyopathy syndrome (CMS) is an inflammatory disease of the heart primarily affecting farmed Atlantic salmon, Salmo salar L. (Ferguson, Poppe & Speare 1990). The disease mainly appears in fish 12–15 months after transfer to sea water, and pathological signs include inflammation of the endocardium and spongiosum of the atrium and ventricle. Highest mortality rates are seen in fish weighing 2–5 kg, and the cause of death is generally rupturing of the atrium or sinus venosus. The disease has been reported from locations all along the Norwegian coastline since the 1980s (Amin & Trasti 1988) and has also been observed in Scotland (Rodger & Turnbull 2000), the Faroe Islands (Sande & Poppe 1995) and Canada (Brocklebank & Raverty 2002). CMS causes substantial financial losses for the fish farming industry (Brun, Poppe, Skrudland & Jarp 2003), and CMS-like lesions have also been observed in wild Atlantic salmon (Poppe & Seierstad 2003). The causative agent is most likely a naked, double-stranded RNA virus related to the Totiviridae group, provisionally named piscine myocarditis virus (PMCV) (Lovoll, Wiik-Nielsen, Grove, WiikNielsen, Kristoffersen, Faller et al. 2010; Haugland, Mikalsen, Nilsen, Lindmo, Thu, Eliassen et al. 2011), and thus far, the virus has been found exclusively in farmed Atlantic salmon sampled from pens with CMS. Using a PMCV-specific real-time PCR assay (Lovoll et al. 2010), we have screened for the presence of PMCV in more than 30 species of marine fish (Table 1) sampled off the central, western coastline of Norway from Trondheim to the Varanger fjord. The set of samples was a representative subset of the catch from a single trawling expedition. Sampling was performed and financed by the project Viral haemorrhagic septicaemia virus (VHSV) in wild and farmed fish in Norway (NFR-190245), and more details on the sampling will be published later along with other results from this project. Organ samples of spleen, kidney and brain were pooled and directly frozen in transport medium (Leibovitz culture medium supplemented with 4 mm l-glutamine and 100 ng mL gentamicin; all from Sigma-Aldrich). Each pool consisted of material from 1 to 5 individuals of the same fish species sampled at the same time and place. For some of the samples included in the pools, kidney tissue from individual fish was also stored on RNAlater (Qiagen AB). Nucleic acids from the organ pools were extracted from 100 uL of tissue homogenate using the NucliSENS easyMAG nucleic acid extraction system (bioMerieux, Inc.) according to the manufacturer s recommendations. RNA from individual fish kidneys was extracted using an RNeasy Mini kit (Qiagen AB). A total of 342 pools (1501 individuals) representing 32 species were screened. The majority of the samples were negative, but 11 of 38 pools from Argentina silus gave a positive PCR result (Table 1). Journal of Fish Diseases 2011, 34, 955–957 doi:10.1111/j.1365-2761.2011.01315.x

Transcriptome profiling of immune responses to cardiomyopathy syndrome (CMS) in Atlantic salmon

BackgroundCardiomyopathy syndrome (CMS) is a disease associated with severe myocarditis primarily in adult farmed Atlantic salmon (Salmo salar L.), caused by a double-stranded RNA virus named piscine myocarditis virus (PMCV) with structural similarities to the Totiviridae family. Here we present the first characterisation of host immune responses to CMS assessed by microarray transcriptome profiling.ResultsUnvaccinated farmed Atlantic salmon post-smolts were infected by intraperitoneal injection of PMCV and developed cardiac pathology consistent with CMS. From analysis of heart samples at several time points and different tissues at early and clinical stages by oligonucleotide microarrays (SIQ2.0 chip), six gene sets representing a broad range of immune responses were identified, showing significant temporal and spatial regulation. Histopathological examination of cardiac tissue showed myocardial lesions from 6 weeks post infection (wpi) that peaked at 8-9 wpi and was followed by a recovery. Viral RNA was detected in all organs from 4 wpi suggesting a broad tissue tropism. High correlation between viral load and cardiac histopathology score suggested that cytopathic effect of infection was a major determinant of the myocardial changes. Strong and systemic induction of antiviral and IFN-dependent genes from 2 wpi that levelled off during infection, was followed by a biphasic activation of pathways for B cells and MHC antigen presentation, both peaking at clinical pathology. This was preceded by a distinct cardiac activation of complement at 6 wpi, suggesting a complement-dependent activation of humoral Ab-responses. Peak of cardiac pathology and viral load coincided with cardiac-specific upregulation of T cell response genes and splenic induction of complement genes. Preceding the reduction in viral load and pathology, these responses were probably important for viral clearance and recovery.ConclusionsBy comparative analysis of gene expression, histology and viral load, the temporal and spatial regulation of immune responses were characterised and novel immune genes identified, ultimately leading to a more complete understanding of host-virus responses and pathology and protection in Atlantic salmon during CMS.

Cardiomyopathy Syndrome of Atlantic Salmon (Salmo salar L.) Is Caused by a Double-Stranded RNA Virus of the Totiviridae Family

Cardiomyopathy syndrome (CMS) of farmed and wild Atlantic salmon (Salmo salar L.) is a disease of yet unknown etiology characterized by a necrotizing myocarditis involving the atrium and the spongious part of the heart ventricle. Here, we report the identification of a double-stranded RNA virus likely belonging to the family Totiviridae as the causative agent of the disease. The proposed name of the virus is piscine myocarditis virus (PMCV). On the basis of the RNA-dependent RNA polymerase (RdRp) sequence, PMCV grouped with Giardia lamblia virus and infectious myonecrosis virus of penaeid shrimp. The genome size of PMCV is 6,688 bp, with three open reading frames (ORFs). ORF1 likely encodes the major capsid protein, while ORF2 encodes the RdRp, possibly expressed as a fusion protein with the ORF1 product. ORF3 seems to be translated as a separate protein not described for any previous members of the family Totiviridae. Following experimental challenge with cell culture-grown virus, histopathological changes are observed in heart tissue by 6 weeks postchallenge (p.c.), with peak severity by 9 weeks p.c. Viral genome levels detected by real-time reverse transcription (RT)-PCR peak earlier at 6 to 7 weeks p.c. The virus genome is detected by in situ hybridization in degenerate cardiomyocytes from clinical cases of CMS. Virus genome levels in the hearts from clinical field cases correlate well with the severity of histopathological changes in heart tissue. The identification of the causative agent for CMS is important for improved disease surveillance and disease control and will serve as a basis for vaccine development against the disease.