Immunohistochemical detection of piscine reovirus (PRV) in hearts of Atlantic salmon coincide with the course of heart and skeletal muscle inflammation (HSMI)

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

Piscine orthoreovirus infection in Atlantic salmon (Salmo salar) protects against subsequent challenge with infectious hematopoietic necrosis virus (IHNV)

Abstract Infectious hematopoietic necrosis virus (IHNV) is endemic in farmed rainbow trout in continental Europe and in various salmonid fish species at the Pacific coast of North America. IHN has never occurred in European Atlantic salmon (Salmo salar) farms, but is considered as a major threat for the European salmon industry. Another virus, Piscine orthoreovirus (PRV), is widespread in the sea phase of Atlantic salmon, and is identified as the causative agent of heart and skeletal muscle inflammation. The aim of this study was to investigate the interactions between a primary PRV infection and a secondary IHNV infection under experimental conditions. A PRV cohabitation challenge was performed with Atlantic salmon. At peak of PRV viremia the fish were challenged by immersion with an IHNV genogroup E isolate. Clinical signs and morbidity were monitored. Target organs were sampled at selected time points to assess viral loads of both pathogens. Antiviral immune response and presence of histopathological findings were also investigated. Whereas the PRV-negative/IHNV positive group suffered significant decrease in survival caused by IHNV, the PRV infected groups did not suffer any morbidity and showed negligible levels of IHNV infection. Antiviral response genes were induced, as measured in spleen samples, from PRV infected fish prior to IHNV challenge. In conclusion, PRV-infection protects Atlantic salmon against IHNV infection and morbidity, most likely by inducing a protective innate antiviral response

Molecular and antigenic characterization of Piscine orthoreovirus (PRV) from rainbow trout (Oncorhynchus mykiss)

Piscine orthoreovirus (PRV-1) causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). Recently, a novel PRV (formerly PRV-Om, here called PRV-3), was found in rainbow trout (Oncorhynchus mykiss) with HSMI-like disease. PRV is considered to be an emerging pathogen in farmed salmonids. In this study, molecular and antigenic characterization of PRV-3 was performed. Erythrocytes are the main target cells for PRV, and blood samples that were collected from experimentally challenged fish were used as source of virus. Virus particles were purified by gradient ultracentrifugation and the complete coding sequences of PRV-3 were obtained by Illumina sequencing. When compared to PRV-1, the nucleotide identity of the coding regions was 80.1%, and the amino acid identities of the predicted PRV-3 proteins varied from 96.7% (λ1) to 79.1% (σ3). Phylogenetic analysis showed that PRV-3 belongs to a separate cluster. The region encoding σ3 were sequenced from PRV-3 isolates collected from rainbow trout in Europe. These sequences clustered together, but were distant from PRV-3 that was isolated from rainbow trout in Norway. Bioinformatic analyses of PRV-3 proteins revealed that predicted secondary structures and functional domains were conserved between PRV-3 and PRV-1. Rabbit antisera raised against purified virus or various recombinant virus proteins from PRV-1 all cross-reacted with PRV-3. Our findings indicate that despite different species preferences of the PRV subtypes, several genetic, antigenic, and structural properties are conserved between PRV-1 and-3

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

Long-term persistence of piscine orthoreovirus-1 (PRV-1) infection during the pre-smolt stages of Atlantic salmon in freshwater

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Piscine Orthoreovirus (PRV)-3, but Not PRV-2, Cross-Protects against PRV-1 and Heart and Skeletal Muscle Inflammation in Atlantic Salmon

Heart and skeletal muscle inflammation (HSMI), caused by infection with Piscine orthoreovirus-1 (PRV-1), is a common disease in farmed Atlantic salmon (Salmo salar). Both an inactivated whole virus vaccine and a DNA vaccine have previously been tested experimentally against HSMI and demonstrated to give partial but not full protection. To understand the mechanisms involved in protection against HSMI and evaluate the potential of live attenuated vaccine strategies, we set up a cross-protection experiment using PRV genotypes not associated with disease development in Atlantic salmon. The three known genotypes of PRV differ in their preference of salmonid host species. The main target species for PRV-1 is Atlantic salmon. Coho salmon (Oncorhynchus kisutch) is the target species for PRV-2, where the infection may induce erythrocytic inclusion body syndrome (EIBS). PRV-3 is associated with heart pathology and anemia in rainbow trout, but brown trout (S. trutta) is the likely natural main host species. Here, we tested if primary infection with PRV-2 or PRV-3 in Atlantic salmon could induce protection against secondary PRV-1 infection, in comparison with an adjuvanted, inactivated PRV-1 vaccine. Viral kinetics, production of cross-reactive antibodies, and protection against HSMI were studied. PRV-3, and to a low extent PRV-2, induced antibodies cross-reacting with the PRV-1 σ1 protein, whereas no specific antibodies were detected after vaccination with inactivated PRV-1. Ten weeks after immunization, the fish were challenged through cohabitation with PRV-1-infected shedder fish. A primary PRV-3 infection completely blocked PRV-1 infection, while PRV-2 only reduced PRV-1 infection levels and the severity of HSMI pathology in a few individuals. This study indicates that infection with non-pathogenic, replicating PRV could be a future strategy to protect farmed salmon from HSMI

Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss)

Abstract 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

Transcriptomics of early responses to purified Piscine orthoreovirus-1 in Atlantic salmon (Salmo salar L.) red blood cells compared to non-susceptible cell lines

Piscine red blood cells (RBC) are nucleated and have been characterized as mediators of immune responses in addition to their role in gas exchange. Salmonid RBC are major target cells of Piscine orthoreovirus-1 (PRV-1), the etiological agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). PRV-1 replicates in RBC ex vivo, but no viral amplification has been possible in available A. salmon cell lines. To compare RBC basal transcripts and transcriptional responses to PRV-1 in the early phase of infection with non-susceptible cells, we exposed A. salmon RBC, Atlantic salmon kidney cells (ASK) and Salmon head kidney cells (SHK-1) to PRV-1 for 24 h. The RNA-seq analysis of RBC supported their previous characterization as pluripotent cells, as they expressed a wide repertoire of genes encoding pattern recognition receptors (PRRs), cytokine receptors, and genes implicated in antiviral activities. The comparison of RBC to ASK and SHK-1 revealed immune cell features exclusively expressed in RBC, such as genes involved in chemotactic activity in response to inflammation. Differential expression analysis of RBC exposed to PRV-1 showed 46 significantly induced genes (≥ 2-fold upregulation) linked to the antiviral response pathway, including RNA-specific PRRs and interferon (IFN) response factors. In SHK-1, PRV induced a more potent or faster antiviral response (213 genes induced). ASK cells showed a differential response pattern (12 genes induced, 18 suppressed) less characterized by the dsRNA-induced antiviral pathway. Despite these differences, the RIG-I-like receptor 3 (RLR3) in the family of cytosolic dsRNA receptors was significantly induced in all PRV-1 exposed cells. IFN regulatory factor 1 (IRF1) was significantly induced in RBC only, in contrast to IRF3/IRF7 induced in SHK-1. Differences in IRF expression and activity may potentially affect viral propagation

Piscine Orthoreovirus-1 Isolates Differ in Their Ability to Induce Heart and Skeletal Muscle Inflammation in Atlantic Salmon (Salmo salar)

Piscine orthoreovirus 1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus is widespread in Atlantic salmon and was present in Norway long before the first description of HSMI in 1999. Furthermore, in Canada the virus is prevalent in farmed Atlantic salmon but HSMI is not and Canadian isolates have failed to reproduce HSMI experimentally. This has led to the hypothesis that there are virulence differences between PRV-1 isolates. In this study we performed a dose standardized challenge trial, comparing six PRV-1 isolates, including two Norwegian field isolates from 2018, three historical Norwegian isolates predating the first report of HSMI and one Canadian isolate. The Norwegian 2018 isolates induced lower viral protein load in blood cells but higher plasma viremia. Following peak replication in blood, the two Norwegian 2018 isolates induced histopathological lesions in the heart consistent with HSMI, whereas all three historical Norwegian and the Canadian isolates induced only mild cardiac lesions. This is the first demonstration of virulence differences between PRV-1 isolates and the phenotypic differences are linked to viral proteins encoded by segment S1, M2, L1, L2 and S4