Characterization of early phases of cardiomyopathy syndrome pathogenesis in Atlantic salmon (Salmo salar L.) through various diagnostic methods.

Since the first description of cardiomyopathy syndrome (CMS) in Atlantic salmon, in 1985, the disease caused by piscine myocarditisvirus (PMCV) has become a common problem in Atlantic salmon farming, not only in Norway, but also in other salmon farming countries like Scotland and Ireland. In the last years, CMS has been ranked as the most important salmon viral disease in Norway regarding both mortality and economic losses. Detailed knowledge of infection and pathogenesis is still lacking, a decade after the causal agent was first described, and there is a need for a wider range of methods/tools for diagnostic and research purposes. In this study, we compared the detection of PMCV- and CMS-related tissue lesions using previously used and well-known methods like histopathology and real-time RT-PCR to immunohistochemistry (IHC), a less used method, and a new method, RNAscope in situ hybridization. Tissue samples of three different cardiac compartments, mid-kidney and skin/muscle tissue were compared with non-lethal parallel samplings of blood and mucus. The development of pathological cardiac lesions observed in this experiment was in accordance with previous descriptions of CMS. Our results indicate a viremic phase 10- to 20-day post-challenge (dpc) preceding the cardiac lesions. In this early phase, virus could also be detected in relatively high amount in mid-kidney by real-time RT-PCR. Plasma and/or mid-kidney samples may, therefore, be candidates to screen for early-phase PMCV infection. The RNAscope in situ hybridization method showed higher sensitivity and robustness compared with the immunohistochemistry and may be a valuable support to histopathology in CMS diagnostics, especially in cases of untypical lesions or mixed infections.

Genome Sequencing of SAV3 Reveals Repeated Seeding Events of Viral Strains in Norwegian Aquaculture.

Understanding the dynamics of pathogen transfer in aquaculture systems is essential to manage and mitigate disease outbreaks. The goal of this study was to understand recent transmission dynamics of salmonid alphavirus (SAV) in Norway. SAV causes significant economic impacts on farmed salmonids in European aquaculture. SAV is classified into six subtypes, with Norway having ongoing epidemics of SAV subtypes 2 and 3. These two viral subtypes are present in largely distinct geographic regions of Norway, with SAV2 present in Trondelag, SAV3 in Rogaland, Sogn og Fjordane, and Hordaland, and Møre og Romsdal having outbreaks of both subtypes. To determine likely transmission routes of Norwegian SAV an established Nanopore amplicon sequencing approach was used in the current study. After confirming the accuracy of this approach for distinguishing subtype level co-infections of SAV2 and SAV3, a hypothetical possibility in regions of neighboring epidemics, twenty-four SAV3 genomes were sequenced to characterize the current genetic diversity of SAV3 in Norwegian aquaculture. Sequencing was performed on naturally infected heart tissues originating from a range of geographic locations sampled between 2016 and 2019. Phylogenetic analyses revealed that the currently active SAV3 strains sampled comprise several distinct lineages sharing an ancestor that existed ∼15 years ago (95% HPD, 12.51-17.7 years) and likely in Hordaland. At least five of these lineages have not shared a common ancestor for 7.85 years (95% HPD, 5.39-10.96 years) or more. Furthermore, the ancestor of the strains that were sampled outside of Hordaland (Sogn of Fjordane and Rogaland) existed less than 8 years ago, indicating a lack of long-term viral reservoirs in these counties. This evident lack of geographically distinct subclades is compatible with a source-sink transmission dynamic explaining the long-term movements of SAV around Norway. Such anthropogenic transport of the virus indicates that at least for sink counties, biosecurity strategies might be effective in mitigating the ongoing SAV epidemic. Finally, genomic analyses of SAV sequences were performed, offering novel insights into the prevalence of SAV genomes containing defective deletions. Overall, this study improves our understanding of the recent transmission dynamics and biology of the SAV epidemic affecting Norwegian aquaculture.

Inactivated Piscine orthoreovirus vaccine protects against heart and skeletal muscle inflammation in Atlantic salmon.

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 in vivo 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 6 weeks 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.

In vitro adaptation of SAV3 in cell culture correlates with reduced in vivo replication capacity and virulence to Atlantic salmon (Salmo salar L.) parr.

Salmonid alphavirus (SAV) is the causative agent of pancreas disease affecting Atlantic salmon and rainbow trout and causes a major burden to the aquaculture industry. This study describes a Norwegian subtype SAV3 virus isolate (SAV3-H10) subjected to serial passages in Chinook salmon embryo cells (CHSE-214) followed by Asian Grouper skin cells (AGK). Two passages from CHSE and one after transfer to AGK cells were chosen for further investigation, based on variation in degree and development of cytopathic effect (CPE). After plaque purification, several in vitro studies were performed. Cell viability after infection, viral replication and ability to cause morphological changes in CHSE and AGK cells was studied for the three isolates. The AGK-transferred isolate was identified with the strongest abilities to reduce cell viability, replicate more and cause more CPE in cell culture when compared with the early and late CHSE-grown isolates. Subsequently, the isolates were tested in an experimental fish challenge, showing higher viral load and higher pathological score for the least cell-cultured isolate. Full-length sequencing of the viral genome of the three isolates revealed divergence in four amino acid positions and the AGK-grown isolate also had a 3 nt deletion in the 3'UTR. In conclusion, we show that cell culture of SAV3-H10 selects for strains inducing earlier CPE in vitro with increased viral replication. In vivo, the effect is reversed, with lower replication levels and lower pathology scores in target organs. This study outlines a path to identify potential virulence motifs of SAV3.

Modification of a salmonid alphavirus replicon vector for enhanced expression of heterologous antigens.

A salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared with terrestrial alphavirus replicons. In this study, we have compared several modifications to a SAV replicon construct and analysed their influence on foreign gene expression. We found that an insertion of a translational enhancer consisting of the N-terminal 102 nt of the capsid gene, together with a nucleotide sequence encoding the foot-and-mouth disease virus (FMDV) 2A peptide, caused a significant increase in EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5' end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at the 3' end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production, which can be applied for immunization of salmonid fish in the future.

A 6K-deletion variant of salmonid alphavirus is non-viable but can be rescued through RNA recombination.

Pancreas disease (PD) of Atlantic salmon is an emerging disease caused by Salmonid alphavirus (SAV) which mainly affects salmonid aquaculture in Western Europe. Although genome structure of SAV has been characterized and each individual viral protein has been identified, the role of 6K protein in viral replication and infectivity remains undefined. The 6K protein of alphaviruses is a small and hydrophobic protein which is involved in membrane permeabilization, protein processing and virus budding. Because these common features are shared across many viral species, they have been named viroporins. In the present study, we applied reverse genetics to generate SAV3 6K-deleted (Δ6K) variant and investigate the role of 6K protein. Our findings show that the 6K-deletion variant of salmonid alphavirus is non-viable. Despite viral proteins of Δ6K variant are detected in the cytoplasm by immunostaining, they are not found on the cell surface. Further, analysis of viral proteins produced in Δ6K cDNA clone transfected cells using radioimmunoprecipitation (RIPA) and western blot showed a protein band of larger size than E2 of wild-type SAV3. When Δ6K cDNA was co-transfected with SAV3 helper cDNA encoding the whole structural genes including 6K, the infectivity was rescued. The development of CPE after co-transfection and resolved genome sequence of rescued virus confirmed full-length viral genome being generated through RNA recombination. The discovery of the important role of the 6K protein in virus production provides a new possibility for the development of antiviral intervention which is highly needed to control SAV infection in salmonids.

Natural infection of Atlantic salmon (Salmo salar L.) with salmonid alphavirus 3 generates numerous viral deletion mutants.

Salmon pancreas disease virus (SPDV) also referred to as salmonid alphavirus (SAV) is a virus causing pancreas disease in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss). Although the virus causes an economically important disease, relatively few full-length genome sequences of SAV strains are currently available. Here, we report full-length genome sequences of nine SAV3 strains from sites farming Atlantic salmon geographically spread along the Norwegian coastline. The virus genomes were sequenced directly from infected heart tissue, to avoid culture selection bias. Sequence analysis confirmed a high level of sequence identity within SAV3 strains, with a mean nucleotide diversity of 0.11 %. Sequence divergence was highest in 6K and E2, while lowest in the capsid protein and the non-structural proteins (nsP4 and nsP2). This study reports for the first time that numerous defective viruses containing genome deletions are generated during natural infection with SAV. Deletions occurred in all virus strains and were not distributed randomly throughout the genome but instead tended to aggregate in certain areas. We suggest imprecise homologous recombination as an explanation for generation of defective viruses with genome deletions. The presence of such viruses, provides a possible explanation for the difficulties in isolating SAV in cell culture. Primary virus isolation was successfully achieved for only two of eight strains, despite extensive attempts using three different cell lines. Both SAV isolates were easily propagated further and concomitant viral deletion mutants present in clinically infected heart tissue were maintained following serial passage in CHH-1 cells.

Gene expression studies of host response to Salmonid alphavirus subtype 3 experimental infections in Atlantic salmon.

Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.

Atlantic salmon reovirus infection causes a CD8 T cell myocarditis in Atlantic salmon (Salmo salar L.).

Heart and skeletal inflammation (HSMI) of farmed Atlantic salmon (Salmo salar L.) is a disease characterized by a chronic myocarditis involving the epicardium and the compact and spongious part of the heart ventricle. Chronic myositis of the red skeletal muscle is also a typical finding of HSMI. Piscine reovirus (PRV) has been detected by real-time PCR from farmed and wild salmon with and without typical changes of HSMI and thus the causal relationship between presence of virus and the disease has not been fully determined. In this study we show that the Atlantic salmon reovirus (ASRV), identical to PRV, can be passaged in GF-1 cells and experimental challenge of naïve Atlantic salmon with cell culture passaged reovirus results in cardiac and skeletal muscle pathology typical of HSMI with onset of pathology from 6 weeks, peaking by 9 weeks post challenge. ASRV replicates in heart tissue and the peak level of virus replication coincides with peak of heart lesions. We further demonstrate mRNA transcript assessment and in situ characterization that challenged fish develop a CD8+ T cell myocarditis.

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.

Alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype 3 replication in vitro.

Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-alpha)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-gamma in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-alpha [rIFN-alpha] and rIFN-gamma) and used for in vitro studies. SAV-3 infection in a permissive salmon cell line (TO cells) results in IFN-alpha and IFN-stimulated gene (ISG) mRNA upregulation. Preinfection treatment (4 to 24 h prior to infection) with salmon rIFN-alpha induces an antiviral state that inhibits the replication of SAV-3 and protects the cells against virus-induced cytopathic effects (CPE). The antiviral state coincides with a strong expression of Mx and ISG15 mRNA and Mx protein expression. When rIFN-alpha is administered at the time of infection and up to 24 h postinfection, virus replication is not inhibited, and cells are not protected against virus-induced CPE. By 40 h postinfection, the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) is phosphorylated concomitant with the expression of the E2 protein as assessed by Western blotting. Postinfection treatment with rIFN-alpha results in a moderate reduction in E2 expression levels in accordance with a moderate downregulation of cellular protein synthesis, an approximately 65% reduction by 60 h postinfection. rIFN-gamma has only a minor inhibitory effect on SAV-3 replication in vitro. SAV-3 is sensitive to the preinfection antiviral state induced by rIFN-alpha, while postinfection antiviral responses or postinfection treatment with rIFN-alpha is not able to limit viral replication.

Decreased expression of TGF-beta, GILT and T-cell markers in the early stages of soybean enteropathy in Atlantic salmon (Salmo salar L.).

This study investigated the early expression of T-cell markers and genes potentially involved in the induction of soybean meal (SBM) enteropathy in the distal intestine (DI) of Atlantic salmon (Salmo salar L.). Quantitative PCR was used to study the expression of CD3, CD8beta, transforming growth factor beta (TGF-beta), interferon-gamma-inducible lysosomal thiol reductase (GILT) and interleukin-1beta (IL-1beta) in salmon fed SBM for 1, 3 and 7 days using fish fed fishmeal as controls. In the same tissue, the morphological development of SBM enteropathy was evaluated by routine histology and the presence of T cells was mapped by immunohistochemistry. TGF-beta was significantly down-regulated on all days of feeding SBM. GILT was significantly down-regulated on days 3 and 7 compared to day 1. A depression in the expression of T-cell markers was observed on day 3 whereas increased densities of T cells were observed at the base of mucosal folds after 7 days of feeding SBM. Down-regulation of GILT and TGF-beta may lead to sensitization of intraepithelial lymphocytes and failure to maintain normal mucosal integrity in the DI. These responses are implicated in the pathogenesis of SBM enteropathy in Atlantic salmon.

Isoflavone-rich extracts from wooly glycine Glycine tomentella inhibits LPS-induced TNF-alpha expression in a macrophage cell line of Atlantic salmon (Salmo salar L.).

The immunomodulatory effects of an isoflavone-rich extract from the root of wooly glycine Glycine tomentella (GTE) were studied in a macrophage-like cell line from Atlantic salmon (TO cells). The TO cell line was stimulated with defined concentrations of lipopolysaccharide (LPS) from Escherichia coli (serotype O127:B8) for defined time periods to induce expression of pro-inflammatory enzymes and cytokines. Cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) were measured by real-time PCR methods and combined with analyses of eicosanoid production in cell extracts and evaluation of molecules of the TNF-alpha cell signaling pathway. The results showed that TNF-alpha was strongly induced by LPS, while GTE (25miicrog/ml) inhibited 67% of the TNF-alpha response when added to the cells together with LPS. Incubation of LPS in combination of GTE in TO cells caused increased intracellular prostaglandin E2 (PGE2), and reduced activation of p38 MAP kinase compared to LPS alone. GTE seemed to arrest NADPH oxidation, the coenzyme for carbonyl reductase and the prostaglandin-E2 9-reductase converting PGE2 to PGF2. We suggest that the mechanism of increased intracellular PGE2 levels following GTE treatment is caused by reduced breakdown of PGE2. GTE did not inhibit the other pro-inflammatory responses in LPS stimulated cells studied herein. IL-1beta and COX-2 showed moderately increased levels of expression likely caused by the increased PGE2.

Differential expression profiles and gene structure of two tumor necrosis factor-alpha variants in Atlantic salmon (Salmo salar L.).

Here we present the gene structure and expression data on two Atlantic salmon TNF-alpha genes. Both genes are approximately 2.0kb in length and organized into four exons and three introns. The open reading frame of both genes translates into 246 amino acid putative peptides, being 91.5% identical at the amino acid level. The upstream regulatory region of both genes were amplified by inverse PCR and analyzed for putative regulatory binding sites. Variant specific gene-expression both in vitro and in vivo revealed that the two variants are differentially regulated. TNF-alpha2 is the dominant variant throughout the experimental period following LPS stimulation of TO-cells. The picture was different in head kidney tissue following vaccination of Atlantic salmon with an experimental multivalent oil-based vaccine. TNF-alpha2 was the most dominant transcript in the non-stimulated controls and in the early phase. A shift from TNF-alpha2 to TNF-alpha1 was however seen, and from day 12 the TNF-alpha1 was most abundant transcript. Implications of variant specific gene-expression are discussed.

Expression profiles of inflammatory and immune-related genes in Atlantic salmon (Salmo salar L.) at early time post vaccination.

Vaccination of Atlantic salmon parr with oil-based vaccines will inevitably cause inflammation at the site of injection, albeit the underlying mechanisms are not very well understood or studied in any detail. Here, we report time-course changes in expression levels, assessed by real-time RT-PCR of IL-1 beta, Mx, two beta-2-microglobulin variants and MHC class II beta, from 2 to 19 days post vaccination with a multi-component oil-adjuvanted vaccine. Highly variable individual responses to vaccination make selection of high responders essential prior to subtractive analysis. Based on the above mentioned expression profiles, high-responding individuals at 2, 8 and 19 days post vaccination, were selected for subtractive analysis. Clustering of clones according to putative function, suggest an initial up-regulation of genes involved in metabolism and cell signalling, before onset of genes involved in inflammation. The lag-time for genes considered as inflammatory markers was more than 48 h, while they were found to constitute the major part of up-regulated transcripts by 8 days post vaccination. By day 19, immune-related genes like immunoglobulin and T cell-receptor genes, comprised a higher proportion of the up-regulated genes than at earlier time points.