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Frequent RNA virus mutations raise concerns about evolving virulent variants. The purpose of this study was to investigate genetic variation in salmonid alphavirus-3 (SAV3) over the course of an experimental infection in Atlantic salmon and brown trout. Atlantic salmon and brown trout parr were infected using a cohabitation challenge, and heart samples were collected for analysis of the SAV3 genome at 2-, 4- and 8-weeks post-challenge. PCR was used to amplify eight overlapping amplicons covering 98.8% of the SAV3 genome. The amplicons were subsequently sequenced using the Nanopore platform. Nanopore sequencing identified a multitude of single nucleotide variants (SNVs) and deletions. The variation was widespread across the SAV3 genome in samples from both species. Mostly, specific SNVs were observed in single fish at some sampling time points, but two relatively frequent (i.e., major) SNVs were observed in two out of four fish within the same experimental group. Two other, less frequent (i.e., minor) SNVs only showed an increase in frequency in brown trout. Nanopore reads were de novo clustered using a 99% sequence identity threshold. For each amplicon, a number of variant clusters were observed that were defined by relatively large deletions. Nonmetric multidimensional scaling analysis integrating the cluster data for eight amplicons indicated that late in infection, SAV3 genomes isolated from brown trout had greater variation than those from Atlantic salmon. The sequencing methods and bioinformatics pipeline presented in this study provide an approach to investigate the composition of genetic diversity during viral infections., (© 2024. The Author(s).)

Infectious hematopoietic necrosis virus (IHNV) severely and lethally infects salmonid fish, including Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) worldwide. Rapid and accurate viral detection is crucial for preventing pathogen spread and minimizing damage. Although several IHNV detection assays have been developed, their analytical and diagnostic performances have not been evaluated and field usability assessments have not been completely validated. Here, we developed a reverse-transcription cross-priming amplification-based lateral flow assay (RT-CPA-LFA) and validated its diagnostic performance. To detect the IHNV, primers were designed based on the consensus sequence of the nucleocapsid (N) gene. Notably, when combined with a lateral flow dipstick, it could visualize the IHNV amplification products within 5 min and the detection limit of the developed RT-CPA-LFA was 3.28×10 5 copies/μL. The diagnostic sensitivity and specificity in fish samples (n=140) were 98.88 % and 96.08 %, respectively. Moreover, the IHNV detection rate by RT-CPA-LFA in dead rainbow trout artificially injected with the virus was 100 %, consistent with to the results obtained from second conventional and real-time PCR, indicating its applicability for rapid IHNV detection and presumptive IHN diagnosis during the endemic period., Competing Interests: Declaration of Competing Interest The author declares no conflicts of interest, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Piscine orthoreovirus-1 (PRV-1) is a prevalent agent in Atlantic salmon (Salmo salar) and the causative agent of heart and skeletal muscle inflammation (HSMI), an important disease in farmed Atlantic salmon. Investigations into the introduction and dissemination routes of PRV-1 in a field setting have been limited. This study aimed to better understand PRV-1 infections and HSMI-associated mortality under field conditions. We tracked introduction and spread of PRV-1 over one production cycle in a geographically isolated region in Norwegian aquaculture. From five sites, a total of 32 virus isolates were sequenced and genogrouped. The results indicated multiple introductions of PRV-1 to the area, but also revealed a high level of genetic homogeneity among the virus variants. The variants differed from that of the previous production cycle at two out of three sites investigated, suggesting that synchronized fallowing can be a useful tool for preventing dissemination of PRV-1 between generations of fish. Exposure to PRV-1 at the freshwater stage was identified as a potential source of introduction. A low level of HSMI-associated mortality was observed at all sites, with the onset of mortality showing some variation across PRV-1 genogroups. However, the study highlighted the complexity of associating viral genogroups with mortality in a field setting. Overall, this study contributes valuable insights into PRV-1 dynamics in a real-world aquaculture setting, offering potential strategies for disease management and prevention., (© 2024 The Author(s). Journal of Fish Diseases published by John Wiley & Sons Ltd.)

The potential for infectious salmon anemia virus (ISAV)-an internationally regulated pathogen of salmon-to transmit vertically from parent to offspring is currently unclear. While the highly virulent ISAV phenotype known as ISAV-HPRΔ has been observed intra-ova, evidence for vertical transmission of the avirulent ISAV phenotype known as ISAV-HPR0 is lacking. In this study, we identified ISAV-HPR0-infected Atlantic salmon broodstock during spawning within a government research recirculating aquaculture facility using qPCR. Eggs and milt from infected brood were used to initiate 16 unique family dam-sire crosses from which 29-60 fertilized eggs per cross were screened for ISAV using qPCR (limit of detection ~100 virus genome copies/egg). A portion of eggs (~300) from one family cross was hatched and further reared in biosecure containment and periodically screened for ISAV by gill clipping over a 2-year period. ISAV was not detected in any of the 781 eggs screened from 16 family crosses generated by infected brood, nor in 870 gill clips periodically sampled from the single-family cohort raised for 2 years in biocontainment. Based on these findings, we conclude that ISAV-HPR0 has a limited likelihood for vertical parent-to-offspring transmission in cultured Atlantic salmon.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Studies of the interplay between metabolism and immunity, known as immunometabolism, is steadily transforming immunological research into new understandings of how environmental cues like diet are affecting innate and adaptive immune responses. The aim of this study was to explore antiviral transcriptomic responses under various levels of polyunsaturated fatty acid. Atlantic salmon kidney cells (ASK cell line) were incubated for one week in different levels of the unsaturated n-3 eicosapentaneoic acid (EPA) resulting in cellular levels ranging from 2-20% of total fatty acid. These cells were then stimulated with the viral mimic and interferon inducer poly I:C (30 ug/ml) for 24 hours before total RNA was isolated and sequenced for transcriptomic analyses. Up to 200 uM EPA had no detrimental effects on cell viability and induced very few transcriptional changes in these cells. However, in combination with poly I:C, our results shows that the level of EPA in the cellular membranes exert profound dose dependent effects of the transcriptional profiles induced by this treatment. Metabolic pathways like autophagy, apelin and VEGF signaling were attenuated by EPA whereas transcripts related to fatty acid metabolism, ferroptosis and the PPAR signaling pathways were upregulated. These results suggests that innate antiviral responses are heavily influenced by the fatty acid profile of salmonid cells and constitute another example of the strong linkage between general metabolic pathways and inflammatory responses., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gjøen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rapid growth in aquaculture has resulted in high-density production systems in ecologically and geographically novel conditions in which the emergence of diseases is inevitable. Well-characterized methods for detection and surveillance of infectious diseases are vital for rapid identification, response, and recovery to protect economic and food security. We implemented a proof-of-concept approach for virus detection using a known high-consequence fish pathogen, infectious salmon anemia virus (ISAV), as the archetypal pathogen. In fish infected with ISAV, we integrated histopathology, virus isolation, whole-genome sequencing (WGS), electron microscopy (EM), in situ hybridization (ISH), and reverse transcription real-time PCR (RT-rtPCR). Fresh-frozen and formalin-fixed tissues were collected from virus-infected, control, and sham-infected Atlantic salmon ( Salmo salar ). Microscopic differences were not evident between uninfected and infected fish. Viral cytopathic effect was observed in cell cultures inoculated with fresh-frozen tissue homogenates from 3 of 3 ISAV-infected and 0 of 4 uninfected or sham-infected fish. The ISAV genome was detected by shotgun metagenomics in RNA extracted from the medium from 3 of 3 inoculated cell cultures, 3 of 3 infected fish, and 0 of 4 uninfected or sham-infected fish, yielding sufficient coverage for de novo assembly. An ISH probe against ISAV revealed ISAV genome in multiple organs, with abundance in renal hematopoietic tissue. Virus was detected by RT-rtPCR in gill, heart, kidney, liver, and spleen. EM and metagenomic WGS from tissues were challenging and unsuccessful. Our proof-of-concept methodology has promise for detection and characterization of unknown aquatic pathogens and also highlights some associated methodology challenges that require additional investigation., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to research, authorship, and/or publication of this article.

Infectious salmon anemia virus (ISAV; Isavirus salaris ) causes an economically important disease of Atlantic salmon ( Salmo salar L.). ISA outbreaks have resulted in significant losses of farmed salmon globally, often with a sudden onset. However, 2 phenotypically distinct variants of ISAV exist, each with divergent disease outcomes, associated regulations, and control measures. ISAV-HPRΔ, also known as ISAV-HPR deleted, is responsible for ISA outbreaks; ISAV-HPR0, is avirulent and is not known to cause fish mortality. Current detection methodology requires genetic sequencing of ISAV-positive samples to differentiate phenotypes, which may slow responses to disease management. To increase the speed of phenotypic determinations of ISAV, we developed a new, rapid multiplex RT-qPCR method capable of 1) detecting if a sample contains any form of ISAV, 2) discriminating whether positive samples contain HPRΔ or HPR0, and 3) validating RNA extractions with an internal control, all in a single reaction. Following assay development and optimization, we validated this new multiplex on 31 ISAV strains collected from North America and Europe (28 ISAV-HPRΔ, 3 ISAV-HPR0). Finally, we completed an inter-laboratory comparison of this multiplex qPCR with commercial ISAV testing and found that both methods provided equivalent results for ISAV detection., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Infectious salmon anaemia virus (ISAV) binds circulating Atlantic salmon erythrocytes, but the relevance of this interaction for the course of infection and development of disease remains unclear. We here characterise ISAV-erythrocyte interactions in experimentally infected Atlantic salmon and show that ISAV-binding to erythrocytes is common and precedes the development of disease. Viral RNA and infective particles were enriched in the cellular fraction of blood. While erythrocyte-associated ISAV remained infectious, erythrocytes dose-dependently limited the infection of cultured cells. Surprisingly, immunostaining of blood smears revealed expression of ISAV proteins in a small fraction of erythrocytes in one of the examined trials, confirming that ISAV can be internalised in this cell type and engage the cellular machinery in transcription and translation. However, viral protein expression in erythrocytes was rare and not required for development of disease and mortality. Furthermore, active transcription of ISAV mRNA was higher in tissues than in blood, supporting the assumption that ISAV replication predominantly takes place in endothelial cells. In conclusion, Atlantic salmon erythrocytes bind ISAV and sequester infective virus particles during infection, but do not appear to significantly contribute to ISAV replication. We discuss the implications of our findings for infection dynamics and pathogenesis of infectious salmon anaemia.

The nonvirulent infectious salmon anaemia virus (ISAV-HPR0) is the putative progenitor for virulent-ISAV, and a potential risk factor for the development of infectious salmon anaemia (ISA). Understanding the transmission dynamics of ISAV-HPR0 is fundamental to proper management and mitigation strategies. Here, we demonstrate that ISAV-HPR0 causes prevalent and transient infections in all three production stages of Atlantic salmon in the Faroe Islands. Phylogenetic analysis of the haemagglutinin-esterase gene from 247 salmon showed a clear geographical structuring into two significantly distinct HPR0-subgroups, which were designated G2 and G4. Whereas G2 and G4 co-circulated in marine farms, Faroese broodfish were predominantly infected by G2, and smolt were predominantly infected by G4. This infection pattern was confirmed by our G2- and G4-specific RT-qPCR assays. Moreover, the HPR0 variants detected in Icelandic and Norwegian broodfish were never detected in the Faroe Islands, despite the extensive import of ova from both countries. Accordingly, the vertical transmission of HPR0 from broodfish to progeny is uncommon. Phylogenetic and statistical analysis suggest that HPR0 persists in the smolt farms as "house-strains", and that new HPR0 variants are occasionally introduced from the marine environment, probably by HPR0-contaminated sea-spray. Thus, high biosecurity-including water and air intake-is required to avoid the introduction of pathogens to the smolt farms.

Cardiomyopathy syndrome (CMS), caused by piscine myocarditis virus (PMCV), is a serious challenge to Atlantic salmon (Salmo salar L.) aquaculture. Regrettably, husbandry techniques are the only tool to manage CMS outbreaks, and no prophylactic measures are available at present. Early diagnosis of CMS is therefore desirable, preferably with non-lethal diagnostic methods, such as serum biomarkers. To identify candidate biomarkers for CMS, the protein content of pools of sera (4 fish/pool) from salmon with a CMS outbreak (3 pools) and from clinically healthy salmon (3 pools) was compared using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Overall, seven proteins were uniquely identified in the sera of clinically healthy fish, while 27 proteins were unique to the sera of CMS fish. Of the latter, 24 have been associated with cardiac disease in humans. These were grouped as leakage enzymes (creatine kinase, lactate dehydrogenase, glycogen phosphorylase and carbonic anhydrase); host reaction proteins (acute-phase response proteins-haptoglobin, fibrinogen, α2-macroglobulin and ceruloplasmin; and complement-related proteins); and regeneration/remodelling proteins (fibronectin, lumican and retinol). Clinical evaluation of the suitability of these proteins as biomarkers of CMS, either individually or as part of a panel, is a logical next step for the development of early diagnostic tools for CMS., (© 2021 John Wiley & Sons Ltd.)

Before seawater transfer, farmed Atlantic salmon are subjected to treatments that may affect the immune system and susceptibility to pathogens. E.g., exposure to constant light (CL) stimulates smoltification, which prepares salmon to life in sea water, but endocrine changes in this period are associated with suppression of immune genes. Salmon are vaccinated towards end of the freshwater period to safeguard that adequate vaccine efficacy is achieved by the time the fish is transferred to sea. In the present study, we investigated how the responses to vaccination and viral infection varied depending on the time of CL onset relative to vaccination. The salmon were either exposed to CL two weeks prior to vaccination (2-PRI) or exposed to CL at the time of vaccination (0-PRI). A cohabitant challenge with salmonid alphavirus, the causative agent of pancreatic disease, was performed 9 weeks post vaccination. The immunological effects of the different light manipulation were examined at 0- and 6-weeks post vaccination, and 6 weeks post challenge. Antibody levels in serum were measured using a serological bead-based multiplex panel as well as ELISA, and 92 immune genes in heart and spleen were measured using an integrated fluidic circuit-based qPCR array for multiple gene expression. The 2-PRI group showed a moderate transcript down-regulation of genes in the heart at the time of vaccination, which were restored 6 weeks after vaccination (WPV). Conversely, at 6WPV a down-regulation was seen for the 0-PRI fish. Moreover, the 2-PRI group had significantly higher levels of antibodies binding to three of the vaccine components at 6WPV, compared to 0-PRI. In response to SAV challenge, transcription of immune genes between 2-PRI and 0-PRI was markedly dissimilar in the heart and spleen of control fish, but no difference was found between vaccinated salmon from the two CL regimens. Thus, by using labor-saving high throughput detection methods, we demonstrated that light regimens affected antibody production and transcription of immune genes in non-vaccinated and virus challenged salmon, but the differences between the light treatment groups appeared eliminated by vaccination., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The feeding behavior in fish is a complex activity that relies on the ability of the brain to integrate multiple signals to produce appropriate responses in terms of food intake, energy expenditure, and metabolic activity. Upon stress cues including viral infection or mediators such as the proinflammatory cytokines, prostaglandins, and cortisol, both Pomc and Npy/Agrp neurons from the hypothalamus are stimulated, thus triggering a response that controls both energy storage and expenditure. However, how appetite modulators or neuro-immune cues link pathogenesis and energy homeostasis in fish remains poorly understood. Here, we provide the first evidence of a molecular linkage between inflammation and food intake in Salmon salar . We show that in vivo viral challenge with infectious pancreatic necrosis virus (IPNV) impacts food consumption by activating anorexic genes such as mc4r, crf, and pomcb and 5-HT in the brain of S. salar . At the molecular level, viral infection induces an overall reduction in lipid content in the liver, favoring the production of AA and EPA associated with the increment of elovl2 gene. In addition, infection upregulates leptin signaling and inhibits insulin signaling. These changes are accompanied by a robust inflammatory response represented by the increment of Il-1b, Il-6, Tnfa, and Pge2 as well as an increased cortisol level in vivo. Thus, we propose a model in which hypothalamic neurons respond to inflammatory cytokines and stress-related molecules and interact with appetite induction/inhibition. These findings provide evidence of crosstalk between pathogenesis-driven inflammation and hypothalamic-pituitary-adrenocortical axes in stress-induced food intake behavior in fish.

Piscine orthoreovirus (PRV-1) infection causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar) . The virus is also associated with focal melanized changes in white skeletal muscle where PRV-1 infection of macrophages appears to be important. In this study, we studied the macrophage polarization into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes during experimentally induced HSMI. The immune response in heart with HSMI lesions was characterized by CD8 + and MHC-I expressing cells and not by polarized macrophages. Fluorescent in situ hybridization (FISH) assays revealed localization of PRV-1 in a few M1 macrophages in both heart and skeletal muscle. M2 type macrophages were widely scattered in the heart and were more abundant in heart compared to the skeletal muscle. However, the M2 macrophages did not co-stain for PRV-1. There was a strong cellular immune response to the infection in the heart compared to that of the skeletal muscle, seen as increased MHC-I expression, partly in cells also containing PRV-1 RNA, and a high number of cytotoxic CD8 + granzyme producing cells that targeted PRV-1. In skeletal muscle, MHC-I expressing cells and CD8 + cells were dispersed between myocytes, but these cells did not stain for PRV-1. Gene expression analysis by RT-qPCR complied with the FISH results and confirmed a drop in level of PRV-1 following the cell mediated immune response. Overall, the results indicated that M1 macrophages do not contribute to the initial development of HSMI. However, large numbers of M2 macrophages reside in the heart and may contribute to the subsequent fast recovery following clearance of PRV-1 infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Malik, Nyman, Wessel, Dahle and Rimstad.)

Infectious salmon anemia virus (ISAV) infection is currently detected by fish sampling for PCR and immunohistochemistry analysis. As an alternative to sampling fish, we evaluated two different membrane filters in combination with four buffers for elution, concentration, and detection of ISAV in seawater, during a bath challenge of Atlantic salmon ( Salmo salar L.) post-smolts with high and low concentrations of ISAV. Transmission of ISAV in the bath challenge was confirmed by a high mortality, clinical signs associated with ISA disease, and detection of ISAV RNA in organ tissues and seawater samples. The electronegatively charged filter, combined with lysis buffer, gave significantly higher ISAV RNA detection by droplet digital PCR from seawater (5.6 × 10 4 ISAV RNA copies/L; p < 0.001). Viral shedding in seawater was first detected at two days post-challenge and peaked on day 11 post-challenge, one day before mortalities started in fish challenged with high dose ISAV, demonstrating that a large viral shedding event occurs before death. These data provide important information for ISAV shedding that is relevant for the development of improved surveillance tools based on water samples, transmission models, and management of ISA.

Multiple cellular components are involved in pathogen-host interaction during viral infection; in this context, the role of miRNAs have become highly relevant. We assessed the expression of selected miRNAs during an in vitro infection of a Salmo salar cell line with Infectious Salmon Anemia Virus (ISAV), the causative agent of a severe disease by the same name. Salmon orthologs for miRNAs that regulate antiviral responses were measured using RT-qPCR in an in vitro time-course assay. We observed a modulation of specific miRNAs expression, where ssa-miR-155-5p was differentially over-expressed. Using in silico analysis, we identified the putative mRNA targets for ssa-miR-155-5p, finding a high prevalence of hosts immune response-related genes; moreover, several mRNAs involved in the viral infective process were also identified as targets for this miRNA. Our results suggest a relevant role for miR-155-5p in Salmo salar during an ISAV infection as a regulator of the immune response to the virus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The sole member of the Piscihepevirus genus (family Hepeviridae ) is cutthroat trout virus (CTV) but recent metatranscriptomic studies have identified numerous fish hepevirus sequences including CTV-2. In the current study, viruses with sequences resembling both CTV and CTV-2 were isolated from salmonids in eastern and western Canada. Phylogenetic analysis of eight full genomes delineated the Canadian CTV isolates into two genotypes (CTV-1 and CTV-2) within the Piscihepevirus genus. Hepevirus genomes typically have three open reading frames but an ORF3 counterpart was not predicted in the Canadian CTV isolates. In vitro replication of a CTV-2 isolate produced cytopathic effects in the CHSE-214 cell line with similar amplification efficiency as CTV. Likewise, the morphology of the CTV-2 isolate resembled CTV, yet viral replication caused dilation of the endoplasmic reticulum lumen which was not previously observed. Controlled laboratory studies exposing sockeye ( Oncorhynchus nerka) , pink ( O. gorbuscha ), and chinook salmon ( O. &nbsp; tshawytscha ) to CTV-2 resulted in persistent infections without disease and mortality. Infected Atlantic salmon ( Salmo salar ) and chinook salmon served as hosts and potential reservoirs of CTV-2. The data presented herein provides the first in vitro and in vivo characterization of CTV-2 and reveals greater diversity of piscihepeviruses extending the known host range and geographic distribution of CTV viruses.

In salmon farming, viruses are responsible for outbreaks that produce significant economic losses for which there is a lack of control tools other than vaccines. Type I interferon has been successfully used for treating some chronic viral infections in humans. However, its application in salmonids depends on the proper design of a vehicle that allows its massive administration, ideally orally. In mammals, administration of recombinant probiotics capable of expressing cytokines has shown local and systemic therapeutic effects. In this work, we evaluate the use of Lactococcus lactis as a type I Interferon expression system in Atlantic salmon, and we analyze its ability to stimulate the antiviral immune response against IPNV, in vivo and in vitro . The interferon expressed in L. lactis , even though it was located mainly in the bacterial cytoplasm, was functional, stimulating Mx and PKR expression in CHSE-214 cells, and reducing the IPNV viral load in SHK-1 cells. In vivo , the oral administration of this L. lactis producer of Interferon I increases Mx and PKR expression, mainly in the spleen, and to a lesser extent, in the head kidney. The oral administration of this strain also reduces the IPNV viral load in Atlantic salmon specimens challenged with this pathogen. Our results show that oral administration of L. lactis producing Interferon I induces systemic effects in Atlantic salmon, allowing to stimulate the antiviral immune response. This probiotic could have effects against a wide variety of viruses that infect Atlantic salmon and also be effective in other salmonids due to the high identity among their type I interferons., Competing Interests: Authors MT and AS were employed by the company IctioBiotic SpA and ActivaQ S.A., respectively. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Muñoz, González-Lorca, Parra, Soto, Valdes, Sandino, Vargas, González and Tello.)

Infectious salmon anaemia virus (ISAV) is the cause of an important waterborne disease of farmed Atlantic salmon. Detection of virus in water samples may constitute an alternative method to sacrificing fish for surveillance of fish populations for the presence of ISA-virus. We aimed to evaluate different membrane filters and buffers for concentration and recovery of ISAV in seawater, prior to molecular detection. One litre each of artificial and natural seawater was spiked with ISAV, followed by concentration with different filters and subsequent elution with different buffers. The negatively charged MF hydrophilic membrane filter, combined with NucliSENS® lysis buffer, presented the highest ISAV recovery percentages with 12.5 ± 1.3% by RT-qPCR and 31.7 ± 10.7% by RT-ddPCR. For the positively charged 1 MDS Zeta Plus® Virosorb® membrane filter, combined with NucliSENS® lysis buffer, the ISAV recovery percentages were 3.4 ± 0.1% by RT-qPCR and 10.8 ± 14.2% by RT-ddPCR. The limits of quantification (LOQ) were estimated to be 2.2 x 103 ISAV copies/L of natural seawater for both RT-qPCR and RT-ddPCR. The ISAV concentration method was more efficient in natural seawater., Competing Interests: The authors have no competing interests.

Salmon Gill Poxvirus Disease (SGPVD) has emerged as a cause of acute mortality in Atlantic salmon ( Salmo salar L. ) presmolts in Norwegian aquaculture. The clinical phase of the disease is associated with apoptotic cell death in the gill epithelium causing acute respiratory distress, followed by proliferative changes in the regenerating gill in the period after the disease outbreak. In an experimental SGPV challenge trial published in 2020, acute disease was only seen in fish injected with hydrocortisone 24 h prior to infection. SGPV-mediated mortality in the hydrocortisone-injected group was associated with more extensive gill pathology and higher SGPV levels compared to the group infected with SGPV only. In this study based on the same trial, SGPV gene expression and the innate and adaptive antiviral immune response was monitored in gills and spleen in the presence and absence of hydrocortisone. Whereas most SGPV genes were induced from day 3 along with the interferon-regulated innate immune response in gills, the putative SGPV virulence genes of the B22R family were expressed already one day after SGPV exposure, indicating a potential role as early markers of SGPV infection. In gills of the hydrocortisone-injected fish infected with SGPV, MX expression was delayed until day 10, and then expression skyrocketed along with the viral peak, gill pathology and mortality occurring from day 14. A similar expression pattern was observed for Interferon gamma ( IFNγ ) and granzyme A ( GzmA ) in the gills, indicating a role of acute cytotoxic cell activity in SGPVD. Duplex in situ hybridization demonstrated effects of hydrocortisone on the number and localization of GzmA -containing cells, and colocalization with SGPV infected cells in the gill. SGPV was generally not detected in spleen, and gill infection did not induce any corresponding systemic immune activity in the absence of stress hormone injection. However, in fish injected with hydrocortisone, IFNγ and GzmA gene expression was induced in spleen in the days prior to acute mortality. These data indicate that suppressed mucosal immune response in the gills and the late triggered systemic immune response in the spleen following hormonal stress induction may be the key to the onset of clinical SGPVD., Competing Interests: ET was employed by Patogen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amundsen, Tartor, Andersen, Sveinsson, Thoen, Gjessing and Dahle.)

The microbial communities that live in symbiosis with the mucosal surfaces of animals provide the host with defense strategies against pathogens. These microbial communities are largely shaped by the environment and the host genetics. Triploid Atlantic salmon (Salmo salar) are being considered for aquaculture as they are reproductively sterile and thus cannot contaminate the natural gene pool. It has not been previously investigated how the microbiome of triploid salmon compares to that of their diploid counterparts. In this study, we compare the steady-state skin and gill microbiome of both diploid and triploid salmon, and determine the effects of salmonid alphavirus 3 experimental infection on their microbial composition. Our results show limited differences in the skin-associated microbiome between triploid and diploid salmon, irrespective of infection. In the gills, we observed a high incidence of the bacterial pathogen Candidatus Branchiomonas, with higher abundance in diploid compared to triploid control fish. Diploid salmon infected with SAV3 showed greater histopathological signs of epitheliocystis compared to controls, a phenomenon not observed in triploid fish. Our results indicate that ploidy can affect the alpha diversity of the gills but not the skin-associated microbial community. Importantly, during a natural outbreak of Branchiomonas sp. the gill microbiome of diploid Atlantic salmon became significantly more dominated by this pathogen than in triploid animals. Thus, our results suggest that ploidy may play a role on Atlantic salmon gill health and provide insights into co-infection with SAV3 and C. Branchiomonas in Atlantic salmon., Competing Interests: The authors have declared that no competing interests exist.

The Tasmanian salmon industry had remained relatively free of major viral diseases until the emergence of pilchard orthomyxovirus (POMV). Originally isolated from wild pilchards, POMV is of concern to the industry as it can cause high mortality in farmed salmon (Salmo salar). Field observations suggest the virus can spread from pen to pen and between farms, but evidence of passive transmission in sea water was unclear. Our aim was to establish whether direct contact between infected and naïve fish was required for transmission, and to examine viral infection dynamics. Atlantic salmon post-smolts were challenged with POMV by either direct exposure via cohabitation or indirect exposure via virus-contaminated sea water. POMV was transmissible in sea water and direct contact between fish was not required for infection. Head kidney and heart presented the highest viral loads in early stages of infection. POMV survivors presented low viral loads in most tissues, but these remained relatively high in gills. A consistent feature was the infiltration of viral-infected melanomacrophages in different tissues, suggesting an important role of these in the immune response to POMV. Understanding POMV transmission and host-pathogen interactions is key for the development of improved surveillance tools, transmission models and ultimately for disease prevention., (© 2020 John Wiley & Sons Ltd.)

Here we have studied the impact of lice ( Lepeophtheirus salmonis ) infestation of donor fish on the ability of isolated peripheral blood monocytes (PMBCs) to control the replication of salmonid alphavirus (SAV) ex vivo. PMBC were collected by Percoll gradients at eight and nine weeks post copepodid infestation of Atlantic salmon post smolt. Uninfested fish were controls. PBMCs were then infected ex vivo with SAV (subtype 3), and samples were collected for analysis at two, four, and six days post virus infection. Virus titer in the supernatant was assayed in CHH-1 cells, and in addition, the relative expression of the virus structural protein E2 and selected host antiviral genes, IRF9, ISG15, Mx, and IFIT5, were assayed using real-time PCR. Significantly higher virus replication was detected in cells collected from lice-infested fish compared to controls. Higher virus titer coincided with an inability to upregulate the expression of different immune genes, IFIT5, IRF9, and Mx. These findings point towards compromised ability of PMBCs from lice-infested fish to control virus replication, and, to our knowledge, is the first report showing the direct effect of lice infestation on the interplay between viruses and immune cells. There is a possible impact on the dynamic spread of viral diseases in the aquatic environment.

Infectious pancreatic necrosis virus (IPNV) infection has been a major problem in salmonid aquaculture. Marker-assisted selection of individuals with resistant genotype at the major IPN quantitative trait locus (IPN-QTL) has significantly reduced mortality in recent years. We have identified host miRNAs that respond to IPNV challenge in salmon fry that were either homozygous resistant (RR) or homozygous susceptible (SS) for the IPN-QTL. Small RNA-sequenced control samples were compared to samples collected at 1, 7, and 20 days post challenge (dpc). This revealed 72 differentially expressed miRNAs (DE miRNAs). Viral load (VL) was lower in RR vs. SS individuals at 7 and 20 dpc. However, analysis of miRNA expression changes revealed no differences between RR vs. SS individuals in controls, at 1 or 7 dpc, while 38 "high viral load responding" miRNAs (HVL-DE miRNAs) were identified at 20 dpc. Most of the HVL-DE miRNAs showed changes that were more pronounced in the high VL SS group than in the low VL RR group when compared to the controls. The absence of differences between QTL groups in controls, 1 and 7 dpc indicates that the QTL genotype does not affect miRNA expression in healthy fish or their first response to viral infections. The miRNA differences at 20 dpc were associated with the QTL genotype and could, possibly, contribute to differences in resistance/susceptibility at the later stage of infection. In silico target gene predictions revealed that 180 immune genes were putative targets, and enrichment analysis indicated that the miRNAs may regulate several major immune system pathways. Among the targets of HVL-DE miRNAs were IRF3, STAT4, NFKB2, MYD88, and IKKA. Interestingly, TNF-alpha paralogs were targeted by different DE miRNAs. Most DE miRNAs were from conserved miRNA families that respond to viral infections in teleost (e.g., miR-21, miR-146, miR-181, miR-192, miR-221, miR-462, miR-731, and miR-8159), while eight were species specific. The miRNAs showed dynamic temporal changes implying they would affect their target genes differently throughout disease progression. This shows that miRNAs are sensitive to VL and disease progression, and may act as fine-tuners of both immediate immune response activation and the later inflammatory processes., (Copyright © 2020 Woldemariam, Agafonov, Sindre, Høyheim, Houston, Robledo, Bron and Andreassen.)

B cell responses are a crucial part of the adaptive immune response to viral infection. Infection by salmonid alphavirus subtype 3 (SAV3) causes pancreas disease (PD) in Atlantic salmon ( Salmo salar ) and is a serious concern to the aquaculture industry. In this study, we have used intraperitoneal (IP) infection with SAV3 as a model to characterize local B cell responses in the peritoneal cavity (PerC) and systemic immune tissues (head kidney/spleen). Intraperitoneal administration of vaccines is common in Atlantic salmon and understanding more about the local PerC B cell response is fundamental. Intraperitoneal SAV3 infection clearly induced PerC B cell responses as assessed by increased frequency of IgM + B cells and total IgM secreting cells (ASC). These PerC responses were prolonged up to nine weeks post-infection and positively correlated to the anti-SAV3 E2 and to neutralizing antibody responses in serum. For the systemic immune sites, virus-induced changes in B cell responses were more modest or decreased compared to controls in the same period. Collectively, data reported herein indicated that PerC could serve as a peripheral immunological site by providing a niche for prolonged maintenance of the ASC response in Atlantic salmon., (Copyright © 2020 Jenberie, Peñaranda, Thim, Styrvold, Strandskog, Jørgensen and Jensen.)

Aquaculture has a long history in many parts of the world, but it is still young at an industrial scale. Marine fish farming in open nets of a single fish species at high densities compared to their wild compatriots opens a plethora of possible infections. Infectious salmon anaemia (ISA) is an example of disease that surfaced after large-scale farming of Atlantic salmon (Salmo salar) appeared. Here, a review of the molecular biology of the ISA virus (ISAV) with emphasis on its pathogenicity is presented. The avirulent HPR0 variant of ISAV has resisted propagation in cell cultures, which has restricted the ability to perform in vivo experiments with this variant. The transition from avirulent HPR0 to virulent HPRΔ has not been methodically studied under controlled experimental conditions, and the triggers of the transition from avirulent to virulent forms have not been mapped. Genetic segment reassortment, recombination and mutations are important mechanisms in ISAV evolution, and for the development of virulence. In the 25 years since the ISAV was identified, large amounts of sequence data have been collected for epidemiologic and transmission studies, however, the lack of good experimental models for HPR0 make the risk evaluation of the presence of this avirulent, ubiquitous variant uncertain. This review summarizes the current knowledge related to molecular biology and pathogenicity of this important aquatic orthomyxovirus., (© 2019 The Society for Applied Microbiology.)

Salmonid alphavirus infection results in pancreas disease causing severe economic losses for Atlantic salmon aquaculture. Knowledge about genes and pathways contributing to resistance is limited. A 54 K SNP panel was used to genotype 10 full-sibling families each consisting of ~ 110 offspring challenged with salmonid alphavirus subtype 3. Relative heart viral load was assessed at 4- and 10-weeks post-infection using quantitative PCR. A moderate genomic heritability of viral load at 4 weeks (0.15-0.21) and a high positive correlation with survival (0.91-0.98) were detected. Positions of QTL detected on chromosome 3 matched those for survival detected by other studies. The SNP of highest significance occurred in the 3' untranslated region of gig1, a fish-specific antiviral effector. Locus B of immunoglobulin heavy chain mapped to an area containing multiple SNPs with genome-wide association. Heart mRNA-seq comparing parr from families with high- versus low-genomic breeding value, and matching sample genotypes for SNPs, identified two eQTL for salmonid alphavirus load. Immune genes associated with trans-eQTL were numerous and spread throughout the genome. QTL regions contained several genes with known or predicted immune functions, some differentially expressed. The putative functional genes and variants identified could help improve marker-based selection for pancreas disease resistance.

Cardiomyopathy syndrome is a viral disease of Atlantic salmon, mostly affecting fish during the late stages of production, resulting in significant losses to the industry. It has been shown that resistance to this disease has a strong genetic component, with quantitative trait loci (QTL) on chromosomes 27 (Ssa27) and Ssa12 to explain most of the additive genetic variance. Here, by analysing animals from a different year-class and a different population, we further aimed to confirm and narrow down the locations of these QTL. The data support the existence of the two QTL and suggest that the causative mutation on Ssa27 is most likely within the 10-10.5 Mbp segment of this chromosome. This region contains a cluster of major histocompatibility complex class I (MHC I) genes with the most strongly associated marker mapped to one of these loci. On Ssa12, the data confirmed the previous finding that the location of the causative mutation is within the 61.3 to 61.7 Mbp region. This segment contains several immune-related genes, but of particular interest are genes related to MHC II. Together, these findings highlight the likely key role of MHC genes in Atlantic salmon following infection with Piscine myocarditis virus (PMCV) and their potential impact on influencing the trajectory of this disease.

The Isavirus is an orthomyxovirus with a genome composed of eight segments of negative single-strand RNA (-ssRNA). It has been proposed that the eight genomic segments of the Isavirus are organized as a ribonucleoprotein (RNP) complex called a minigenome, which contains all the viral RNA segments, a viral heterotrimeric polymerase and multiple copies of the viral nucleoprotein (NP). Here, we develop an Isavirus minigenome system and show the importance of the formation of active RNPs and the role of viral NP R189, R194, R302 and K325 residues in the NP RNA-binding domain in the context of RNPs. The results indicate it is possible to generate a minigenome in salmon cells, a composite ISAV RNPs with EGFP-based chimeric vRNA with heterotrimeric polymerase (PB1, PB2, PA) and NP protein using CMV-based auxiliary plasmids. It was also shown that NP R189, R194, R302 and K325 residues are important to generate viral mRNA from the constituted RNPs and a detectable reporter protein. This work is the first salmon cell-based minigenome assay for the Isavirus, which was evaluated by a bioinformatic and functional study of the NP protein in viral RNPs, which showed that correct NP-vRNA interaction is key to the functioning of RNPs., (© 2019 John Wiley & Sons Ltd.)

Pancreas disease caused by salmonid alphaviruses leads to severe losses in Atlantic salmon aquaculture. The aim of our study was to gain a better understanding of the biological differences between salmon with high and low genomic breeding values (H-gEBV and L-gEBV respectively) for pancreas disease resistance. Fish from H- and L-gEBV families were challenged by intraperitoneal injection of salmonid alphavirus or co-habitation with infected fish. Mortality was higher with co-habitation than injection, and for L- than H-gEBV. Heart for RNA-seq and histopathology was collected before challenge and at four- and ten-weeks post-challenge. Heart damage was less severe in injection-challenged H- than L-gEBV fish at week 4. Viral load was lower in H- than L-gEBV salmon after co-habitant challenge. Gene expression differences between H- and L-gEBV manifested before challenge, peaked at week 4, and moderated by week 10. At week 4, H-gEBV salmon showed lower expression of innate antiviral defence genes, stimulation of B- and T-cell immune function, and weaker stress responses. Retarded resolution of the disease explains the higher expression of immune genes in L-gEBV at week 10. Results suggest earlier mobilization of acquired immunity better protects H-gEBV salmon by accelerating clearance of the virus and resolution of the disease.

Cardiomyopathy syndrome is a severe, viral disease of Atlantic salmon that mostly affects farmed animals during their late production stage at sea. Caused by piscine myocarditis virus (PMCV), over the past few years outbreaks due to this disease have resulted in significant losses to the aquaculture industry. However, there is currently no vaccine that has proven effective against this virus. In this study, using a challenge model, we investigated the genetic variation for resistance to PMCV, by screening a large number of animals using a 55 K SNP array. In particular, we aimed to identify genetic markers that are tightly linked to higher disease resistance and can potentially be used in breeding programs. Using genomic information, we estimated a heritability of 0.51 ± 0.06, suggesting that resistance against this virus, to a great extent, is controlled by genetic factors. Through association analysis, we identified a significant quantitative trait locus (QTL) on chromosome 27, explaining approximately 57% of the total additive genetic variation. The region harboring this QTL contains various immune-related candidate genes, many of which have previously been shown to have a different expression profile between the naïve and infected animals. We also identified a suggestive association on chromosome 12, with the QTL linked markers located in 2 putatively immune-related genes. These results are of particular interest, as they can readily be implemented into breeding programs, can further assist in fine-mapping the causative mutations, and help in better understanding the biology of the disease and the immunological mechanisms underlying resistance against PMCV., (© The American Genetic Association. 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Piscine myocarditis virus (PCMV) is a double-stranded RNA virus structurally similar to the Totiviridae family. PCMV is the causative agent of cardiomyopathy syndrome (CMS), a severe cardiac disease that affects farmed Atlantic salmon (Salmo salar). A recent study characterized the host immune response in infected salmon through a transcriptome immune profiling, which confirmed a high regulation of immune and anti-viral genes throughout infection with PCMV. Previously we developed a novel model based on repeated non-lethal blood sampling, enabling the individual monitoring of salmonids during an infection. In the present work, we used this model to describe the host immune response in the blood cells of Atlantic salmon after intramuscular infection with PCMV-containing tissue homogenate over a 77-day period. At the final stage heart samples were also collected to verify the PCMV load, the pathological impact of infection and to compare the transcript profiles to blood. The expression level of a range of key immune genes was determined in the blood and heart samples by real-time PCR. Results indicated selected immune genes (mx, cd8α and γip) were up-regulated in the heart tissue of infected animals at the terminal time point, in comparison to the non-infected fish. When analyzing the blood samples over the course of infection, a significant n up-regulation of mx gene was also observed. The time and number of peaks in the kinetics of expression was different between individuals. The PCMV load and CMS pathology was verified by real-time PCR and histopathology, respectively. No pathogen and no pathology could be detected during the course of the experiment except at the terminal stage (viral load by qPCR and pathology by histology). This study emphasizes the value of non-lethal monitoring for evaluating the health status of fish at early stages of infection and in the absence of clinical signs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Piscine orthoreovirus (PRV-1) can cause heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon ( Salmo salar ). The virus targets erythrocytes in the acute peak phase, followed by cardiomyocytes, before the infection subsides into persistence. The persistent phase is characterized by high level of viral RNA, but low level of viral protein. The origin and nature of persistent PRV-1 are not clear. Here, we analyzed for viral persistence and activity in various tissues and cell types in experimentally infected Atlantic salmon. Plasma contained PRV-1 genomic dsRNA throughout an 18-week long infection trial, indicating that viral particles are continuously produced and released. The highest level of PRV-1 RNA in the persistent phase was found in kidney. The level of PRV-1 ssRNA transcripts in kidney was significantly higher than that of blood cells in the persistent phase. In-situ hybridization assays confirmed that PRV-1 RNA was present in erythroid progenitor cells, erythrocytes, macrophages, melano-macrophages and in some additional un-characterized cells in kidney. These results show that PRV-1 establishes a productive, persistent infection in Atlantic salmon and that erythrocyte progenitor cells are PRV target cells.

Pancreas disease (PD), caused by several subtypes of salmonid alphavirus (SAV), is associated with significant economic losses in European salmonid aquaculture. In this retrospective cohort study, we investigate the impact of PD caused by SAV subtype 2 (SAV2) on growth, feed conversion, and mortality in farmed Atlantic salmon (Salmo salar L.). The study was based on harvest data from a large salmon farming company operating in the SAV2 endemic area of Norway. Mixed-effect regression analyses showed a severe impact on both growth and feed conversion when PD appeared late in the production cycle. In a scenario with fixed slaughter time the estimated impact corresponded to a growth reduction of 0.7 kg and 0.07 points increase in feed conversion ratio. No effect on mortality was observed in this data set. In conclusion, the most important consequences of PD caused by SAV2 infection is reduced growth and feed conversion in large Atlantic salmon. The lack of effect on mortality in this study may be due to other factors overshadowing the impact of PD., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Losses due to cardiomyopathy syndrome (CMS) keep increasing in salmon-producing countries in the North-Atlantic. Recently, Piscine myocarditis virus (PMCV) has been detected in post-smolts shortly after sea-transfer, indicating a possible carry-over from the hatcheries. In addition, there are reports of prevalences of PMCV as high as 70%-90% in certain groups of broodfish, and a recent outbreak of CMS in the Faroe Islands has been linked to the importation of eggs from a CMS-endemic area. Thus, there is a need to investigate whether PMCV can be transmitted vertically from infected broodstock to their progeny. In the present study, samples from eggs, larvae, fingerlings and presmolt originating from PMCV-positive broodstock from two commercial Atlantic salmon producers were tested for PMCV. The prevalence of PMCV in the broodstock was 98% in the hearts, 69% in the roe and 59% in the milt. Piscine myocarditis virus was detected in all stages of the progeny until and including the 40 g stage. Piscine myocarditis virus was also detected in presmolt sampled for tissue tropism. This provides farmers with several options for minimizing the risk of transfer of PMCV from broodstock to progeny, including screening of broodstock and aiming to use only those that are negative for PMCV or have low levels of virus., (© 2019 The Authors. Journal of Fish Diseases Published by John Wiley & Sons Ltd.)

Melanized focal changes in skeletal muscle of farmed Atlantic salmon (Salmo salar) are a major quality problem. The aetiology is unknown, but infection with Piscine orthoreovirus (PRV) has been associated with the condition. Here, we addressed the pathogenesis of red and melanized focal changes and their association with PRV. First, a population of farmed fish (PRV-negative prior to sea transfer) was sequentially investigated throughout the seawater period. The fish were autopsied and tested for PRV infection. Muscular changes were described by macroscopy and histology, and a classification system was established. Second, in an experimental infection trial, PRV was injected intramuscularly to induce changes. The farmed fish was gradually infected with PRV. Red focal changes occurred throughout the observation period with a low prevalence regardless of PRV status. Melanized changes were highly diverse and their prevalence increased during the trial. Changes of low macroscopic grade and histological category were more prevalent in PRV-negative fish. Diffuse granulomatous melanized changes only occurred after PRV infection. No muscular changes were observed in the experimentally challenged fish. Our studies do not indicate that PRV infection causes red focal changes, but seems important in the development of granulomatous melanized changes., (© 2019 The Authors. Journal of Fish Diseases Published by John Wiley & Sons Ltd.)

Heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon ( Salmo salar) was first diagnosed in Norway in 1999. The disease is caused by Piscine orthoreovirus -1 (PRV-1). The virus is prevalent in farmed Atlantic salmon, but not always associated with disease. Phylogeny and sequence analyses of 31 PRV-1 genomes collected over a 30-year period from fish with or without HSMI, grouped the viral sequences into two main monophylogenetic clusters, one associated with HSMI and the other with low virulent PRV-1 isolates. A PRV-1 strain from Norway sampled in 1988, a decade before the emergence of HSMI, grouped with the low virulent HSMI cluster. The two distinct monophylogenetic clusters were particularly evident for segments S1 and M2. Only a limited number of amino acids were unique to the association with HSMI, and they all located to S1 and M2 encoded proteins. The observed co-evolution of the S1-M2 pair coincided in time with the emergence of HSMI in Norway, and may have evolved through accumulation of mutations and/or segment reassortment. Sequences of S1-M2 suggest selection of the HSMI associated pair, and that this segment pair has remained almost unchanged in Norwegian salmon aquaculture since 1997. PRV-1 strains from the North American Pacific Coast and Faroe Islands have not undergone this evolution, and are more closely related to the PRV-1 precursor strains not associated with clinical HSMI.

Type I interferons (IFNs) have been shown to play an important role in shaping adaptive immune responses in addition to their antiviral properties in immune cells. To gain insight into the impact of IFN-I-induced pathways involved in early adaptive immune responses, i.e., antigen-presenting pathways, in an Atlantic salmon-derived ( Salmo salar L.) macrophage cell line (TO-cells), we used a comparative de novo transcriptome analysis where cells were treated with IFN-I or kept untreated and concurrently infected with salmonid alphavirus subtype 3 (SAV3). We found that concurrent treatment of TO-cells with IFN-I and SAV3 infection (SAV3/IFN + ) significantly enriched the major histocompatibility complex class I (MHC-I) pathway unlike the non-IFN-I treated TO-cells (SAV3/IFN - ) that had lower expression levels of MHC-I pathway-related genes. Genes such as the proteasomal activator ( PA28 ) and β-2 microglobulin ( β2M ) were only differentially expressed in the SAV3/IFN + cells and not in the SAV3/IFN - cells. MHC-I pathway genes like heat shock protein 90 ( Hsp90 ), transporter of antigen associated proteins ( TAPs ) and tapasin had higher expression levels in the SAV3/IFN + cells than in the SAV3/IFN - cells. There were no MHC-II pathway-related genes upregulated in SAV3/IFN + -treated cells, and cathepsin S linked to the degradation of endosomal antigens in the MHC-II pathway was downregulated in the SAV3/IFN - cells. Overall, our findings show that concurrent IFN-I treatment of TO-cells and SAV3 infection enriched gene expression linked to the MHC-I antigen presentation pathway. Data presented indicate a role of type I IFNs in strengthening antigen processing and presentation that may facilitate activation particularly of CD8+ T-cell responses following SAV3 infection, while SAV3 infection alone downplayed MHC-II pathways.

The infectious salmon anaemia virus (ISAV) is an important pathogen on farmed salmon in Europe. The virus occurs as low- and high virulent variants where the former seem to be a continuous source of new high virulent ISAV. The latter are controlled in Norway by stamping out infected populations while the former are spreading uncontrolled among farmed salmon. Evidence of vertical transmission has been presented, but there is still an ongoing discussion of the importance of circulation of ISAV via salmon brood fish. The only known wild reservoirs are in trout (Salmo trutta) and salmon (Salmo salar). This study provides the first ISAV sequences from wild salmonids in Norway and evaluates the importance of this reservoir with respect to outbreaks of ISA among farmed salmon. Phylogenetic analyses of the surface protein hemagglutinin-esterase gene from nearly all available ISAV from Norway, Faeroe Islands, Scotland, Chile and wild salmonids in Norway show that they group into four major clades. Including virulent variants in the analysis show that they belong in the same four clades supporting the hypothesis that there is a high frequency of transition from low to high virulent variants in farmed populations of salmon. There is little support for a hypothesis suggesting that the wild salmonids feed the virus into farmed populations. This study give support to earlier studies that have documented local horizontal transmission of high virulent ISAV, but the importance of transition from low- to high virulent variants has been underestimated. Evidence of vertical transmission and long distance spreading of ISAV via movement of embryos and smolt is presented. We recommend that the industry focus on removing the low virulent ISAV from the brood fish and that ISAV-free brood fish salmon are kept in closed containment systems (CCS)., Competing Interests: M. Karlsen is employed by Pharmaq AS, but the company had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Pharmaq AS provided support in the form of salaries for the author, M. Karlsen, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the author contributions section. Our cooperation with M. Karlsen does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Piscine orthoreovirus (PRV) is an emergent virus in salmon aquaculture belonging to the family Reoviridae. PRV is associated with a growing list of pathological conditions including heart and skeletal inflammation (HSMI) of farmed Atlantic salmon. Despite widespread PRV infection in commercially farmed Atlantic salmon, information on PRV prevalence and on the genetic sequence variation of PRV in Atlantic salmon on the north Pacific Coast is limited., Methods: Feral Atlantic salmon caught in Washington State and British Columbia following a large containment failure at a farm in northern Puget Sound were sampled. Fish tissues were tested for PRV by RT-qPCR assay for segment L1 and conventional RT-PCR for PRV segment S1. The PCR products were sequenced and their relationship to PRV strains in GenBank was determined using phylogenetic analysis and nucleotide and amino acid homology comparisons., Results: Following the escape of 253,000 Atlantic salmon from a salmon farm in Washington State, USA, 72/73 tissue samples from 27 Atlantic salmon captured shortly after the escape tested PRV-positive. We estimate PRV-prevalence in the source farm population at 95% or greater. The PRV found in the fish was identified as PRV sub-genotype Ia and very similar to PRV from farmed Atlantic salmon in Iceland. This correlates with the source of the fish in the farm. Eggs of infected fish were positive for PRV indicating the possibility of vertical transfer and spread with fish egg transports., Conclusions: PRV prevalence was close to 100% in farmed Atlantic salmon that were caught in Washington State and British Columbia following a large containment failure at a farm in northern Puget Sound. The PRV strains present in the escaped Atlantic salmon were very similar to the PRV strain reported in farmed Atlantic salmon from the source hatchery in Iceland that was used to stock commercial aquaculture sites in Washington State. This study emphasizes the need to screen Atlantic salmon broodstock for PRV, particularly where used to supply eggs to the global Atlantic salmon farming industry thereby improving our understanding of PRV epidemiology.

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., (© 2019 The Authors. Journal of Fish Diseases Published by John Wiley & Sons Ltd.)

Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon and sometimes associated with disease - most notably, Heart and Skeletal Muscle Inflammation (HSMI). However, PRV is also widespread in non-diseased fish, particularly in Pacific Canada, where few cases of severe heart inflammation have been documented. To better understand the mechanisms behind PRV-associated disease, this study investigated the infection dynamics of PRV from Pacific Canada and the potential for experimental passage of putatively associated heart inflammation in Pacific-adapted Mowi-McConnell Atlantic salmon. Regardless of the PRV source (fish with or without HSMI-like heart inflammation), infections led to high-load viremia that induced only minor focal heart inflammation without significant transcriptional induction of inflammatory cytokines. Repeated screening of PRV dsRNA/ssRNA along with histopathology and gene expression analysis of host blood and heart tissues identified three distinct phases of infection: (1) early systemic dissemination and replication without host recognition; (2) peak replication, erythrocyte inclusion body formation and load-dependent host recognition; (3) long-term, high-load viral persistence with limited replication or host recognition sometimes accompanied by minor heart inflammation. These findings contrast previous challenge trials with PRV from Norway that induced severe heart inflammation and indicate that strain and/or host specific factors are necessary to initiate PRV-associated disease.

Infectious salmon anemia virus (ISAV) is an Orthomyxovirus challenging salmon production, with a particular impact in Chile. During 2007-2010 a devastating and of unexpected consequences epizootic event almost destroyed a blooming industry in the country. The event was caused by an aggressive variant with a distinctive deletion in Segment 6, one of the eight genomic segments of the virus. After the outburst, although the infective viral variant seemed to have disappeared, a non-infective variant, not previously reported, was discovered and is characterized by a complete, non-deleted coding segment 6, which has prevailed in the fish population until now. This variant, known as HPR0, appears to be the ancestor strain of ISAV from which novel infective variants are generated. Additional variations in segment 5 have also been associated with the virulence observed in the field, an analysis of the differences in these two protein coding segments has been performed. It appears to us that a combinatorial effect exists between the features displayed by segments 5 and 6 which modulate the intensity of viral outbursts. As a result, a theoretical integrative model is presented which explains the different degree of virulence observed in the field based only on molecular data, this could help estimating the intensity of damage a given variant might exert over a productive farm., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The role of parasitic sea lice (Siphonostomatoida; Caligidae), especially Lepeophtheirus salmonis, in the epidemiology of Infectious Salmon Anemia Virus (ISAv) has long been suspected. The epidemiological studies conducted during the 1998 major Infectious Salmon Anaemia (ISA) outbreak in Scotland demonstrated a strong correlation between sea lice presence and ISAv positive sites or subsequent clinical outbreaks of ISA. The question posed from this observation was "do sea lice infestations on Atlantic salmon make them more susceptible to viral infections?" This study investigated the role that sea lice infestations have on the severity of ISAv infections and disease mortality in experimental populations of farmed Atlantic salmon (Salmo salar). A series of experiments was carried out that investigated the potential of sea lice to modify the outcome of an ISAv infection. Experimental populations of Atlantic salmon were established that had: no lice and no ISAv, a single infection with either ISAv or lice and a co-infection with lice then ISAV. The results were quite clear, the process of infestation by the parasite prior to ISAv exposure significantly increased the mortality and death rates of Atlantic salmon, when compared to uninfected controls and ISAv infected groups only. This was consistent over two source strains of Atlantic salmon (Pennobscot and Saint John River), but the severity and timing was altered. Immunological responses were also consistent in that pro-inflammatory genes were induced in lice only and co-infected fish, whereas the anti-viral response, Mx, MH class I β, Galectin 9 and TRIM 16, 25 genes were down-regulated by lice infection prior to and shortly after co-infection with ISAv. It is concluded that the sea lice settlement on Atlantic salmon and the parasite's subsequent manipulation of the host's immune system, which increases parasite settlement success, also increased susceptibility to ISAv., Competing Interests: The authors have declared that no competing interests exist.

Salmonid red blood cells are the main target cells for Piscine orthoreovirus (PRV). Three genotypes of PRV (PRV-1,2,3) infect Atlantic salmon ( Salmo salar ), Chinook salmon ( Onchorhynchus tshawytscha ), Coho salmon ( Oncorhynchus kisutch ), rainbow trout ( Onchorhynchus mykiss ) and brown trout (Salmo trutta), and can cause diseases like heart and skeletal muscle inflammation (HSMI), jaundice syndrome, erythrocyte inclusion body syndrome (EIBS) and proliferative darkening syndrome (PDS). Purified PRV administrated to fish has proven the causality for HSMI and EIBS. During the early peak phase of infection, salmonid erythrocytes are the main virus-replicating cells. In this initial phase, cytoplasmic inclusions called "virus factories" can be observed in the erythrocytes, and are the primary sites for the formation of new virus particles. The PRV-infected erythrocytes in Atlantic salmon mount a strong long-lasting innate antiviral response lasting for many weeks after the onset of infection. The antiviral response of Atlantic salmon erythrocytes involves upregulation of potential inhibitors of translation. In accordance with this, PRV-1 protein production in erythrocytes halts while virus RNA can persist for months. Furthermore, PRV infection in Coho salmon and rainbow trout are associated with anemia, and in Atlantic salmon lower hemoglobin levels are observed. Here we summarize and discuss the recently published findings on PRV infection, replication and effects on salmonid erythrocytes, and discuss how PRV can be a useful tool for the study of innate immune responses in erythrocytes, and help reveal novel immune functions of the red blood cells in fish.

Piscine orthoreoviruses (PRVs) are emerging pathogens causing circulatory disorders in salmonids. PRV-1 is the etiological cause of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar), characterized by epicarditis, inflammation and necrosis of the myocardium, myositis and necrosis of red skeletal muscle. In 2017, two German breeding farms for Atlantic salmon and rainbow trout (Oncorhynchus mykiss) experienced disease outbreaks with mortalities of 10% and 20% respectively. The main clinical signs were exhaustion and lethargic behaviour. During examinations, PRV-1 in salmon and PRV-3 in trout were detected for the first time in Germany. Further analyses also indicated the presence of Aeromonas salmonicida in internal tissues of both species. While PRV-1 could be putatively linked with the disease in Atlantic salmon, most of the rainbow trout suffered from an infection with A. salmonicida and not with PRV-3. Interestingly, the sequence analysis suggests that the German PRV-3 isolate is more similar to a Chilean PRV-3 isolate from Coho salmon (Oncorhynchus kisutch) than to PRV-3 from rainbow trout from Norway. This indicates a wide geographic distribution of this virus or dispersal by global trade. These findings indicate that infections with PRVs should be considered when investigating disease outbreaks in salmonids., (© 2018 Blackwell Verlag GmbH.)

A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land-based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real-time RT-PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re-isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%-50% of the cohabiting lumpfish survived after 4 weeks. 80%-92% of the Atlantic salmon survived, but no viral RNA was detected by real-time RT-PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters., (© 2018 The Authors. Journal of Fish Diseases Published by John Wiley & Sons Ltd.)

Salmonid alphavirus (SAV) causes pancreas disease (PD) in farmed Atlantic salmon (Salmo salar L.), and exocrine pancreas tissue is a primary target of the virus. Digestive enzymes secreted by the exocrine pancreas break down macromolecules in feed into smaller molecules that can be absorbed. The effect of SAV infection on digestion has been poorly studied. In this study, longitudinal observations of PD outbreaks caused by SAV subtype 2 (SAV2) in Atlantic salmon at two commercial sea sites were performed. The development of PD was assessed by measurement of SAV2 RNA load and evaluation of histopathological lesions typical of PD. Reduced digestion of both protein and fat co-varied with the severity of PD lesions and viral load. Also, the study found that during a PD outbreak, the pen population comprise several subpopulations, with different likelihoods of being sampled. The body length of sampled fish deviated from the expected increase or steady state over time, and the infection status in sampled fish deviated from the expected course of infection in the population. Both conditions indicate that disease status of the individual fish influenced the likelihood of being sampled, which may cause sampling bias in population studies., (© 2018 John Wiley & Sons Ltd.)

Infectious salmon anemia virus (ISAV) is the causative agent of infectious salmon anemia (ISA), a relatively novel disease primarily affecting farmed salmon species, primarily in Salmo salar specimens, causing severe outbreaks in most producer countries. Although ISAV has been extensively studied at the molecular level, not much is known about the host/cell interaction at the small RNA level. MicroRNAs (miRNAs) are small, non-coding RNA that regulate mRNA expression at the post-transcriptional level. In recent years, the putative role of these molecules in host-pathogen interactions has drawn particular attention because of their pivotal involvement as regulatory elements in a number of eukaryotic organisms. Given the importance of the salmon industry in Chile, a deep understanding of the interaction between ISAV and its hosts is of importance. In the present work, we studied the kinetic expression of selected miRNAs during ISAV infection, both in vitro and in vivo. Based on initial experimental data derived from a small RNA-Seq analysis, a group of miRNAs that were differentially expressed in infected cells were selected for analysis. As a result, two miRNAs, miR-462a-5p and miR-125 b-5p, showed increased and decreased expression, respectively, during ISAV infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)