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.

Infectious salmon anaemia virus (ISAV) can cause severe systemic infection in Atlantic salmon (Salmo salar L.), and a timely diagnosis is critical. Conventional real-time reverse transcription PCR (RT-qPCR) assays target unspliced RNA from either ISAV segment 7 or 8 and provide data on viral load. Here, we evaluate a TaqMan one-step RT-qPCR assay that detects explicitly a spliced messenger RNA (mRNA) of ISAV segment 7, thus providing evidence of active viral transcription. Assay performance was comparable with existing unspliced segment 7 and segment 8 assays. PCR efficiency as evaluated from dilutions of a synthetic DNA fragment was 98% (R² = 1.00). The assay also performed well on clinical heart samples with PCR efficiency of 108% (R² = 1.00). Finally, evaluation on kidney samples from experimental infection revealed higher levels of active transcription for high-virulent compared to low-virulent ISAV. At early, peak, and late infection, mean ratios of spliced to unspliced segment 7 RNA were 3.0 (±0.7), 1.7 (±0.3), and 1.5% (±0.1) for the low virulent and 9.4 (±2.2), 4.7 (±0.8), and 6.2% (±0.1) for the high virulent isolate, respectively. By detection and quantification of active ISAV transcription, this assay may provide a more detailed understanding of ISAV infection dynamics. DATA AVAILABILITY STATEMENT: All relevant data are within the paper and its supplementary materials (Appendix A).

Piscine myocarditis virus (PMCV) is the causative agent of cardiomyopathy syndrome (CMS), a significant disease in farmed Atlantic salmon (Salmo salar L.). Although an increasing number of CMS outbreaks have been recorded in the Faroe Islands since the reemergence of CMS in 2013, overall PMCV genetic diversity, transmission pathways and evolutionary trajectories remain elusive. Here, we present a fast amplicon-based whole-genome sequencing method of PMCV directly from field samples and disclose 48 novel genomes, adding to the single genome currently available. Phylogenetic analysis revealed that genomes with a broad spatiotemporal representation of Faroese farmed salmon formed a homogenous monophyletic cluster compared to Norwegian and Irish PMCV genomes. Homogeneity of the Faroese genomes was substantiated with principal component analyses, where no spatiotemporal clustering of genotypes was found, nor any clustering based on roe or smolt origin. One genome from a returning wild salmon differed considerably from all the rest and formed an outgroup. All three ORFs exhibited signs of purifying selection, although ORF3 displayed a comparatively lower degree of selective constraint. Furthermore, no virulence-determining amino acid substitutions were identified in the Faroese genomes as no association was found between CMS cases and specific amino acid substitutions or motifs. Our data suggest that PMCV was introduced into the Faroe Islands from Norway, where brood fish is known to be infected. However, despite a steadily increasing import of Norwegian roe, our results show no continuous reintroduction of persisting PMCV strains to Faroese farmed salmon.