Background: Cardiomyopathy syndrome (CMS) is a severe disease affecting large farmed Atlantic salmon. Mortality often appears without prior clinical signs, typically shortly prior to slaughter. We recently reported the finding and the complete genomic sequence of a novel piscine reovirus (PRV), which is associated with another cardiac disease in Atlantic salmon; heart and skeletal muscle inflammation (HSMI). In the present work we have studied whether PRV or other infectious agents may be involved in the etiology of CMS. Results: Using high throughput sequencing on heart samples from natural outbreaks of CMS and from fish experimentally challenged with material from fish diagnosed with CMS a high number of sequence reads identical to the PRV genome were identified. In addition, a sequence contig from a novel totivirus could also be constructed. Using RT-qPCR, levels of PRV in tissue samples were quantified and the totivirus was detected in all samples tested from CMS fish but not in controls. In situ hybridization supported this pattern indicating a possible association between CMS and the novel piscine totivirus. Conclusions: Although causality for CMS in Atlantic salmon could not be proven for either of the two viruses, our results are compatible with a hypothesis where, in the experimental challenge studied, PRV behaves as an opportunist whereas the totivirus might be more directly linked with the development of CMS.

Most commercial vaccines offered to the aquaculture industry include inactivated antigens (Ag) formulated in oil adjuvants. Safety concerns are related to the use of oil adjuvants in multivalent vaccines for fish, since adverse side effects (e.g., adhesions) can appear. Therefore, there is a request for vaccine formulations for which protection will be maintained or improved, while the risk of side effects is reduced. Here, by using an inactivated salmonid alphavirus (SAV) as the test Ag, the combined use of two Toll-like receptor (TLR) ligand adjuvants, CpG oligonucleotides (ODNs) and poly I:C, as well as a genetic adjuvant consisting of a DNA plasmid vector expressing the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) was explored. VHSV-G DNA vaccine was intramuscularly injected in combination with intraperitoneal injection of either SAV Ag alone or combined with the oil adjuvant, Montanide ISA763, or the CpG/polyI:C combo. Adjuvant formulations were evaluated for their ability to boost immune responses and induce protection against SAV in Atlantic salmon, following cohabitation challenge. It was observed that CpG/polyI:C-based formulations generated the highest neutralizing antibody titres (nAbs) before challenge, which endured post challenge. nAb responses for VHSV G-DNA- and oil-adjuvanted formulations were marginal compared to the CpG/poly I:C treatment. Interestingly, heat-inactivated sera showed reduced nAb titres compared to their non-heated counterparts, which suggests a role of complement-mediated neutralization against SAV. Consistently elevated levels of innate antiviral immune genes in the CpG/polyI:C injected groups suggested a role of IFN-mediated responses. Co-delivery of the VHSV-G DNA construct with either CpG/polyI:C or oil-adjuvanted SAV vaccine generated higher CD4 responses in head kidney at 48 h compared to injection of this vector or SAV Ag alone. The results demonstrate that a combination of pattern recognizing receptor (PRR) ligands, such as CpG/polyI:C, increases both adaptive and innate responses and represents a promising adjuvant strategy for enhancing the protection of future viral vaccines.

Atlantic halibut Hippoglossus hippoglossus, age 8 mo and weighing 20 g, were challenged by either intraperitoneal injection (i.p.) or by bath exposure using nodavirus isolated from Atlantic halibut. Fish were sampled at intervals over a 41 d period, starting on Day 5 post-challenge. Although no clinical disease or mortality was recorded, the data show that nodavirus did successfully propagate in i.p.-challenged fish. Using conventional end-point reverse transcription (RT)-PCR, nodavirus was detected in the kidney of all examined i.p.-challenged fish, and further in the head, heart, liver and posterior intestine of most of these individuals. Quantitative real-time RT-PCR revealed that the amount of virus in head samples from the i.p.-challenged group increased during the experiment. The presence of nodavirus in nervous tissue of i.p.-challenged fish was detected by immunohistochemistry from Day 13 post-challenge. In the retina, virus positive cells were found adjacent to the circumferential germinal zone at the ciliary margin towards the iris. In the brain, a few positive cells were detected in the tectum opticum. An ELISA was developed to detect anti-nodavirus activity in plasma. The method included an optimized coating procedure, which allowed the use of non-purified nodavirus as the coating antigen in a simple indirect ELISA. An anti-nodavirus antibody response was detected from Day 19 post-challenge in i.p.-challenged fish, while a response was not detected in the bath-challenged or control fish. This experiment demonstrates a subclinical nodavirus infection in Atlantic halibut at a post-juvenile stage induced by i.p. injection of virus.

Piscine orthoreovirus (PRV) is a ubiquitous virus in Norwegian salmon farms associated with the disease heart and skeletal muscle inflammation (HSMI). Experimental challenge has shown that the virus replicates in circulating red blood cells of Atlantic salmon prior to infecting heart myocytes. The infection route from water to blood is however still unknown. The related mammalian orthoreovirus primarily infects the lungs and gastrointestinal (GI) tract and is proposed to spread mainly through the faecal–oral route. To investigate the role of the salmonid GI tract in PRV-infection, oral and anal administration of virus was compared to intraperitoneal (i.p.) injection. When administered anally, PRV was transferred to blood 4 days post challenge (dpc) and levels peaked at 42 dpc, similar to i.p. injected fish. PRV was detected in heart and faeces with corresponding kinetics, and inflammatory heart lesions consistent with HSMI were observed from 49 dpc. The orally intubated group showed slower virus kinetics in both blood and heart, and no signs of HSMI. Compared to the oral and i.p. administration routes, leakage of virus inoculate by anal intubation was minor and challenge was restricted to the mid- and distal intestine. These findings show that anal intubation is an efficacious method for PRV delivery to the GI tract and demonstrates that PRV can establish infection through the intestine with the potential for transmission via faeces.