Viral infections are still a major concern for the aquaculture industry. For salmonid fish, even though breeding strategies and vaccine development have reduced disease outbreaks, viral diseases remain among the main challenges having a negative impact on the welfare of fish and causing massive economic losses for the industry. The main entry port for viruses into the fish is through mucosal surfaces including that of the gastrointestinal tract. The contradictory functions of this surface, both creating a barrier towards the external environment and at the same time being responsible for the uptake of nutrients and ion/water regulation make it particularly vulnerable. The connection between dietary components and viral infections in fish has been poorly investigated and until now, a fish intestinal in vitro model to investigate virus-host interactions has been lacking. Here, we established the permissiveness of the rainbow trout intestinal cell line RTgutGC towards the important salmonid viruses-infectious pancreatic necrosis virus (IPNV), salmonid alphavirus (subtype 3, SAV3) and infectious salmon anemia virus (ISAV)-and explored the infection mechanisms of the three different viruses in these cells at different virus to cell ratios. Cytopathic effect (CPE), virus replication in the RTgutGC cells, antiviral cell responses and viral effects on the barrier permeability of polarized cells were investigated. We found that all virus species infected and replicated in RTgutGC cells, although with different replication kinetics and ability to induce CPE and host responses. The onset and progression of CPE was more rapid at high multiplicity of infection (MOI) for IPNV and SAV3 while the opposite was true of ISAV. A positive correlation between the MOI used and the induction of antiviral responses was observed for IPNV while a negative correlation was detected for SAV3. Viral infections compromised barrier integrity at early time points prior to observations of CPE microscopically. Further, the replication of IPNV and ISAV had a more pronounced effect on barrier function than SAV3. The in vitro infection model established herein can thus provide a novel tool to generate knowledge about the infection pathways and mechanisms used to surpass the intestinal epithelium in salmonid fish, and to study how a virus can potentially compromise gut epithelial barrier functions.

In order to obtain an insight into genomic changes and associated evolution and adaptation of Infectious Pancreatic Necrosis Virus (IPNV), the complete coding genomes of 57 IPNV isolates collected from Scottish aquafarms from 1982 to 2014 were sequenced and analysed. Phylogenetic analysis of the sequenced IPNV strains showed separate clustering of genogroups I, II, III and V. IPNV isolates with genetic reassortment of segment A/B of genogroup III/II were determined. About 59 % of the IPNV isolates belonged to the persistent type and 32 % to the low-virulent type, and only one highly pathogenic strain (1.79 %) was identified. Codon adaptation index calculations indicated that the IPNV major capsid protein VP2 has adapted to its salmonid host. Under-representation of CpG dinucleotides in the IPNV genome to minimize detection by the innate immunity receptors, and observed positive selection in the virulence determination sites of VP2 embedded in the variable region of the main antigenic region, suggest an immune escape mechanism driving virulence evolution. The prevalence of mostly persistent genotypes, together with the assumption of adaptation and immune escape, indicates that IPNV is evolving with the host.

IPNV is a salmonid birnavirus that possesses the ability to establish asymptomatic persistent infections in a number of valuable fish species. The presence of IPNV may interfere with subsequent infection by other viruses. In the present study we show that an IPNV-carrier cell line (EPCIPNV) can induce an antiviral state in fresh EPC by co-cultivating both cell types in three different ways: a \"droplet\" culture system, a plastic chamber setup, and a transmembrane (Transwell®) system. All three cell co-culture methods were proven useful to study donor/target cell interaction. Naïve EPC cells grown in contact with EPCIPNV cells develop resistance to VHSV superinfection. The transmembrane system seems best suited to examine gene expression in donor and target cells separately. Our findings point to the conclusion that one or more soluble factors produced by the IPNV carrier culture induce the innate immune response within the target cells. This antiviral response is associated to the up-regulation of interferon (ifn) and mx gene expression in target EPC cells. To our knowledge this is the first article describing co-culture systems to study the interplay between virus-carrier cells and naive cells in fish.