We have reported the toxic effect of an ORF3 encoded protein of PMCV upon plasmid transfection and protein expression. We have also cloned the gene into a "carrier virus" or hybrid virus, VHSV, where we showed that the hybrid virus becomes virulent ("toxic") for cells in culture. Since then, we have found that the ORF3 protein undergoes a degradation process to become virulent and this also happens for atoxic variants of the protein. Some variants are almost non-toxic to cells when expressed in vitro. An "attenuated" variant of the ORF3 protein has been cloned into the VHSV genome to investigate potential loss of or reversion to low virulence when mutated variants of ORF3 are used. In vivo studies have been carried out where Atlantic salmon have been infected with PMCV-positive material. Examination of heart samples 6, 8 and 10 weeks after infection shows typical changes, and studies of the regeneration processes seem to indicate that PMCV "turns off" regenerative properties of the cardiomyocytes and downregulates large parts of the immune response. NGS analysis of heart samples from various times after infection has identified differential regulation of genes that are strongly associated with susceptibility/resistance to infection with PMCV, and it is particularly MHC1-related genes that can be linked to susceptibility or resistance to the disease, which are also differentially regulated by an infection. Initial studies of local inflammatory responses to expression of ORF3 protein in muscles of Atlantic salmon, and studies of inflammatory responses to local expression of ORF3 protein, have also been carried out.
Better understanding of cardiac responses to PMCV infection.
Developed tools to be used for assessment of antibody responses in salmon post infection and vaccination (pseudovirus).
Documented responses to ORF3 protein expression in situ which can have impact on infection and methods of prevention.
Piscine myocarditis virus (PMCV) is the first member of Totivirdae that infects a vertebrate host, causing cardiomyopathy syndrome (CMS). The salmon heart has a good ability to regenerate from virus-induced damage after HSMI and PD disease, but for CMS there are few signs of regeneration and scarring and ruptur is the ultimate consequence. The question is why a PMCV infection a 'kiss of death' while PRV and SAV infections are not? If the heart lesions causing HSMI and PD are of a character and degree that enables regeneration, why is regeneration impaired or blocked in CMS affected hearts? Our approach to this is to study the mechanisms of cardiomyocyte regeneration or inhibition of these after infection of the three viruses. We hypothesize that PMCV infection results in production of a protein toxic to the infected myocytes and this protein remains in the cardiomyocytes as aggregates. The damage caused to the cardiomyocytes is so severe that regeneration is impossible while for SAV (PD) and PRV (HSMI) infection damage is reversible and regeneration occurs. Better knowledge of underlying mechanisms will facilitate better intervention and optimisation of management based routines, i.e. risk management. As part of this we will also explore the potential of non-invasive methods for early diagnosis of viral myocarditis from sequential sampling of serum from salmon experimentally infected with SAV, PRV or PMCV. Highly sensitive reporter cell lines will be used to assess inflammatory responses to infection and tissue damage. This project will fill knowledge gaps related to the viral agent causing CMS. The results will open up for disease limiting measures as therapy and vaccination.