Virulence motifs of piscine myocarditis virus causing CMS in Atlantic salmon

Piscine myocarditis virus (PMCV) is the first viral variant in this family to infect vertebrates. Infection causes the disease cardiomyopathy syndrome (CMS) in Atlantic salmon. Today we do not know when the fish are infected, but there are indications that salmon smolt are exposed to the virus after transmission to seawater. We know that it takes a long time (months) for the infected fish to show clinical signs, i.e. PMCV gives a chronic infection and the fish will carry the virus for an extended period. Clinical signs of disease are seen when the fish are handled / transported / treated for lice infestation and the only symptoms seen are sudden death with few previous signs of disease, typically during handling or transport. Groups of fish suffering from CMS die from a rupture of the heart wall, which leads to bleeding to the pericardial sac. The virus shares similarities in the genome with viruses that infect fungi, parasites, insects and shrimp, and recently a similar virus has been found in other fish species, in freshwater and seawater. Fish infecting viruses from this group have a unique gene that encodes a unique protein that can destroy fish cells within a few days when they are expressed in cell cultures. The virus cannot be grown in the laboratory, and in this study we will test many different cell lines, including cells that we have modified so that they do not respond to virus infection using CRISPR / cas9 methods. This can provide increased opportunities to grow viruses in vitro. We will also use methods where we take the entire virus genome, insert it into a plasmid and deliver it to naive salmon with the aim of facilitating the virus to multiply in target organs. This can provide opportunities for more detailed studies of infection mechanisms and at the same time provide an opportunity for the development of a vaccine. We will also conduct studies in which viruses from field outbreaks are sequenced in connection with disease outbreaks with high and low mortality, with the intention of demonstrating connections with genetic fingerprints in viruses and mortality in disease outbreaks.

Piscine myocarditis virus (PMCV) is the first member of the Totiviridae family shown to infect a vertebrate host. It is the causative agent of cardiomyopathy syndrome (CMS) of Atlantic salmon. The sequence of events during an infection is not well understood, indications are that primary replication occurs in headkidney and spleen, while the cardiomyocyte is infected at later stage. Infection persists and fish will carry the virus for extended periods. The atrium and the spongious part of the heart ventricle take a hard hit from the infection resulting in necrosis and severe inflammatory changes. PMCV differs from other members of Totiviridae family in the sense that its genome has a 3rd open reading frame (ORF3) that encodes a protein that has acute cytotoxic effects when expressed in vitro in fish cell lines. ORF3 sequences obtained from clinical cases of CMS show variation and we have found that certain variants of the ORF3 protein are non-toxic when expressed in vitro. We will use this information to design full length constructs encoding the entire genome and inject plasmids into parr and postsmolt of salmon, and follow the development of virus replication and formation of progeny over periods of 30 weeks. The need for long term studies come from cohabitation studies in fingerlings/parr showing that 24 weeks are needed for an infection to be established. For in vitro culture, cell lines will be gene edited using CRISPR/Cas9 methods and sensors and effectors of the antiviral genes will be knocked out, i.e. TLR3, PKR, and downstream genes like STAT1/2 or Mx. Edited cell lines will be infected with material from clinical cases of CMS and also from the challenge experiment described. Finally, lab scale studies will be validated through field screening and sequencing of virus strains associated with high and low mortality outbreaks of disease, to possibly link controlled studies with real life situations.