In vivo and in vitro studies of Salmon Pancreas Disease Virus (SPDV) in Atlantic salmon (Salmo salar L.)

Salmon Pancreas Disease Virus (SPDV) is the only viral species of the genus Alphavirus, family Togaviridae, affecting fish. SPDV induces two conditions historically recognised independently as Pancreas disease (PD) and Sleeping disease (SD), affecting Atlantic salmon (Salmo salar L) and rainbow trout (Oncorhynchus mykiss), respectively. Infection by SPDV can lead to clinical disease with characteristic acinar pancreatic necrosis and a range of myopathies of the skeletal and heart muscle. Mortality is not a necessary outcome of the disease and usually is not significant. However, affected fish stop eating and therefore present a reduced growth rate and the disease can also leave visible lesions at the fillet level that lead to downgrading at slaughter. SPDV can affect in the fresh and sea water environments, but a higher and most relevant impact reported in the latter. Historically, PD has posed a significant challenge to the Atlantic salmon farming industry in the UK, as well as in other salmon producing countries. This thesis was developed and conducted at Marine Scotland Science (MSS), the Scottish National Reference Laboratory, with the aim to contribute to knowledge gaps identified by the industry and research communities. The focus was on development and improvement of in vivo and in vitro infection models to assist with host pathogen interaction studies. In vivo work was to establish an experimental challenge model to induce SPDV infection in a more natural way than by intra-peritoneal (IP) injection. The first step involved selection of an infective SPDV isolate through a comparative IP challenge study. An infective isolate was then used to establish a co-habitation challenge model in “post smolts”, the sea-water stage predominantly affected by PD. Additionally, during this experiment assessment of viral tissue tropism along time and potential intra-subtype differences in infectivity was undertaken. In vitro work accounted for the more innovative part of this thesis with the development, optimization and application of an ex vivo cardiac primary culture originated from Atlantic salmon embryos. While fish origin aggregates of self-contracting cardiomyocytes had been previously isolated and suggested as a robust tool on human biomedical research and pharmacological and toxicology testing, paradoxically very little has been done to explore the approach of ex vivo primary cultures as a disease model with the specific goal for health issues affecting fish. The work involved an adaptation and refinement to produce salmon cardiac primary cultures (SCPCs). Once this was achieved, SCPCs could be kept under laboratory conditions with minimal maintenance for periods up to 6 months. Following this work, SCPCs were successfully challenged with different SPDV isolates as well as another cardiotropic viral agent (Infectious Salmon Anaemia, ISA). The kinetics of SPDV and ISA viral infection and one element of the immune response (i.e. expression of mx gene) were studied. As part of this study, the comparative response of SCPCs of diverse genetic backgrounds (i.e. IPN resistant vs. IPN sensitive) was also assessed. Differences were observed, which highlights potential usefulness of SCPCs to examine genotype-based differences in response to viral disease. Finally, SCPCs were used to examine the SPDV infection cycle ultrastructure by transmission electron microscopy (TEM). This work resulted in novel insights on the replication cycle of SPDV, drawing from the extensive literature in mammalian alphavirus work. With SPDV and other virus associated myocarditis severely affecting Atlantic salmon aquaculture at present, I believe that the SCPCs model represents the most relevant contribution of this PhD.