| Summary: | Mastergradsoppgave i næringsrettet bioteknologi, Avdeling for lærerutdanning og naturvitenskap, Høgskolen i Hedmark, 2014. Master of applied and commercial biotechnology. Heart and skeletal muscle inflammation (HSMI) is a challenging disease of farmed Atlantic salmon, and was first discovered in farms situated in the coastal areas of Norway. Piscine orthoreovirus (PRV) is the causative agent of HSMI but the mechanism of PRV infection in salmon is still unclear. The NS and NS proteins of PRV are predicted to be involved in the formation of viral factories. The aim of the present study was to investigate the co-localization of the NS and NS proteins in salmon cells. However, several attempts to amplify and clone the NS and NS genes into the bicistronic mammalian pIRES vector were unsuccessful. The PRV NS gene was successfully amplified from cDNA and was cloned into a pCR vector for further amplification and analysis. We failed, however, to amplify the NS gene after several attempts with different primers. The NS gene, previously cloned into a pcDNA 3.1 vector, was therefore used and transfected into CHSE cells to identify the localisation of the NS gene product in these cells. The results showed that the NS protein localized both to the nucleus and to the cytoplasm of CHSE cells. In the present study we also investigated the presence of PRV and immune gene regulation in the spleen tissue of infected salmon using the experimental cohabitation challenge mode. In studies performed in freshwater (challenge study1) and seawater (challenge study 2), we quantified PRV and the regulation of immune genes using the RT-qPCR technique. In this study we determined the regulation of CD8α, granzyme, perforin 1a, perforin 1b and viral gene copy numbers in freshwater salmon injected with PRV and in their cohabitants, and also in cohabitants in the corresponding study performed in seawater. In challenge study1, we determined that CD8α was gradually increased in spleen tissue of both injected fish and cohabitants. We found that the granzyme up-regulation was associated with an increased copy number of viruses in both cohabitant salmon and virus injected salmons, and then both the viral copies and granzyme level decreased gradually in spleen. The up regulation of CD8α, perforin A1 and B1 and granzyme indicates the activation of CD8 cytotoxic T lymphocytes in Atlantic salmon in both challenge studies. These findings revealed new information about HSMI and its pathogen in Atlantic salmon.
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