Immune response dynamics and cardiac mitochondrial morphology during piscine myocarditis virus infection in Atlantic salmon

Sammendrag

Piscine myocarditis virus (PMCV) is a double-stranded RNA virus that causes systemic infection in Atlantic salmon. Infection with PMCV can result in deadly cardiomyopathy syndrome (CMS), considered one of the top threats to salmon aquaculture in Norway. CMS primarily targets the atrium and spongy ventricle of the heart, but the mechanisms driving the immune response and determining virulence remain poorly understood at both the systemic and molecular levels. In this thesis, we conducted a long-term 16-week experimental PMCV infection to investigate the progression of the disease at the cellular and tissue levels. We report that while both the ventricle and atrium exhibit similar patterns of pro-inflammatory and T cell marker expression, the atrium consistently shows earlier and higher upregulation, partially mirroring virus RNA expression levels. Notably, tissue-specific upregulation of ifna, ifng, and t-bet was observed in the atrium but not in the ventricle, suggesting a stronger pro-inflammatory environment in the atrium. This heightened immune response likely explains the more severe histopathological changes in the atrium, as reported in this and previous studies on PMCV-induced CMS. Virus RNA levels peaked earlier in the lymphoid organs, spleen, and head kidney. While antiviral genes peak together with virus RNA levels in the heart and the lymphoid organs, lymphoid organs had a second peak in expression of T-cell markers, which was concurrent with the immune response in the heart, from 6-9 WPI, indicating their involvement in the immune response in the heart. Following the peak phase of infection, we observed a decline in virus RNA levels in the atrium and ventricle, showing a trend towards clearance, together with a contraction of the immune response. In contrast to the heart, virus RNA levels in lymphoid tissues did not show a trend towards clearance and may be a source of virus particles, which may lead to resurgence. Furthermore, elevated cd8b in the heart sets the stage for sequelae and persistent CMS. At the subcellular level, fluorescence microscopy (FM) of mitochondria in ventricular cardiomyocytes revealed altered mitochondrial dynamics during the early and peak phases of the infection. Mitochondria exhibited swollen and elongated morphologies, alongside upregulation of prkn, pink1 and mtfp1, indicating infection-induced mitochondrial damage and remodeling. These changes likely reflect the antiviral and cytotoxic immune response in the heart. Transmission electron microscopy further confirmed these findings showing that intermyofibrillar mitochondria were more strongly affected to PMCV infection than perinuclear mitochondria. This suggests that the cardiomyocyte energy supply may be disrupted, which could be the basis for circulatory problems associated with CMS. This is the first study to document the effects of virus infection on mitochondrial dynamics in Atlantic salmon cardiomyocytes. Additionally, we characterized the expression of two key pattern recognition receptor pathways in the head kidney (HK) tissue, HK leukocytes, and atrium - Rig-I-like and Toll-like receptors. This study revealed that tlr3 and tlr7 were transiently upregulated in all three compartments with higher expression during phases of increased virus RNA levels, while tlr22 was only upregulated in the atrium. Likewise, cytosolic RNA sensors rig-i and mda5 showed higher and consistent upregulation in the atrium compared to HK, reflecting robust cytoplasmic RNA sensing in atrium. PRR adaptor molecules mavs, ticam1, and myd88 showed tissue specific activation, with the atrium exhibiting highest upregulation, reflecting robust immune activation, whereas, mavs and ticam1 were downregulated in the head kidney, indicating limited activation of RNA sensing pathways in head kidney due to virus-induced immunosuppression or immune contraction. Our experiments revealed that the virus first replicated in HK tissue (1 WPI), before spreading to HK leukocytes (2 WPI), and finally to the heart (2-4 WPI). While the atrium and HK leukocytes showed a trend of clearance, the infection appears persistent in the HK tissue. Throughout the infection, we observed consistently higher expression of PMCV ORF3 compared to the other ORFs (ORF1 and 2) in HK tissue and atrium. Between 7-16 WPI in HK tissue, there was a significant trend of lower ORF1 expression compared to the other ORFs, which we also observed from 12-16 WPI in HK leukocytes. This study raises important questions about the impact of PMCV infection on lymphoid tissues and the role of individual ORFs in shaping the overall virulence profile of PMCV.

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NVA : hdl.handle.net/11250/5527939