Disease development of wild-type and Rag1 mutant zebrafish was evaluated after challenge with Snakehead Rhabdovirus (SHRV), a novirhabdovirus. Rag1 mutants lack T and B lymphocytes and thus lack lymphocyte-based acquired immunity. Wild-type zebrafish became more disease resistant as they aged (4 months and older) and at an elevated temperature (28°C) but mutants remained sensitive at all ages and temperatures tested. Quantitative reverse transcription polymerase chain reaction (RT qPCR) demonstrated that interferon gamma and MxA expression significantly increased in both types of fish at 2 days post-infection with subsequent dwindling of expression. The high interferon gamma expression suggests activation of natural killer cells (and/or T lymphocytes in wild-type fish), and the up-regulation of MxA expression indicated an activation of type 1 interferon response. The development of protection in virus exposed fish was evaluated by lethal challenge at 3 weeks post vaccination. Vaccinated wild-type fish showed significant protection and while most of the mutant groups showed no protection. One vaccination treatment group of the mutants demonstrated a significantly slower mortality and less overall mortality. The results suggest that lymphocyte based immunity imparts a robust protective response to SHRV while low-level protection can develop in the absence of lymphocytes. A cell mediated cytotoxicity assay was established. Cell lines were developed from the inbred fish populations and class I MHC U lineage genes were compared. The mhc1uba and mhc1uca genes were found in the mutant and cells but no class I MHC U lineage genes were detected in the wild-type fish or cells. These cells were used as targets in cytotoxicity assays. Kidney-marrow cells of vaccinated mutant or wild-type zebrafish killed more SHRV infected target cells than did those from non-vaccinated fish with the wild-type effectors showing higher cytotoxicity. The lymphocyte component appears responsible for the temperature and age associated resistance. This helps explain why novirhabdoviruses cause higher losses in young fish and at low temperatures. Further studies are needed to understand the relative contribution of the cellular components that play important role in SHRV resistance, but the establishment of cytotoxicity assays is an important first step in dissecting the cellular defenses in zebrafish.

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