A novel genotype (IVb) of the highly pathogenic fish virus, viral hemorrhagic septicemia virus (VHSV), emerged in the Great Lakes basin more than a decade ago. Over time, VHSV-IVb has spread throughout the basin, evidenced by mass mortality events involving numerous fish species. With a common goal of learning more about this emerging freshwater genotype, researchers and managers united to investigate pathogenicity, host range and the comparative susceptibility of Great Lakes fishes. However, there remains a lack of knowledge on the host immune response against VHSV-IVb and are still no approved preventative measures against this virus. This dissertation seeks to address these knowledge gaps and investigate protective measures for eliciting immunity against VHSV-IVb. My studies focused primarily on muskellunge (Esox masquinongy) due to their high susceptibility and vigorous immune response following VHSV-IVb exposure. To elucidate the role of the muskellunge humoral immune response against VHSV-IVb, I first developed a monoclonal antibody (mAb), designated 3B10, against muskellunge immunoglobulin (Ig). The development of the 3B10 mAb allowed for the creation of a muskellunge-specific indirect ELISA to detect anti-VHSV antibodies. I demonstrate the use of this indirect ELISA assay in both a serosurveillance capacity to determine previous viral exposure and to study the humoral immune response following immunization. Following immunization with a DNA plasmid containing the VHSV glycoprotein (G) gene, muskellunge anti-VHSV binding antibody levels peaked after approximately seven weeks. Knowing that the VHSV-IVb G gene can indeed elicit antibody production, I then compared the protective efficacy of two plasmids, differing only in their promoter sequence. One preparation (designated pVHSivb-G) conferred significant protection in muskellunge, resulting in 95% mean relative percent survival (RPS) following a single intramuscular administration, while the other (designated pβ-VHSivb-G) conferred less than 25% mean RPS. However, I found that immunized-protected muskellunge could still harbor and shed VHSV following viral challenge. Building from these results, I showed that the pVHSivb-G preparation could also elicit significant protection in three salmonid species: rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) and lake trout (Salvelinus namaycush). Following these successes with the pVHSivb-G preparation, I examined whether recombinant technology could provide similar protection. I expressed and purified a eukaryotic VHSV glycoprotein in cabbage looper (Trichoplusia ni) larvae using a recombinant baculovirus. After confirming the correct three-dimensional shape of this recombinant glycoprotein, the protective efficacy was assessed in muskellunge and resulted in 80% mean RPS. The culmination of my studies used the successful vaccination regimen and assays that I developed to investigate whether immunized fish can provide indirect protection to naïve fish. To accomplish this, I designed a flow-through system utilizing viral shedding as the viral exposure source. Co-mingled immunized muskellunge indeed conferred indirect protection to naïve muskellunge, resulting in a decrease in mean mortality from 80.2% to 36.5% when compared to naïve muskellunge housed alone. No protective effect was observed when naïve muskellunge were housed with Chinook salmon (Oncorhynchus tshawytscha), a semi-resistant species, indicating that immunity rather than resistance is important in this protective effect. The end result of this dissertation is an increased knowledge of the immunity against VHSV-IVb, as well as repeatable methods for eliciting this immunity.
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