Viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb) is a recently emerged pathogen of Great Lakes fishes in North America. Following initial fish kills in Lake St. Clair, Michigan (MI) in 2006, VHSV-IVb went undetected for nearly three years there until another die-off ensued in the summer of 2009. This prompted questions regarding host immunity to the virus as well as the usefulness of the virus isolation assay as a surveillance tool. Herein, I describe evidence of humoral immunity to VHSV-IVb in several populations of wild fishes and the development of antibodies by muskellunge (Esox masquinongy) following experimental challenge and vaccination. Using a 50% plaque neutralization test (PNT) and a competitive enzyme-linked immunosorbent assay (cELISA), the latter developed as part of these studies, antibodies were detected in Lake St. Clair fish during all sampling years between 2004-2011 (six summers). In contrast, VHS virus was only detected during the summer of 2006 and 2009, and in both cases, fish were sampled during or immediately following mortality events. Neutralizing (PNT) and/or binding (cELISA) antibodies were detected in 5 of 13 fish species including muskellunge, northern pike (E. lucius), freshwater drum (Aplodinotus grunniens), smallmouth bass (Micropterus dolomieu) and channel catfish (Ictalurus punctatus). The greatest overall seroprevalence and antibody titers were detected in muskellunge. Antibodies were also detected by cELISA in muskellunge from Lower Fox River/Green Bay, Wisconsin and from Thornapple Lake, Michigan. Muskellunge experimentally infected with VHSV-IVb developed neutralizing antibodies by 5 - 7 weeks (385 - 539 degree days) post-challenge. The development of neutralizing antibodies corresponded to a decrease in virus presence and titers in sera, indicating their role in limiting VHSV-IVb infection. A reduced neutralizing antibody response was mounted by fish exposed with the lowest dose of the virus, suggesting a threshold level of infection is necessary for induction. In surviving fish, neutralizing antibodies were detectable for a longer period of time after infection then was the virus in tissues (assessed in a parallel study). To better understand host immune response in this species, a DNA vaccine encoding the glycoprotein (G) gene of VHSV-IVb was developed. DNA vaccination primed the adaptive immune response to respond to VHSV-IVb as evident by high levels of neutralizing antibodies in 100% of surviving muskellunge by only 4 weeks (310 degree days) after challenge. Compared to plasmid control fish, pVHSivb-G-vaccinated fish were significantly protected (relative percent survival = 45.2%) following lethal virus challenge 7 weeks (539 degree days) post-vaccination. Surviving fish from the pVHSivb-G vaccinated group also had a significantly lower infection prevalence and significantly reduced tissue viral loads compared to control fish. The cELISA and PNT are non-lethal immunological tools that will find multiple applications in future studies for assessment of adaptive immunity to VHSV-IVb.
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