Field-based safety evaluation of rVHSV-GΔTM in aquaculture and verification of vaccine efficacy through freeze-dried prototyping in olive flounder.

Single-cycle viruses hold great promise as fish viral vaccines due to their high protective efficacy. Although the efficacy of the vaccine in olive flounder and rainbow trout has been proven through previous research, safety must be additionally proven considering the environment of use for commercialization. This study comprehensively assesses the safety of rVHSV-GΔTM and its impact on both the host and the surrounding environment, including the coastal habitat of nearby species and seawater. We constructed an experimental tank geographically mimicking coastal conditions and evaluated its effects on co-reared olive flounder and other organisms. Results showed no detectable viral presence in co-reared organisms or concentrated culturing seawater, and the virus demonstrated limited proliferation under varying salinity conditions. The survival rate of olive flounder immunized with rVHSV-GΔTM was significantly higher (up to 90 % protection against wild-type VHSV) compared to non-immunized fish. Additionally, we developed a prototype vaccine of rVHSV-GΔTM through freeze-drying. Immunization of olive flounder with the prototype vaccine conferred significantly higher protection against wild-type VHSV. These findings underscore the safety of rVHSV-GΔTM as a commercial vaccine, unaffected by the host or surrounding environment, and highlight the efficacy of freeze-drying in delivering single-cycle viruses.