Construction and Immunological Evaluation of a Bivalent DNA Vaccine Targeting Micropterus salmoides Rhabdovirus and Largemouth Bass Ranavirus

The largemouth bass (Micropterus salmoides) is persistently threatened by Micropterus salmoides rhabdovirus (MSRV) and largemouth bass ranavirus (LMBV), highlighting the need for combined immunization strategies. Here, a bivalent DNA vaccine pEGFP-G-MCP was constructed by concatenating the highly immunogenic fragments of MSRV glycoprotein (MSRV-G) and LMBV main capsid protein (LMBV-MCP) using a flexible linker peptide, and its immunogenicity and protective effect were systematically evaluated. Fish were immunized by intramuscular injection of 200 ng of plasmid on day 1, followed by a booster immunization on day 8. Blood and tissues were collected at 10, 20, and 30 d post-last immunization. The results showed that pEGFP-G-MCP immunization induced G- and MCP-specific antibodies and upregulated the expression of immunity-related genes. The bivalent vaccine achieved a relative percent survival (RPS) of 93.3% against MSRV and 90.0% against LMBV, which were higher than those of the monovalent vaccine groups. Additionally, the bivalent vaccine reduced viral mRNA and alleviated pathological damage in MSRV- or LMBV-infected fish. These results indicate that pEGFP-G-MCP confers immune protection against MSRV and LMBV, providing a foundation for combined prevention and control of MSRV and LMBV.