Comparative analysis of immunogenicity for viral hemorrhagic septicemia virus (VHSV) vaccines inactivated by different methods.

Viral hemorrhagic septicemia virus (VHSV) belonging to the genus Novirhabdovirus and family Rhabdoviridae is a major viral disease that causes economic loss in the olive flounder (Paralichthys olivaceus). In this study, we produced formalin-, binary ethylenimine (BEI)-, β-propiolactone (BPL)-, and heat-treated, inactivated VHSV vaccines; and observed the glycoprotein changes in the inactivated VHSV vaccines and confirmed their correlation with vaccine efficacy. The control group showed a 100 % cumulative mortality rate, but the groups immunized with formalin- and BPL-inactivated VHSV vaccines had high survival rates of 80 % and 90 %, and the virus copy numbers was significantly low. Conversely, groups immunized with BEI- and heat-inactivated VHSV vaccines had low survival rates of 20 % and 30 %, and high virus copy numbers. Fish immunized with formalin- and BPL-inactivated VHSV vaccines significantly increased VHSV-specific immunoglobulin (Ig) M titers in serum, and showed significant upregulation of several immune genes (Mx, ISG15, IL-10, IFNγ, and CD8α) in the spleen. Upon VHSV challenge, the BPL group displayed significant upregulation of IL-2, CD8α, and IgM along with downregulation of Mx, IL-10, IL-17A, and IFNγ compared to the virus infected control group. When evaluating the antigenic change of VHSV glycoprotein (G) protein using 4 different VHSV G monoclonal antibodies, formalin- and BPL-inactivated VHSV conserved VHSV monomer G similar to the non-inactivated VHSV, whereas BEI- and heat-inactivated VHSV monomer G exhibited changes. These results suggest that the conserved disulfide bond-dependent G protein structure of the inactivated VHSV may induce significant immune responses in fish, resulting in high protective efficacy.