Efficacy of a formalin-killed cell vaccine against infectious spleen and kidney necrosis virus (ISKNV) and immunoproteomic analysis of its major immunogenic proteins.

Infectious spleen and kidney necrosis virus (ISKNV), the type species of genus Megalocytivirus in the family Iridoviridae, is the most important etiological agents in mandarin fish industry in China. Since its first occurrence in China in the early 1990s, there is no effective method to prevent and control this virus. Here we report the successful development of a formalin-killed cell-cultured (FKC) vaccine again ISKNV. Immuno-protection experiments showed that greater than 90% of fish immunized with the FKC vaccine were protected against virulent ISKNV. Sera derived from the immunized fish markedly inhibited the virus infection both in vitro and in vivo. Purified IgM from the immunized fish sera also showed efficient neutralization effects in vivo, strongly suggesting that antibody-mediated immunity may play an important role in the FKC vaccine. Using FKC-immunized fish sera as first antibody, a two-dimensional gel electrophoresis mass spectrometry analysis-based immuno-proteomic method was performed to identify the immunogenic proteins. ORF006L (the major capsid protein), ORF054L, ORF055L, ORF101L, ORF117R, and ORF125R were found to be the major immunogenic proteins of ISKNV. Antibodies generated from these six recombinant viral proteins were able to recognize specifically the corresponding protein fractions from purified virions by Western blot analysis, and five of them (excluding ORF125) showed positive reactions by indirect immunofluorescence assay. In summary, the present study first developed an effective vaccine against ISKNV, and the major immunogenic proteins of ISKNV are also identified. The reported ISKNV immunogenic proteins are important for further development of diagnostic regents and genetic vaccine candidates for all megalocytivirus.