Infectious pancreatic necrosis virus induces apoptosis in vitro and in vivo independent of VP5 expression

Infectious pancreatic necrosis virus (IPNV), the causative agent of a highly infectious disease in salmonid fish, encodes a small non-structural protein designated VP5. This protein contains Bcl-2 homologous domains and inhibits apoptosis when expressed in cell culture. We have previously reported the generation of three VP5 mutants of IPNV-Sp serotype, using reverse genetics (Santi, N., Song, H., Vakharia, V.N., Evensen, Ø., 2005. Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence. J. Virol. 79 (14), 9206-9216). The wild-type rNVI15 virus encodes a truncated 12-kDa VP5 protein, rNVI15-15K encodes a full-length 15-kDa VP5, whereas rNVI15-ΔVP5 is deficient in VP5 expression. In the present report, the role of VP5 in apoptosis was assessed both in vitro and in vivo, using the recombinant IPNV strains. Apoptosis was observed in hepatocytes of Atlantic salmon post-smolts challenged with all three VP5 mutant viruses. Using a double-labeling technique to detect apoptotic cells and IPNV antigens, we found that viral antigen and apoptotic cells co-distributed. In addition, numerous double-positive cells were seen. The recombinant viruses also induced apoptosis in infected cell cultures, and the morphology and membrane integrity of infected cells at different time points was similar. In summary, these results indicate that IPNV induces apoptosis in infected cell cultures and in fish, independent of VP5 expression. However, substitutions of putative functionally important amino acids in the BH2 domain of VP5 of IPNV-Sp strains were identified, which might influence the anti-apoptosis effect of the protein, and partly explain the apparent absence of this specific function.