Live fish vaccines: history and perspectives.

In the development of live vaccines against enzootic fish viral diseases, the conventional approaches, although somewhat successful, failed finally to deliver efficient, safe, and tagged strains for vaccine application. The genetically-engineered vaccine approach also gave similarly disappointing results. Faced with these realities during our work with VHS, we turned our research effort towards understanding the molecular basis of virulence and antigenicity of the virus. Using sequence analysis of neutralization-escape mutants, we identified several amino acid positions on the glycoprotein which seemed to be involved in the pathological process. The attenuated phenotype was consistently associated with simultaneous mutations at two distant regions, 125-140 and 430-433 of the glycoprotein. We also demonstrated that reversion to virulence was accompanied by the loss of the concurrent mutations, which confirmed their involvement in virulence. The importance of these two regions of the glycoprotein was confirmed by the finding that laboratory or naturally attenuated variants had mutations within these regions. Using the same methodology, we selected mutants from an attenuated temperature resistant variant (tr25), which had previously been developed in our laboratory. Virulence, antigenicity and protective activity of the further attenuated mutants were evaluated in fish of different size by intramuscular (i.m.) or water bath administration. Strains having an additional mutation at position 139 were completely non-virulent for fish of 1000-1400 degree-days (dxd) by bath. These mutants retained their immunogenicity and had a thermo-resistant, an antigenic, and five genetic markers. Thus, they will constitute ideal candidates for live vaccine development, once their protective activity and containment have been confirmed in field trials.