Reintroduction of gene(s) into an attenuated deletion mutant of vaccinia virus strain IHD-W.

To evaluate individual genes responsible for virulence of vaccinia virus, a shuttle vector containing a viral fusion fragment from a deletion mutant (Z-19) was constructed. The vector also included a beta-galactosidase cassette which contained the LacZ under the control of the vaccinia late promoter p11 to screen recombinants. The gene in question was reinserted into the attenuated mutant by homologous recombination. The vaccinia growth factor and the vaccinia complement-binding protein genes were inserted into this shuttle vector and transferred by recombination into Z-19. The presence of the inserted gene was examined by Southern hybridization and its expression by Northern blot hybridization. The virulence of the recombinants was investigated by inoculation into mice. Results indicate that the recombinants contained and expressed the inserted gene. Although virulence was recovered, it did not reach the level of the wild-type, either when the recombinant viruses were inoculated alone or in combination. Construction of the shuttle vector allows reintroduction of genes into Z-19 which is completely attenuated in vivo and could be useful as a vaccine vector.