Attenuated deletion mutant of vaccinia virus IHD-W recovered virulence by reinsertion of a terminal restriction fragment.

Since vaccinia virus is being considered as a potential immunization vector, it is important to identify genes responsible for pathogenicity. One approach to identify virulence genes is the use of attenuated deletion mutants with a null background to reintroduce regions of the deleted genome material. We have previously described an attenuated deletion mutant of vaccinia virus strain IHD-W (Z-19) that lost 21 kb from the left terminus comprising fragments HindIII C and N, displays rearrangements at the right terminus and is unable to grow in vivo. To establish whether the loss of genes present at the left terminus is the basis for the attenuated phenotype, rescue experiments were performed to reintroduce fragment HindIII C, using growth in vivo as a selection system to isolate recombinant viruses. Several recombinants were isolated and molecularly and biologically characterized. The results indicate that recovery of virulence in mice was correlated with the presence and expression of two genes: vaccinia growth factor and C4b binding protein, which are located at the left terminus. Serpin I and II genes, located at the right terminus, were not affected in Z-19 and seem not to play a role in virulence in the IHD-W strain; interestingly, a copy of the serpin I gene was found at the left terminus. Moreover, a gene involved in virulence in the WR strain, the 13.8 kDa virokine gene (N1L) was found to be located in fragment HindIII G in the IHD-W strain, and therefore did not contribute to the attenuated phenotype.