Dominant host range selection of vaccinia recombinants by rescue of an essential gene

We report the rescue of a defective vaccinia virus, forming the basis for a stringent selection protocol to generate replicating recombinant virus without the need for marker cassettes and selection agents. Plaques of recombinant virus could be isolated solely by their ability to grow in wild-type cells normally supporting the growth of vaccinia virus. All growth-competent clones analyzed contained the gene of interest in the intended genomic locus and displayed foreign gene expression to the same levels as was seen with classical recombinants obtained by insertion into the vaccinia virus thymidine kinase locus. The system is based on a defective vaccinia virus, expressing exclusively early genes, termed eVAC-1, and an insertion plasmid vector providing the essential function, the uracil DNA glycosylase gene. In addition, the defective virus is free of selection and color marker genes, thus also representing a basic vector for the generation of defective recombinants.