Selection of recombinant vaccinia viruses on the basis of plaque formation.

We developed a procedure for isolation of recombinant vaccinia viruses (re-VV) based solely on plaque formation, without a requirement for specific cell lines, selective medium or special staining. The system consists of two components: (i) a mutant non-plaque-forming VV and (ii) a plasmid vector that, through homologous recombination, can simultaneously introduce a foreign gene and repair mutation in the VV genome. The mutant VV contains a deletion of the vp37 gene, encoding a 37-kDa protein component of the viral outer envelope that is required for efficient viral spread on cell monolayers. The plasmid vector contains a functional vp37, a strong synthetic VV early/late promoter, unique restriction sites for gene insertion, and flanking segments of VV DNA for homologous recombination. Following infection and transfection of cells with the mutant VV and plasmid vector, respectively, re-VV are identified and isolated by their ability to form plaques. To evaluate the system, a re-VV that expresses the gene encoding influenza virus hemagglutinin (HA) was isolated simply by picking visible plaques.