Figure 1. Construction of a recombinant vaccinia virus by homologous recombination. In a first step, a eukaryotic host cell is infected with a wild-type vaccinia virus. Next, the plasmid transfer vector is transfected into the cells. This vector usually provides the foreign cDNA (ORF) controlled by a vaccinia promoter (P) and a selection marker (S) flanked by viral sequences (F_L, left flank; F_R, right flank). The recombinant virus is generated in vivo in the cytoplasm of the host cell. The flanking virus DNA on the plasmid vector directs the foreign gene cassette into the homologous site in the virus genome. Virus assembly results in infectious recombinant virus, which is released by budding into the extracellular space. Plaque purifications, usually under selective conditions, allow the isolation of pure recombinant viruses.

Figure 2. Identification of recombinant viruses by selection and staining techniques. (a) Viruses containing, for instance, a gpt selection marker are plated on to indicator cells under selective conditions in low melting agarose. After 2 days, viral plaques are visualized by staining the indicator cells with a second agarose overlay containing neutral red. (b) Wild-type viruses grown under the same conditions form minute plaques that are not visible by neutral red staining. (c) A recombinant virus with a lacZ marker gene can be identified by addition of the chromogenic substrate X-Gal to the neutral red overlay; local -galactosidase expression results in blue plaques.

Figure 3. Nonreplicating vaccinia viruses growing in complementing cell lines. A permanent cell line expressing a vaccinia gene essential for viral multiplication (complementing host cell) permits the construction and propagation of recombinant vaccinia viruses lacking the essential gene (recombinant defective vaccinia virus). The design of the plasmid transfer vector may be chosen so that a desired foreign gene substitutes the viral essential gene. The recombinant defective viruses still infect normal hosts (cells or animals), and produce viral proteins including the recombinant antigens, but do not multiply and spread. Multiplication of the virus is limited to an engineered complementing host cell line.