Construction of virus recombinants by combined positive and negative selection.

A. A vaccinia virus expression plasmid encoding an ORF of interest (ORF-X) is cloned downstream of a bacteriophage T7 promoter (pT7) under the additional transcriptional control of a Lac operator (LacO), an encephalomyocarditis (EMC) virus leader sequence and a bacteriophage transcriptional stop sequence (TT7). A positive selection marker (GPT behind a 7.5 early/late vaccinia virus promoter) is included on the plasmid. Sequences on the left and right sides of the vaccinia virus hemagglutinin gene (HA-L and HA-R) are used in vivo to recombine with the MVA-Koom virus which encodes T7 RNA polymerase, GFP as well as the GYR-PKR and the mCherry-NeoR fusion genes. Replacement of the fusion genes by OFF-X is selected by the ability of the recombinant virus to multiply in the presence of MPA and coumermycin resulting in loss of mCherry expression. Note that the sequences are represented according to the standard orientation of the virus genome. B. Analysis of recombinant virus plaques by light and epifluorescence microscopy. BHK 21 cells were infected in the presence of IPTG with MVA-Koom or an MVA-T7 recombinant virus derived from MVA-Koom by insertion of a polyprotein sequence encoding YFP at its 3’ end (see Fig 4). One representative virus plaque for each infection was observed with an EVOS Cell Imaging System using from top to bottom a white light cube (top panel), a GFP light cube (470/22, 510/42), a YFP light cube 500/24, 524/27 or an RFP light cube (531/40, 593/40).