Schematic representation of targeted recombination to construct EGFP-expressing MHVs. The replacement of ORFs 4a and 4b with the EGFP gene was carried out by using recombinant technology, as described previously in [114]. (a) a schematic of the fMHV genome; it encodes the ectodomain of the feline infectious peritonitis spike in the background of the A59 genome. (b) shows the synthetic RNA transcribed from the vector pMH5 (MHV-A59 S gene) or pMHV- (MHV-2 S gene) [81, 114]. The curved line between the genome and the pMH54 RNA indicates the region in which the crossover must have occurred. The restriction sites relevant to the introduction of the EGFP gene are shown. The enlargement of the gene 4 region shows the modifications in which most of ORFs 4a and 4b are replaced by the enhanced green fluorescent protein (EGFP) gene. The IGS (intergenic sequence) is the site of initiation of transcription of mRNA 4. (c) and (d) show the resulting EGFP-expressing viruses: (c) expressing the MHV-A59 S gene and (d) RSMHV expressing the MHV-2 S gene. (adapted from the work of [114]).
