A schematic
diagram of the genome of this virus (RA59-gfp/gp33) as
well as a previously described recombinant MHV-A59 expressing
the EGFP protein alone (RA59-gfp), is shown in Fig. 1. In
both genomes the nonessential gene 4 is replaced by the foreign
sequences. Selection of RA59-gfp was described previously
(5). RA59-gfp/gp33 was selected using techniques similar
to those used in the selection of RA59-gfp and is described in
the Materials and Methods. Briefly, recombination occurred
between fMHV and an RNA encoding, in place of gene 4, a
recombinant protein containing 9 amino acids from ORF4a
and 9 amino acids of gp33 fused in frame with the EGFP gene.
Such recombinants were selected and plaque purified on murine
L2 cells all as previously described (26) and were characterized
further.