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. Fig.1.1. 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.