The role of the non-coding region between ORF 62 and ORF 63 was examined by construction of recombinant viruses and analyzing their virological properties in the comparison with the wild type. An EHV-1 mutant, Ab4-GFP, was constructed by inserting a green fluorescent protein (GFP) expression cassette flanked by loxP into the intergenic region between ORF 62 and ORF 63 in order to determine the roles of this region in the virus life cycle. The targeted insertion site was inside the 1584 bp intergenic space located between ORF 62 and ORF 63, specifically between nucleotides 110055 and 110056 of the Ab4p genome (1254 bp upstream from the initiating codon of ORF 62 and 330 bp downstream from the termination of ORF 63). EHV-1 Ab4p was inoculated into MDBK cells. The infected MDBK cells were transfected with the recombinant plasmid (pEGr) where the homologous recombination occurred. After three rounds of selection and plaque purification, one recombinant virus was cloned and designated Ab4-GFP. A loxP-containing mutant was also constructed by excision of the GFP expression cassette from the viral genome using cre recombination. The cloned virus without the GFP was designated as Ab4-loxP.
The growth kinetics and plaque sizes of Ab4-GFP and Ab4-loxP were compared to those of Ab4p in MDBK cells in order to determine whether the insertion of GFP and the presence of loxP sequence in the intergenic region had any effect on viral growth. In multi-step growth kinetics, the growth patterns of Ab4p and Ab4-loxP were similar. The extracellular and intracellular virus titers of Ab4p and Ab4-loxP reached their peak values at 40 and 32 hr post-inoculation, respectively. On the other hand, both the extracellular and intracellular titers of Ab4-GFP were lower than those of Ab4p and Ab4-loxP. The peak extracellular and intracellular titers of Ab4-GFP were observed at 40 hr post-inoculation. The peak titer of Ab4-GFP was significantly (p |
