구조유전자 (G gene, P gene)를 knockout 시킨 재조합 viral hemorrhagic septicemia virus (VHSV) 제작 및 특성

Abstract

Generation of recombinant viruses lacking an essential gene for viral replication would be a way to produce safety-enhanced live viral vaccines. In the present study, we produced a recombinant viral hemorrhagic septicemia virus (VHSV) that lacks G gene or P gene in the genome using reverse genetics system, and analyzed the characteristics of the recombinant viruses. To produce a recombinant VHSV that lacks G gene in the genome (rVHSV-ΔG), we constructed a G gene-deleted recombinant VHSV vector and transfected to the cells expressing G protein by pcDNA3.1(+) plasmid. Using this system, although we could get the rVHSV-ΔG, the titer measured by plaque assay was too low to be used in other experiments despite lots of trials to increase viral titer. To solve this problem, we used phiC31 Integrase Vector System which inserts the expression cassette into the cell chromosome, and could produce rVHSV-ΔG with a higher titer. In the semi-quantitative RT-PCR analysis, the amount of expressed G gene in the cells having the expression cassette in the chromosome was clearly higher than cells with plasmids, suggesting that G protein expressing efficiency might be the cause of the different viral titers. Through in vitro and in vivo experiments, the present rVHSV-ΔG could not produce infective viruses without trans-supply of G protein. The P gene ORF contains 7 methionine codons including start codon in the same reading frame, and among them, we destroyed the first, second, and third methionine codon successively (change “ATG” to “TAG”) by site-directed mutagenesis, and named P1, P2, and P3, respectively. Recombinant VHSVs that could not express intact P protein (rVHSV-P1, -P2, and –P3) were successfully generated by trans-supply of the intact P protein. The rVHSV-Ps were not generated from cells expressing truncated P protein (P1, P2 or P3 protein), suggesting intact P protein is needed for infective virus production. However, interestingly, we isolated rVHSV-P1 that could produce infective viruses without P protein supply. The nucleotide sequence of the rVHSV-P1 revealed that a mutation occurred at the nucleotide 4 base before the second ATG codon, which changed isoleucine into methionine without frame shift, suggesting that strong selection pressure might exert on the mutation occurrences in the VHSV genome. In conclusion, the present rVHSVs can infect cells only a single round and cannot spread to other cells because of lacking ability to express G or P protein, which can guarantee the safety of the recombinant virus-based vaccines. The availability of these recombinant viruses as attenuated viral vaccines will be evaluated in further studies.