Construction and identification of recombinant human cytomegalovirus with ganciclovir-resistance associated mutations in UL97 gene / 中华传染病杂志

ABSTRACT

Objective To construct drug-resistant variant recombinant human cytomegalovirus (rHCMV)and identify drug susceptibility by phenotypic and genotypic assays.Methods The UL97 fragments containing Pine I recongnition site and resistant mutation were introduced by site-directed mutagenesis using gene splicing by overlap extension polymerase chain reaction(PCR),and blended with human cytomegalo-virus(HCMV)standard strain ADl 69 genomic DNA proportionally,then the DNA mixture were transfected into MRC-5 fibroblasts by the vector of liposomes.HCMV-PP65 antigen was detected by indirect-immunofluorescent assay to verify rHCMV infection of MRC-5 fibroblasts.When the eytopathic effect(CPE)of homologous recombinant virus reached 100%,the virus was harvested.The purified target virus was screened by plague with different concentrations of ganciclovir(GCV)and the recombinant virus was identified by plague reduction test and DNA sequencing of drug-resistant genes(UL97 and UL54).Results The UL97 fragments containing intended mutations for transfection were constructed successfully.After cotransfected with AD169DNA mixture for 7 days,rHCMV formed cytopathology was obviously visible,which was verified as rHCMV infected focus by HCMV-PP65 antigen test.The UL97 genotypic analysis of recombinant virus obtained by cloning was as expected.No mutation was found by UL54 gene sequencing.The GCV susceptibility of rHCMV positive clone was 15 μmol/L (50% inhibiting concentration),which was 12-fold of standard AD169 strain and was drug-resistant phenotype.Conclusions The rHCMV containing intended mutations is constructed successfully by cotransfeetion into MRC-5 cells and the recombinant virus strain is obtained by GCV screening and plaque purifying.The establishment of this method provides technique platform for identifications of new drug-resistant mutations of HCMV during anti-viral therapy.