Detection and identification of U69 gene mutations encoded by ganciclovir-resistant human herpesvirus 6 using denaturing high-performance liquid chromatography.
A denaturing high-performance liquid chromatography (dHPLC) assay was developed to detect antiviral drug-resistance mutations of human herpesvirus 6 (HHV-6). Recombinant baculoviruses were created that contained wild-type and mutant forms of the HHV-6 U69 gene, which determines sensitivity to the antiviral drug ganciclovir (GCV). The mutations causing GCV resistance in HHV-6 U69 were single-base mutations adapted from known GCV-resistant DNA sequences of HCMV, and their ability to confer GCV resistance on recombinant baculoviruses was confirmed. Six characterized mutant sequences, including one reported previously that encodes a GCV-sensitive kinase-activity mutant, were used. DNA was extracted, and the levels of homoduplex and heteroduplex DNA in the PCR products from mixed wild-type and mutant viral DNAs were determined using dHPLC. The optimized assay could distinguish the different mutants, and could detect mutants representing only 10% of the DNAs. The new assay with dHPLC readout permitted the rapid (4 h), objective, and reproducible detection of HHV-6 drug-resistance mutations.
Nakano
Kazushi
K
Department of Infectious Disease Control, G-5, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
Nishinaka
Kazuko
K
Tanaka
Tatsuya
T
Ohshima
Atsushi
A
Sugimoto
Nakaba
N
Isegawa
Yuji
Y
D000998:Antiviral Agents / Q000627:therapeutic use*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D015774:Ganciclovir / Q000627:therapeutic use*; D005814:Genes, Viral; D015654:Herpesvirus 6, Human / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D015203:Reproducibility of Results; D019349:Roseolovirus Infections / Q000188:drug therapy / Q000821:virology*; D012680:Sensitivity and Specificity
D000998:Antiviral Agents; D004279:DNA, Viral; D015774:Ganciclovir