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J Virol Methods. 2009 Nov;161(2):223-30. doi: 10.1016/j.jviromet.2009.06.016. Epub 2009 Jun 25.

Detection and identification of U69 gene mutations encoded by ganciclovir-resistant human herpesvirus 6 using denaturing high-performance liquid chromatography.

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1
Department of Infectious Disease Control, G-5, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

Abstract

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.

PMID:
19559728
DOI:
10.1016/j.jviromet.2009.06.016
[Indexed for MEDLINE]
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