Detection and identification of U69 gene mutations encoded by ganciclovir-resistant human herpesvirus 6 using denaturing high-performance liquid chromatography.
J Virol Methods
; 161(2): 223-30, 2009 Nov.
Article
en En
| MEDLINE
| ID: mdl-19559728
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Antivirales
/
Ganciclovir
/
Mutación Puntual
/
Herpesvirus Humano 6
/
Infecciones por Roseolovirus
/
Farmacorresistencia Viral
/
Genes Virales
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
J Virol Methods
Año:
2009
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Países Bajos