title: Rapid detection of cytomegalovirus strains resistant to ganciclovir through mutations within the gene UL97.
abstract: The human cytomegalovirus (HCMV) gene UL97 product was shown to play an important role in phosphorylation of ganciclovir (GCV) in HCMV-infected cells. The deletion of the 4 amino-acid sequence AACR confers resistance to a laboratory mutant. The aim of this study was to develop a rapid and simple method to detect mutations within the 12 base pair (bp) fragment encoding AACR, from isolates and clinical specimens (urine, bronchoalveolar lavage, cerebral spinal fluid samples). A target region encompassing this 12bp sequence was amplified by a single-step PCR assay from HCMV isolates and reference strains, and a two-step procedure from clinical specimens. Reaction products were submitted to restriction enzyme analysis and dot-blot hybridization assay. Two biotinylated probes were used: one probe (DL) overlapping the 12bp region; and a control probe with similar length and GC content. Hybridization was performed under conditions allowing the detection of one bp deletion (HCMV strain susceptibility to GCV was determined by a rapid late antigen synthesis reduction assay.) The control probe hybridized to the UL97 sequence amplified from all 23 tested isolates and the reference strains. The DL probe gave a positive signal with GCV-susceptible strains; no signal was obtained for five out of seven resistant isolates, and for a laboratory mutant derived from the strain AD169. Restriction analysis of amplification products showed different patterns suggesting this region can be involved in various DNA changes.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Service de Bactériologie-Virologie, Hôpital Lariboisière, Université Paris VII, France.
authors.forename: S
authors.initials: S
authors.lastname: Alain
authors.forename: M C
authors.initials: MC
authors.lastname: Mazeron
authors.forename: J M
authors.initials: JM
authors.lastname: Pépin
authors.forename: F
authors.initials: F
authors.lastname: Morinet
authors.forename: L
authors.initials: L
authors.lastname: Raskine
authors.forename: M J
authors.initials: MJ
authors.lastname: Sanson-Le Pors
mesh_terms: D001483:Base Sequence; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology / Q000382:microbiology; D002555:Cerebrospinal Fluid / Q000166:cytology / Q000382:microbiology; D003587:Cytomegalovirus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000382:microbiology*; D015342:DNA Probes; D004352:Drug Resistance, Microbial / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D017245:Foscarnet / Q000494:pharmacology; D015774:Ganciclovir / Q000493:pharmacokinetics / Q000494:pharmacology*; D005814:Genes, Viral; D006801:Humans; D008658:Inactivation, Metabolic; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014556:Urine / Q000166:cytology / Q000382:microbiology; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics*
chemical_list: D015342:DNA Probes; D014764:Viral Proteins; D015678:Viral Structural Proteins; D017245:Foscarnet; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D015774:Ganciclovir