Rapid detection of cytomegalovirus strains resistant to ganciclovir through mutations within the gene UL97.
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)
Alain
S
S
Service de Bactériologie-Virologie, Hôpital Lariboisière, Université Paris VII, France.
Mazeron
M C
MC
Pépin
J M
JM
Morinet
F
F
Raskine
L
L
Sanson-Le Pors
M J
MJ
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*
D015342:DNA Probes; D014764:Viral Proteins; D015678:Viral Structural Proteins; D017245:Foscarnet; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D015774:Ganciclovir