Rapid detection by reverse hybridization of mutations in the UL97 gene of human cytomegalovirus conferring resistance to ganciclovir.
BACKGROUND OF STUDY: Diseases due to human cytomegalovirus (HCMV) infection constitute a major threat in marrow and solid organ transplant recipients. Ganciclovir (GCV) is widely used in prophylaxis and pre-emptive therapy of active HCMV infection. Resistance to ganciclovir (GCV) may arise at variable frequency under GCV therapy and is conferred by mutations (i) in the UL97 gene (codons 460, 520, and 591-607) encoding a phosphotransferase which is essential for monophosphorylation of GCV and, to a lesser extent, (ii) in the UL54 gene coding for the DNA polymerase of HCMV.


OBJECTIVE
The purpose was to develop a rapid assay to screen for emerging GCV resistance mutations in the UL97 gene of HCMV whereby avoiding virus isolation and nucleotide sequencing procedures.


METHODS
A nested PCR (nPCR) amplifying UL97 codons 450-672 was developed. Nested amplicons were subsequently sequenced directly. Oligonucleotides for use in a reverse hybridization assay were designed to detect relevant non-synonymous mutations at codons UL97 460, 520, 603 and 607. Strain AD169 served as a wild-type control.


RESULTS
UL97-specific nPCR amplicons were obtained from 18 EDTA blood samples of ten transplant recipients receiving GCV for more than 30 days. In three consecutive samples from a single patient a GCV resistance mutation at codon 603 (C-->W) was detected. In addition, two out of four cell culture-adapted HCMV isolates known to exhibit GCV resistance in vitro revealed mutations at codons 460 (M-->V) and 607 (C-->Y), respectively. By reverse hybridization a discrimination of single nucleotide changes at codons 460, 520, 603 and 607 was possible whereby matching exactly the results of the nucleotide sequence analysis for all 23 amplicons examined.


CONCLUSIONS
Reverse hybridization appeared to be a rapid and convenient alternative to nucleotide sequencing when screening the UL97 gene of HCMV for selected markers of GCV resistance.
Department of Microbiology, Zhejiang Medical University, Hangzhou, People's Republic of China.
L
L
Zhou
T C
TC
Harder
U
U
Ullmann
P
P
Rautenberg
D000998:Antiviral Agents / Q000494:pharmacology*; D015331:Cohort Studies; D003587:Cytomegalovirus / Q000235:genetics*; D003586:Cytomegalovirus Infections / Q000821:virology; D004352:Drug Resistance, Microbial / Q000235:genetics; D015774:Ganciclovir / Q000494:pharmacology*; D016041:Heart-Lung Transplantation; D006801:Humans; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D000998:Antiviral Agents; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D015774:Ganciclovir