TY - JOUR
T1 - Sequencing of cytomegalovirus UL97 gene for genotypic antiviral resistance testing
AU - Lurain, N. S.
AU - Weinberg, A.
AU - Crumpacker, C. S.
AU - Chou, S.
PY - 2001
Y1 - 2001
N2 - The widespread use of ganciclovir (GCV) to treat cytomegalovirus (CMV) infections in immunosuppressed patients has led to the development of drug resistance. Phenotypic assays for CMV drug resistance are presently too time-consuming to be therapeutically useful. To support the development of genotypic assays for GCV resistance, the complete sequences of the UL97 phosphotransferase genes in 28 phenotypically GCV-sensitive CMV clinical isolates were determined. The gene was found to be highly conserved, with nucleotide sequence identity among strains ranging from 98.6 to 100% and amino acid sequence identity of >99%. Primers for a genotypic assay were designed to amplify codons 400 to 707, because all known UL97 mutations conferring drug resistance occur at three sites within this region. This part of the UL97 gene was amplified from over 50 clinical isolates, and two sequencing reactions for the coding strand were successfully used to identify GCV resistance mutations. This genotypic assay can be performed in 48 h using genomic DNA extracted from cell monolayers at very low levels of virus infectivity, thus rapidly providing therapeutically useful results.
AB - The widespread use of ganciclovir (GCV) to treat cytomegalovirus (CMV) infections in immunosuppressed patients has led to the development of drug resistance. Phenotypic assays for CMV drug resistance are presently too time-consuming to be therapeutically useful. To support the development of genotypic assays for GCV resistance, the complete sequences of the UL97 phosphotransferase genes in 28 phenotypically GCV-sensitive CMV clinical isolates were determined. The gene was found to be highly conserved, with nucleotide sequence identity among strains ranging from 98.6 to 100% and amino acid sequence identity of >99%. Primers for a genotypic assay were designed to amplify codons 400 to 707, because all known UL97 mutations conferring drug resistance occur at three sites within this region. This part of the UL97 gene was amplified from over 50 clinical isolates, and two sequencing reactions for the coding strand were successfully used to identify GCV resistance mutations. This genotypic assay can be performed in 48 h using genomic DNA extracted from cell monolayers at very low levels of virus infectivity, thus rapidly providing therapeutically useful results.
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U2 - 10.1128/AAC.45.10.2775-2780.2001
DO - 10.1128/AAC.45.10.2775-2780.2001
M3 - Article
C2 - 11557468
AN - SCOPUS:0034812453
SN - 0066-4804
VL - 45
SP - 2775
EP - 2780
JO - Antimicrobial agents and chemotherapy
JF - Antimicrobial agents and chemotherapy
IS - 10
ER -