Sequencing of cytomegalovirus UL97 gene for genotypic antiviral resistancetesting

The widespread use of ganciclovir (GCV) to treat cytomegalovirus (CNV) infe ctions in immunosuppressed patients has led to the development of drug resi stance. Phenotypic assays for CW drug resistance are presently too time-con suming to be therapeutically useful. To support the development of genotypi c assays for GCV resistance, the complete sequences of the UL97 phosphotran sferase genes in 28 phenotypically GCV-sensitive CMV clinical isolates were determined. The gene was found to be highly conserved, with nucleotide seq uence identity among strains ranging from 98.6 to 100% and amino acid seque nce identity of > 99%. Primers for a genotypic assay were designed to ampli fy codons 400 to 707, because all known UL97 mutations conferring drug resi stance 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 mut ations. This genotypic assay can be performed in 48 h using genomic DNA ext racted from cell monolayers at very low levels of virus infectivity, thus r apidly providing therapeutically useful results.