Phenotyping of Cytomegalovirus Drug Resistance Mutations by Using Recombinant Viruses Incorporating a Reporter Gene

Treatment of cytomegalovirus (CMV) infections with current antiviral drugs ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV) may select for viral drug resistance mutations. CMV UL97 phosphotransferase gene mutations at codons 460, 520, and 590 to 607 have been shown to confer ganciclovir resistance (1, 4), while an increasingly diverse set of viral UL54 DNA polymerase (pol) mutations confer varying degrees of resistance and cross-resistance to the three drugs (2, 3). Resistance of CMV to antiviral drugs may be diagnosed genotypically, by looking for known viral resistance mutations in UL97 or pol, or phenotypically, by applying various drug concentrations to a growing virus in cell culture. Genotypic analysis is rapid and can be performed without the need for viral isolation, but mutations of indeterminate significance for resistance continue to be reported without phenotypic validation (8) and the degree of resistance conferred by combinations of mutations may be difficult to deduce by genotypic testing alone. Phenotypic analysis detects drug resistance without the need for genetic information, but even if a CMV isolate is available for testing, the standard plaque reduction assay is slow, technically demanding, and shows a high degree of interlaboratory variability (6). For diagnosis of CMV drug resistance in a clinical setting, genotypic methods are more practical, but this requires the continuing study of phenotypes corresponding to new mutations encountered in clinical isolates. The drug resistance phenotype corresponding to specific viral mutations can be determined by transferring them to a known drug-sensitive reference strain and testing for the resulting change in drug sensitivity. In the past few years this process, known as marker transfer or recombinant phenotyping, has been facilitated by the development of CMV strains containing unique restriction sites in UL97 or pol that permit the rapid generation of recombinant viruses containing desired mutations (2, 4). Phenotyping of the resulting recombinant viruses then becomes the rate-limiting step, especially when performed by the standard plaque reduction assay. Here, we report the construction of CMV strains containing unique restriction sites in UL97 and pol, along with a secreted alkaline phosphatase (SEAP) reporter gene inserted at the nonessential gene region US6. This enabled the generation of recombinant viruses containing desired mutations in either or both UL97 and pol, which could be quantitated by simply measuring the SEAP activity in the culture supernatant. Reduction in SEAP activity under drug was used to determine drug sensitivity. Results of transferring specific mutations were compared with results previously obtained using traditional phenotyping assays, and the effects of combinations of mutations were studied for the first time. (Presented in part at the Infectious Diseases Society of America Annual Meeting, Boston, Mass., October 2004 [abstract 641] and the 44th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, October 2004 [abstract 615].)