Phenotypic diversity of cytomegalovirus DNA polymerase gene variants observed after antiviral therapy

Background: Cytomegalovirus UL54 DNA polymerase mutations observed in clinical specimens are of diagnostic significance if confirmed to affect antiviral drug susceptibility. Objectives: Validate an updated recombinant phenotyping method to determine the degree of drug resistance conferred by previously uncharacterized UL54 sequence variants, in comparison with known resistance-related mutations. Study design: Bacterial artificial chromosome clones of viral DNA were mutagenized by recombination, transfected to produce live virus and phenotyped by standardized reporter-based yield reduction assays. Results: Sixteen recombinant viruses were constructed, representing baseline sequences, known resistance-related mutations and amino acid changes of unproven significance from clinical specimens. Phenotypes of baseline strains and known mutants were comparable to results from prior methods and helped to resolve some published inconsistencies. Mutations F412L, F412S, L545W were newly confirmed to confer ganciclovir and cidofovir resistance, while Q578H conferred ganciclovir and foscarnet resistance with borderline cidofovir resistance. Some foscarnet-resistant mutants were appreciably growth-retarded. Conclusions: Results add to known exonuclease domain mutations that confer ganciclovir-cidofovir cross-resistance, polymerase domain mutations that confer foscarnet resistance with variably decreased ganciclovir/cidofovir susceptibility, and increase the list of sequence variants with no measurable impact on drug susceptibility. The phenotypic diversity of similar UL54 genotypic variants complicates the interpretation of genotypic resistance testing. Technical improvements are facilitating the phenotyping of remaining unknown sequence variants.