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Human cytomegalovirus (HCMV) resistance to antivirals is a major problem in immunocompromised patients. Drug resistance is characterized by phenotypic testing or genotypic analysis of the phosphotransferase (UL97) and DNA polymerase (UL54) genes. However, genotypic assays require further characterization of unknown mutations in the drug resistance phenotype. Here, we describe a novel method for generating single or multiple mutations anywhere in the HCMV genome and for studying their effects on drug susceptibility. This method is based on cloning of the reference AD169 strain in a bacterial artificial chromosome and the use of “en passant” mutagenesis in bacteria to introduce mutations in recombinant HCMV without scar sequences. We also used this methodology to introduce theGaussia luciferase reporter gene into the genome of the recombinant virus. To validate our system, the well-characterized single mutants with UL97 A594V and UL54 E756K mutations as well as the undescribed A594V/E756K double mutant were generated and their drug susceptibility profiles were determined by measuring the luciferase activity in cell culture supernatants. Drug susceptibility phenotypes for the A594V (8.2-fold increase in ganciclovir 50% effective concentration [EC50 ]) and E756K (1.9-, 3.9-, and 3.0-fold increases in ganciclovir, foscarnet, and cidofovir EC50 s, respectively) mutants were similar to those previously reported, while the double mutant exhibited 10.8-, 4.1-, and 2.0-fold increases in ganciclovir, foscarnet, and cidofovir EC50 s, respectively. The combination of theGaussia luciferase reporter-based assay with the markerless “en passant” mutagenesis methodology constitutes an efficient system for studying phenotypes with single or multiple HCMV mutations.

journal of clinical microbiology

Novel Method Based on “En Passant” Mutagenesis Coupled with a Gaussia Luciferase Reporter Assay for Studying the Combined Effects of Human Cytomegalovirus Mutations