The use of flow cytometry to detect antiviral resistance in human cytomegalovirus.

A method for detecting the antiviral susceptibility of human cytomegalovirus (HCMV) isolates to antiviral agents using flow cytometry was developed. This method has been used to detect the resistance phenotype of HCMV isolates to ganciclovir (GCV). The procedure involves infecting MRC-5 cells with 10(4) pfu HCMV for 120 h, then fixing and permeabilising the cells to allow intracellular labelling of the HCMV early and late antigens. The percentage reduction in the fluorescence positive population of HCMV-infected MRC-5 cells treated with GCV at concentrations of 20 or 50 microM compared with control cultures without GCV was determined. The IC50 defined as a < 50% reduction in the fluorescence positive population in cells infected in the presence of 20 microM GCV or an IC90 defined as a < 90% reduction in the fluorescence-positive population in cells infected in the presence of 50 microM GCV, correlated with resistance determined by a plaque reduction assay. The FACS assay is a rapid and reproducible method for detecting antiviral resistance of HCMV.