A flow cytometric assay has been developed for the rapid detection and quantitation of MRC-5 cells infected with human cytomegalovirus (HCMV). The ACTG Flow Cytometry/Virology Team has used this technique to develop a standardized method for the rapid determination of the susceptibility or resistance of HCMV clinical isolates to ganciclovir. MRC-5 cells were infected with either cell-free or cell-associated HCMV at an MOI of 0.01 to 1 infectious unit per cell. After incubation at 37C for 72 to 96 hr, in the presence or absence of various concentrations of ganciclovir, the cells were harvested, permeabilized with methanol, and treated with an FITC-labeled Mab to a nuclear HCMV IE antigen and a PE-labeled Mab to a cytoplasmic HCMV late antigen followed by FACS analysis. Using this assay, the ICSO of ganciclovir for AD169, the ganciclovir-sensitive strain of HCMV was between 1.0 and 3.0 micromolar, and the IC50 for each of two ganciclovir-resistant strains of HCMV, XbaF and D6/3/1, was greater than 12 micromolar. These results are in good agreement with the IC50 of the AD169, D6/3/1,and XbaF strains of HCMV obtained by the standard plaque reduction assay which takes approximately 14 days. The flow cytometric assay is rapid, taking only three to four days to complete, the data is collected mechanically as opposed to manual enumeration of stained foci using a microscope, and the assay is easily reproduced among laboratories. This assay should be useful for determining the sensitivity or resistance of HCMV to antiviral compounds that block viral DNA synthesis.

Antibodies, Monoclonal/IMMUNOLOGY Antigens, Viral/IMMUNOLOGY Antiviral Agents/*PHARMACOLOGY Cell Separation Cytomegalovirus/*DRUG EFFECTS/GENETICS/PHYSIOLOGY DNA Replication/DRUG EFFECTS DNA, Viral Flow Cytometry Human Microbial Sensitivity Tests Reproducibility of Results ABSTRACT

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