Novel method based on real-time cell analysis for drug susceptibility testing of herpes simplex virus and human cytomegalovirus

Abstract

The plaque reduction assay (PRA) is the gold standard phenotypic method to determine herpes simplex virus (HSV) and human cytomegalovirus (HCMV) susceptibilities to antiviral drugs. However, this assay is subjective and labor intensive. Here, we describe a novel antiviral phenotypic method based on real-time cell analysis (RTCA) that measures electronic impedance over time. The effective drug concentrations that reduced by 50% (EC50s) the cytopathic effects induced by HSV-1 and HCMV were evaluated by both methods. The EC50s of acyclovir and foscarnet against a reference wild-type (WT) HSV-1 strain in Vero cells were, respectively, 0.5 +Mand 32.6 +Mby PRA and 0.8 +Mand 93.6 +Mby RTCA. The EC50 ratios for acyclovir against several HSV-1 thymidine kinase (TK) mutants were 101.8, 73.4, 28.8, and 35.4(PRA) and 18.0, 52.0, 5.5, and 87.8 (RTCA) compared to those for the WT. The EC50 ratios for acyclovir and foscarnet against the HSV-1 TK/DNA polymerase mutant were 182.8and 9.7(PRA) and>125.0and 10.8(RTCA) compared to the WT. The EC50s of ganciclovir and foscarnet against WT HCMV strain AD169 in fibroblasts were, respectively, 1.6 +Mand 27.8 +Mby PRA and 5.0 +Mand 111.4 +Mby RTCA. The EC50 ratios of ganciclovir against the HCMV UL97 mutant were 3.8(PRA) and 8.2(RTCA) compared to those for the WT. The EC50 ratios of ganciclovir and foscarnet against the HCMV UL97/DNA polymerase mutant were 17.1and 12.1 (PRA) and 14.7and 4.6(RTCA) compared to those for the WT. RTCA allows objective drug susceptibility testing of HSV and HCMV and could permit high-throughput screening of new antivirals.