Improved DNA hybridization method for detection of acyclovir-resistant herpes simplex virus.

Abstract

A simplified DNA hybridization method was developed to detect acyclovir-resistant isolates of herpes simplex virus. Herpes simplex virus-infected cell cultures in microtiter plates were treated with concentrations of acyclovir ranging from 8 to 0.015 micrograms/ml. At 48 h postinfection, infected cells were lysed by a one-step procedure and lysates were absorbed to membranes. Without further treatment, membranes were hybridized by using a herpes simplex virus-specific radioiodinated probe. The membranes were then washed and counted in a gamma counter. The elapsed time for assay performance was 4 h. Parallel plaque reduction assays were performed for comparison. The mean 50% inhibitory dose of in vivo- and in vitro-derived acyclovir-resistant, thymidine kinase-negative isolates was greater than 2 micrograms/ml by DNA hybridization. The 50% inhibitory dose of acyclovir-susceptible, thymidine kinase-positive isolates ranged from 0.01 to 1.1 micrograms/ml. This assay is simple and objective and should facilitate antiviral susceptibility testing in diagnostic laboratories.