Real-time PCR as a versatile tool for investigating the susceptibility of human herpesvirus 6 to antiviral agents

Abstract

A quantitative real-time PCR assay was developed for the determination of antiviral drug susceptibility and growth kinetics of human herpesvirus 6. The susceptibility and fitness of a sensitive strain, HST, and its ganciclovir-resistant derivative, GCVR1, were then characterized, leading us to conclude that the mutations of this latter virus did not alter its fitness significantly.

FIG. 1.

Growth curves of HST and GCVR1 in individual infections. MT4 cells were infected either by a standardized inoculum of HST (A) or GCVR1 (B). Infection was carried out in the absence of drug (open symbols) and in the presence of 4 μM (gray symbols) or 32 μM (black symbols) GCV. Every 2 days postinfection, cells were collected, and HHV-6 DNA was quantified by real-time PCR. The number of cells in samples was determined by the same approach, and the final result was expressed as the number of HHV-6 equivalent genome copies per cell (EqCop/cell).

FIG. 2.

Growth curves of HST and GCVR1 in coculture experiments. MT4 cells were simultaneously infected with HST (shaded columns) and GCVR1 (open columns). Incubation was performed in the absence of drug (A) and in the presence of 4 μM (B) or 32 μM (C) GCV. At the indicated times, overall viral load was measured (upper lines with triangles) as described in the legend for Fig. 1 and expressed as the number of HHV-6 equivalent genome copies per cell (EqCop/cell). The ratio of the two strains was measured by quantitative analysis of restriction fragment length polymorphism and expressed for each strain as the percentage of total virus present (histograms in the lower part of each panel).