Application of Real-Time PCR for Determination of Antiviral Drug Susceptibility of Herpes Simplex Virus

Abstract

A quantitative real-time PCR (TaqMan) assay was developed for determination of antiviral drug susceptibility of herpes simplex virus (HSV). After short-time culture of the virus, the antiviral drug susceptibility of HSV isolates for acyclovir (ACV) was determined by measuring the reduction of the HSV type 1 (HSV-1) DNA levels in culture supernatants using real-time PCR. The 50% inhibitory concentration was reported as the concentration of antiviral drug that reduced the number of HSV-1 DNA copies by 50%. A total of 15 wellcharacterized ACV-sensitive or -resistant strains and clinical isolates were used for assay evaluation. The new assay with real-time PCR readout permitted rapid (3 days), objective, and reproducible determination of HSV-1 drug susceptibilities with no need for stringent control of initial multiplicity of infection. Furthermore, the real-time PCR assay results showed good correlation (r ‫؍‬ 0.86) with those for the plaque reduction assay. In conclusion, the real-time PCR assay described here is a suitable quantitative method for determination of antiviral susceptibility of HSV-1, amenable for use in the routine diagnostic virology laboratory.Extensive use of acyclovir and other antiviral drugs for prophylaxis and treatment of herpes simplex virus (HSV) infections exerts a continuous selection pressure on the HSV virus population. HSV antiviral drug resistance occurs relatively frequently especially in immunocompromised patients such as those undergoing bone marrow (6 to 12%) or solid organ transplantation (ϳ4%) or AIDS patients (ϳ6%) Several phenotypic assays have been described, and some of them are used in clinical practice, with the plaque reduction assay (PRA) as the most frequently used drug susceptibility assay. Although this technique is laborious and time-consuming, it still remains the "gold standard" method by which other tests are evaluated (24). The majority of alternative susceptibility assays is based on reduction in cytopathic effect (CPE), which is either microscopically evaluated or colorimetrically detected (7,13,15,19,23,29). Assays based on enzyme-linked immunosorbent assay (ELISA) include the sandwich ELISA (33) and the microplate in-situ ELISA (MISE-test) (16,21,25). The latter has been shown to correlate well with PRA. Other currently used antiviral susceptibility assays involve the use of DNA hybridization (9,30,31), flow cytometric analysis (20) and transgenic HSV inducible reporter cells (32).With the increasing numbers of immunocompromised individuals, there is a need for the widespread routine availability of antiviral drug susceptibility assays, which would be rapid, reproducible and clinically relevant. Currently used methods, except for the MISE-test, suffer from certain pitfalls, which preclude their routine use. Most of the assays are time-consuming and labor-intensive; some may have subjective endpoints, require special equipment or trained laboratory personnel. Therefore we set out to develop an assay, which would overcome most of the aforementioned restrictio...

Antiviral Activity of Myticin C Peptide from Mussel: an Ancient Defense against Herpesviruses

“…Because the highest concentration of Myt-Tat used in these assays did not allow calculation of the CC 50 value, it can only be suggested that the CC 50 would be higher than 32.6 M. Therefore, the putative SI values for Myt-Tat would be Ͼ8.21 against HSV-1 and Ͼ10.5 against HSV-2. The EC 50 s obtained for ACV, which was used as a positive control in the antiviral activity assay (EC 50 s, 0.9 g/ml for HSV-1 and 1.1 g/ml for HSV-2), are in agreement with previous studies (32,33) and suggested that both the HSV-1 and HSV-2 isolates used throughout this study are sensitive to acyclovir (EC 50 Ͻ 2 g/ml) (34).…”

Real-Time PCR as a Versatile Tool for Investigating the Susceptibility of Human Herpesvirus 6 to Antiviral Agents

“…According to preliminary data, the overall efficiency of PCR assay was not modified when either a 10-foldhigher MOI or primary HHV-6 isolates growing on peripheral blood mononuclear cells were used (not shown). This reflected its robustness and adaptability, confirming similar recent results obtained with a herpes simplex virus real-time PCR assay (13).…”

In vitro anti-herpetic activity of an aqueous extract from the plant Phyllanthus orbicularis

“…The ACV EC 50 values obtained were 2.8 mg/ml for HSV-1 strain 8ACV, previously described as ACV-resistant; 1.2 mg/ml for the ACV-sensitive HSV-1 strain 2677, and 5.1 mg/ml for the ACV-resistant HSV-2 clinical isolate 2373. It should be mentioned that an EC 50 value of 2 mg/ml or greater, is considered as indicative of ACV resistance of a HSV strain, as has been previously reported (Stranska et al 2002).…”