No abstract

A colorimetric yield reduction assay, ELVIRA (enzyme-linked virus inhibitor reporter assay) HSV, was developed to determine the antiviral drug susceptibilities of herpes simplex virus (HSV). It uses an HSVinducible reporter cell line. This simple and rapid assay has an objective readout, low inoculum size, and good reproducibility. The results correlate well with those of the plaque reduction assay.The prevalence of herpes simplex virus (HSV) infections caused by a drug-resistant virus in immunocompromised patients has been demonstrated to be significant (3.5 to 7.1%) (1-4). This underlines the clinical importance of HSV drug susceptibility determinations for this patient group. The "gold standard," the plaque reduction assay (PRA), is laborious and time-consuming and has a subjective endpoint, and the results are often obtained too late to play a role in therapeutic decision making (5). There has been a considerable effort to develop less laborious and more rapid assays (8). One of the strategies was a modified PRA, which used a transgenic cell line expressing ␤-galactosidase upon infection with HSV and microscopic counting of blue plaques as a readout (9, 10).We describe a rapid, quantitative colorimetric antiviral drug susceptibility assay, ELVIRA (enzyme-linked virus inhibitor reporter assay) HSV (Diagnostic Hybrids, Inc.). The assay is based on the HSV-inducible reporter cell line BHKICP6 LacZ-5 (ELVIRA cells) stably transformed with the Escherichia coli lacZ gene under the control of the HSV type 1 (HSV-1) early promoter ICP6, which expresses ␤-galactosidase upon HSV infection (6). A yield reduction assay was set up in which virus is inoculated on human fibroblasts in the presence of antiviral drug. Subsequently, reporter ELVIRA cells, which represent an overlay readout cell line, are added. The ␤-galactosidase activity in the cell lysates reflects the number of infected reporter cells and, thereby, the yield of infectious virus after drug action.Confluent HFF cells were inoculated in triplicate with 0.1 ml of virus suspension and 0.1 ml of culture medium containing antiviral drugs (acyclovir [ACV] and foscarnet [PFA]) at different concentrations (7). After centrifugation (700 ϫ g)-enhanced virus adsorption for 1 h and incubation overnight at 37°C, a suspension of reporter ELVIRA cells (Diagnostic Hybrids, Inc., Athens, Ohio) was prepared from frozen stocks (final concentration, 29,000 cells/ml). The culture supernatant was aspirated, and 0.2 ml of the ELVIRA cell suspension was added and was allowed to settle. After overnight incubation, the culture supernatant was aspirated, 0.15 ml of 0.03% sodium desoxycholate solution was added, and cell cultures were lysed for 30 min. The ␤-galactosidase activity in the lysates was determined spectrophotometrically (optical density at 570 nm) after incubation for 15 to 90 min at 37°C with 0.1 ml of substrate solution (chlorophenol red-␤-D-galactopyranoside monosodium salt [3 mg/ml; Roche Diagnostics, Almere, The Netherlands] and 4.35 mM magnesium chloride in phosphat...

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...