ELVIRA HSV, a Yield Reduction Assay for Rapid Herpes Simplex Virus Susceptibility Testing

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...

Spectrum of antiviral activity of o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS)

“…While the IC 50 of ACV on HSV-1 KOS replication was 7.7 M, the KOS-derived DP mutant HSV-1 strains PAA r 5, F891C, PFA r 2 and PFA r 5 and the TK mutant HSV strains 98.14742-PE/1, O24, and 97-12961 showed a 2-13-fold decrease in susceptibility to ACV when compared with the parental HSV-1 KOS strain (Table 3). These results correlate well with previous studies [30]. The replication of the DP mutant HSV-1 strains PAA r 5, F891C, PFA r 2 and PFA r 5, the TK mutant HSV-1 strains 98.14742-PE/1 and 97-12961 and the TK mutant HSV-2 strain O24 was inhibited by APHS at concentrations ranging from 24 to 191 M. The IC 50 values of APHS for all ACV-resistant HSV strains tested were statistically …”

“…This assay was performed as described previously [30]. Briefly, HFF cells were seeded at a concentration of 6 × 10 4 cells/ml and incubated for 2 days at 37 • C and 5% CO 2 .…”

Establishment of a Cell-Based Assay for Screening of Compounds Inhibiting Very Early Events in the Cytomegalovirus Replication Cycle and Characterization of a Compound Identified Using the Assay

“…Similar cell lines have been described for several other viruses (34). We and others generated reporter cell lines for herpesviruses, including herpes simplex virus, B virus, HHV-8, and VZV (7,17,34,42,47). Two reporter cell lines for HCMV that depend on the activation of the UL54 (DNA polymerase) promoter were also published previously (13,45).…”

Identification and characterization of acyclovir-resistant clinical HSV-1 isolates from children

“…Genotypic tests were mainly performed through direct sequencing of tk and DNA pol to determine viral susceptibility based on databases of mutations related to ACV resistance. 5,7,8,[12][13][14][15][16] On the other hand, phenotypic evaluations, including plaque reduction assay (PRA), 17 dye uptake assay, and virus yield reduction assay, [18][19][20][21][22] were largely conducted on HSV infections that induced the cytopathic effect. These assays are time consuming, laborious, and hence inconvenient for routine clinical screening.…”