A rapid assay was developed to screen for herpes simplex virus (HSV) isolates that are resistant to acyclovir and other antiviral agents. The assay is a modified plaque reduction assay (PRA) in which the number of plaques seen in the absence of acyclovir was compared with that seen in the presence of a single cutoff concentration of acyclovir (2 microg/mL). This assay utilizes a cell line that expresses beta-galactosidase only after infection with HSV. Since histochemically stained plaques are easily visualized, small plaques can be easily enumerated. This allows the assay to be performed on dilutions of untitered specimens in the small wells of a 24-well plate and allows the results to be read only 2 days after inoculation of the virus. The assay performed well compared with a standard PRA and should be a valuable tool in identifying drug-resistant HSV in a timely manner.
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...
Cryopreserved cell monolayers are a new cell culture technology intended to ensure the availability of cells in the laboratory for virus detection. Two cryopreserved cell monolayers, ELVIS for the detection of herpes simplex virus (HSV) and R-Mix for the detection of influenza virus, were evaluated. The results indicated that fresh and cryopreserved cell monolayers are comparable in sensitivity for the detection of HSV and influenza virus. The cells retain the same level of sensitivity for up to 4 months at ؊80°C.A diagnostic virology laboratory generally receives fresh cells from commercial sources once or twice a week. Receipts of commercially prepared cells can be compromised by shipping delays, mishandling of packages, and exposure to temperature stress, which may lead to suboptimal performance. Additionally, because these shipments are estimated standing orders, shortage or overage is common. Finally, the quality of cell monolayers is variable from lot to lot and difficult to control and standardize. These fundamental shortcomings of commercially prepared cells are generally accepted by clinical virology laboratories because the only functional alternative, i.e., preparing one's own cells each week, is generally impractical for technical and/or economic reasons.Recently, Diagnostic Hybrids Inc. (DHI, Athens, Ohio) developed a cryopreservation method that addresses the practical issues cited above. In this study, we compare two sensitive cell culture systems, ELVIS cells for the rapid detection of herpes simplex virus (HSV) (5, 7) and R-Mix (mixture of A549 and mink lung) cells for the detection of influenza viruses A and B (1, 2, 3, 4, 6), in both frozen and nonfrozen monolayer formats, to determine whether frozen monolayers can match the virus detection performance of fresh, commercially prepared monolayers.The cryopreserved ELVIS and R-Mix cell monolayers (ready cell frozen monolayers [RCFM]) were provided on a glass coverslip in shell vials by DHI. RCFM were shipped on dry ice and transferred quickly to storage in a Ϫ80°C chest freezer. The nonfrozen cell equivalents were commercially produced and shipped by express courier, as is routinely done. Prior to inoculation with clinical specimens, cryopreserved ELVIS RCFM and R-Mix RCFM vials were removed from the Ϫ80°C freezer and placed in an empty 24-well cluster plate. The plate was gently placed in a 35 to 37°C water bath such that the water level was just high enough to flood the plate. The vials were incubated for 4 min (Ϯ15 s) without any agitation. The thawed vials were gently removed from the water bath, and the freeze medium was immediately removed from the vials by gentle aspiration.For ELVIS RCFM, 1 ml of ELVIS replacement medium (DHI) was added to each vial and 0.2 ml of clinical specimen was inoculated into both RCFM and fresh cells. All shell vials were centrifuged at 700 ϫ g for 60 min at room temperature and incubated at 35 to 37°C for 20 to 24 h. ELVIS cells were fixed and stained for HSV detection and typing by using the ELVIS HSV ID/typing...
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