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Antimicrobial Agents and Chemotherapy, June 2004, p . 2331-2333, Vol . 48, No . 6
ELVIRA HSV, a Yield Reduction Assay for Rapid Herpes Simplex Virus Susceptibility Testing
R  ena Stránská,1* Rob Schuurman,1 David R . Scholl,2 Joseph A . Jollick,2 Carl J . Shaw,2 Caroline Loef,1 Merjo Polman,1 and Anton M . van Loon1
Department of Virology, Eijkman-Winkler Center, University Medical Center Utrecht, Utrecht, The Netherlands,1
Diagnostic Hybrids, Inc., Athens, Ohio2
Received 6 September 2003/
Returned for modification 6 November 2003/
Accepted 20 February 2004
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 HSV-inducible 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 ß-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 BHKICP6LacZ-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 ß-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 ß-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 x 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 ß-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-ß-D-galactopyranoside monosodium salt [3 mg/ml; Roche Diagnostics, Almere, The Netherlands] and 4.35 mM magnesium chloride in phosphate-buffered saline) (Fig . 1) .
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FIG . 1 . Schematic overview of ELVIRA HSV procedure . RT, room temperature; OD, optical density.
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The optimal incubation period of the culture amplification step was determined with virus inocula ranging from 37.5 to 600 PFU/ml (multiplicities of infection [MOI], 0.00013 to 0.00200 PFU/cell) and by addition of ELVIRA cells after 12, 16, 20, and 24 h . An increased incubation period postinfection resulted in an increased sensitivity of the assay (Fig . 2) . Similar data were obtained for HSV-2 (data not shown) . An incubation period of 22 to 24 h was chosen for further use . The incubation period of the detection system was based on previously determined ß-galactosidase expression kinetics (6, 9) . The ELVIRA HSV had a linear range from approximately 70 to 300 PFU/ml ( MOI, 0.0002 to 0.0010 PFU/cell) .
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FIG . 2 . The effect of duration of infection of HFF cells on sensitivity of ELVIRA HSV . HFF cell monolayers were infected with a range of HSV-1 inocula; and ELVIRA reporter cells were added after 12, 16, 20, and 24 h . Results are representative of four independent experiments performed in triplicate with HSV-1; error bars indicate SDs . OD, optical density.
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The utility of the assay for susceptibility testing was evaluated (in parallel with PRA) with well-characterized ACV- and PFA-sensitive and -resistant HSV strains (n = 6) and clinical isolates (n = 16) with various susceptibilities to ACV and PFA (Table 1; Fig . 3) . The antiviral effects of ACV and PFA measured by ELVIRA HSV corresponded to the results of PRA . The data in Table 1 clearly demonstrate the ability of ELVIRA HSV to discriminate between HSV strains sensitive and resistant to both drugs examined . The ACV 50% inhibitory concentrations (IC50s) obtained by ELVIRA HSV showed a good correlation with the results of PRA (rACV = 0.94) . The IC50s for ACV-sensitive strains obtained by ELVIRA HSV were lower than those obtained by PRA (P = 0.002) . For ACV-resistant strains, ELVIRA HSV showed higher mean IC50s than PRA (P = 0.0005) . The PFA IC50s obtained by ELVIRA HSV did not differ from those obtained by PRA for sensitive (P = 0.21) and resistant strains (P = 0.75); however, the number of strains tested was small .
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TABLE 1 . Results of ACV and PFA susceptibility testing by ELVIRA HSV and PRA with well-characterized reference strains and clinical isolates
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FIG . 3 . Susceptibilities to ACV and PFA of well-characterized HSV strains and clinical isolates and clinical isolates from untreated patients determined by ELVIRA HSV (the results represent data combined from Table 1 and the Results).
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The interassay variability of the IC50s, expressed as a coefficient of variation (CV), ranged from 4 to 33% (Table 1), with similar mean CVs for sensitive and resistant strains (21.2 and 20.6%, respectively) .
The natural variation in the susceptibilities of HSV strains to ACV and PFA was tested with HSV-1 clinical isolates from untreated patients . Isolates from 18 patients were tested for their susceptibilities to both drugs, and additional isolates from 12 and 15 patients were tested for their susceptibilities to ACV and PFA, respectively . The median ACV IC50 was 0.17 µg/ml (range, 0.01 to 0.57 µg/ml; standard deviation [SD], 0.12 µg/ml), and the median PFA IC50 was 20.98 µg/ml (range, 9 to 47.1 µg/ml; SD, 8.5 µg/ml) . On the basis of the susceptibility data for the clinical isolates from untreated patients, the well-characterized strains, and clinical isolates (Table 1) and on the basis of the reproducibility of ELVIRA HSV, preliminary ACV and PFA IC50 cutoffs were set at 0.8 and 60 µg/ml, respectively .
ELVIRA HSV demonstrated a 100% correlation with PRA for the discrimination between ACV- and PFA-sensitive and -resistant HSV of both serotypes . The reproducibility of the assay was acceptable, with interassay CVs ranging from 4 to 43% . The early expression of ß-galactosidase in reporter ELVIRA cells results in rapid (pre-cytopathic effect [CPE]) virus detection (6) . The whole assay is thus more rapid than CPE- or DNA-based assays, which require more replication cycles . In a diagnostic laboratory setting, the ELVIRA HSV susceptibility result can be available within 5 days from the time of specimen arrival, which also includes the time needed for determination of the virus titer in the specimen .
In conclusion, we describe the development and evaluation of a novel colorimetric, transgenic cell-based assay, ELVIRA HSV, that can be used for rapid determination of HSV drug susceptibility and also for evaluation of new antiviral compounds . The assay can be used to determine the susceptibilities of HSV-1 and HSV-2 to antiviral drugs even in specimens with low virus titers . The short turn-around time, easy-to-use format, objective endpoint, and good reproducibility make ELVIRA HSV amenable for use in the routine diagnostic virology laboratory . The testing of larger number of sensitive and resistant clinical isolates is warranted for further validation of the assay .
We thank M . Aymard (Universite Claude Bernard, Lyon, France), D . M . Coen (Harvard Medical School, Boston, Mass.), and A . Linde (Swedish Institute for Infectious Disease Control, Solna, Sweden) for providing HSV control strains and clinical isolates .
* Corresponding author . Mailing address: Department of Virology, G04.614, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands . Phone: 31 30 250 6526 . Fax: 31 30 250 5426 . E-mail: r.stranska{at}azu.nl .
- Chen, Y., C . Scieux, V . Garrait, G . Socie, V . Rocha, J . M . Molina, D . Thouvenot, F . Morfin, L . Hocqueloux, L . Garderet, H . Esperou, F . Selimi, A . Devergie, G . Leleu, M . Aymard, F . Morinet, E . Gluckman, and P . Ribaud. 2001 . Resistant herpes simplex virus type 1 infection: an emerging concern after allogeneic stem cell transplantation . Clin . Infect . Dis . 31:927-935.
- Christophers, J., J . Clayton, J . Craske, R . Ward, P . Collins, M . Trowbridge, and G . Darby. 1998 . Survey of resistance of herpes simplex virus to acyclovir in northwest England . Antimicrob . Agents Chemother . 42:868-872.
- Danve-Szatanek, C., M . Aymard, D . Thouvenot, F . Morfin, G . Agius, I . Bertin, S . Billaudel, B . Chanzy, M . Coste-Burel, L . Finkielsztejn, H . Fleury, T . Hadou, C . Henquell, H . Lafeuille, M . E . Lafon, A . Le Faou, M . C . Legrand, L . Maille, C . Mengelle, P . Morand, F . Morinet, E . Nicand, S . Omar, B . Picard, B . Pozzetto, J . Puel, D . Raoult, C . Scieux, M . Segondy, J . M . Seigneurin, R . Teyssou, and C . Zandotti. 2004 . Surveillance network for herpes simplex virus resistance to antiviral drugs: 3-year follow-up . J . Clin . Microbiol . 42:242-249.
- Morfin, F., and D . Thouvenot. 2003 . Herpes simplex virus resistance to antiviral drugs . J . Clin . Virol . 26:29-37.
- Safrin, S., T . Elbeik, L . Phan, D . Robinson, J . Rush, A . Elbaggari, and J . Mills. 1994 . Correlation between response to acyclovir and foscarnet therapy and in vitro susceptibility result for isolates of herpes simplex virus from human immunodeficiency virus-infected patients . Antimicrob . Agents Chemother . 38:1246-1250.
- Stabell, E . C., and P . D . Olivo. 1992 . Isolation of a cell line for rapid and sensitive histochemical assay for the detection of herpes simplex virus . J . Virol . Methods 38:195-204.
- Stranska, R., A . M . van Loon, R . G . Bredius, M . Polman, E . Nienhus, M . F . C . Beersma, A . C . Lankester, and R . Schuurman. 2004 . Sequential switching of DNA polymerase and thymidine kinase mediated HSV-1 drug resistance in an immunocompromised child . Antivir . Ther . 9:99-104.
- Swierkosz, E . M. 2000 . Antiviral susceptibility testing, p . 154-168 . In S . Specter, R . L . Hodinka, and S . A . Young (ed.), Antiviral drug susceptibility testing . ASM Press, Washington, D.C.
- Tebas, P., D . Scholl, J . Jollick, K . McHarg, M . Arens, and P . D . Olivo. 1998 . A rapid assay to screen for drug-resistant herpes simplex virus . J . Infect . Dis . 177:217-220.
- Tebas, P., E . C . Stabell, and P . D . Olivo. 1995 . Antiviral susceptibility testing with a cell line which expresses ß-galactosidase after infection with herpes simplex virus . Antimicrob . Agents Chemother . 39:1287-1291.
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