Evaluation of Susceptibility of Human Herpesvirus 8 to Antiviral Drugs by Quantitative Real-Time PCR

Abstract

A new in vitro system based on real-time PCR was developed for evaluation of human herpesvirus 8 susceptibility to antiviral agents. Cidofovir had the greatest inhibitory activity against HHV-8 (50% inhibitory concentration [IC 50 ], 0.43 M) followed by ganciclovir (2.61 M), adefovir (18.00 M), acyclovir (31.00 M), and foscarnet (34.15 M). The potential therapeutic efficacy for HHV-8 (i.e., peak serum drug level/IC 50 ) is highest for cidofovir (167) and foscarnet (22).The human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus, is a new member of the ␥-herpesvirinae subfamily that has been associated with human immunodeficiency virus (HIV)-and non-HIV-related Kaposi's sarcoma (KS) as well as with multicentric Castleman's disease and primary effusion lymphoma (5,6,17,25). Despite the decrease in HIV-related KS with the advent of highly active antiretroviral therapy, there is still an interest in evaluating strategies to inhibit the early stages of HHV-8 replication.Several in vitro systems have been reported for determination of HHV-8 susceptibility to antiviral drugs (10,16,20,22). Because most B cells are latently infected by the virus, inducing agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or sodium butyrate have been used to promote lytic viral replication (23). Also, due to the absence of viral plaques in infected lymphoma cell lines, most susceptibility systems were designed to measure viral DNA synthesis. However, the existing assays are very cumbersome, as they require gel electrophoresis and isotopic hybridization. In this report, we describe the development of a rapid and objective in vitro susceptibility system for HHV-8 based on real-time quantitative PCR.The BCBL-1 cell line, which is a B-cell line latently infected by HHV-8 (23), was kindly provided by Benoît Barbeau (Centre de Recherche en Infectiologie, Ste-Foy, Québec, Canada). The cells were maintained as described previously (16). On day 1, 10 ml of BCBL-1 cells at 2 ϫ 10 5 cells/ml (in RPMI 1640 medium [Life Technologies, Burlington, Ontario, Canada] supplemented with 10% heat-inactivated fetal bovine serum) were pelleted at 250 ϫ g for 10 min and then washed with 2 ml of phosphate-buffered saline. The cell pellet was then resuspended in an equal volume of medium with TPA (without TPA for the negative control) at a final concentration of 20 ng/ml in 25-cm 2 flasks (BD Biosciences, Oakville, Ontario, Canada). Serial concentrations of antivirals, i.e., acyclovir (zovirax; GlaxoSmithKline), foscarnet (Sigma, Oakville, Ontario, Canada), ganciclovir (cytovene; Hoffman La Roche), cidofovir (vistide; Gilead Sciences, Foster City, Calif.), and adefovir (kindly provided by Tomas Cihlar; Gilead Sciences) were made in triplicate and added to culture medium. On day 2, 20 h after TPA stimulation, the cells were repelleted as described above, washed with phosphate-buffered saline, and resuspended in 10 ml of fresh medium containing the same antiviral drugs but without TPA. On day 4 (3 days after the addition of TPA...

Reply to “Calibration Technologies for Correct Determination of Epstein-Barr Virus, Human Herpesvirus 6 (HHV-6), and HHV-8 Antiviral Drug Susceptibilities by Use of Real-Time-PCR-Based Assays”

“…The 50% effective concentrations (EC 50 s) followed the same trend (i.e., cidofovir Ͻ ganciclovir Ͻ acyclovir Ͻ foscarnet) using the two different assays. Moreover, the trends of EC 50 s obtained for the four antivirals against HHV-6, HHV-8, and EBV are in agreement with those published in the literature (9,11,12).…”

“…The 50% effective concentrations (EC 50 s) followed the same trend (i.e., cidofovir Ͻ ganciclovir Ͻ acyclovir Ͻ foscarnet) using the two different assays. Moreover, the trends of EC 50 s obtained for the four antivirals against HHV-6, HHV-8, and EBV are in agreement with those published in the literature (9,11,12).In summary, these real-time-PCR assays for HHV-6, HHV-8, and EBV were not developed to determine viral DNA load in clinical specimens but to evaluate antiviral drug susceptibilities on cell culture supernatants, and as such, they do not require extensive calibration technologies. Indeed, by following appropriate treatments (centrifugation and DNase I), these samples are less prone to contamination with cellular and unencapsidated viral DNA and to the presence of PCR inhibitors, as is the case with clinical specimens.…”

“…Following incubation with nontoxic drug concentrations [as determined by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (8)], aliquots of cell culture supernatants are centrifuged to remove cell-associated virus (either latent or replicating) as well as cellular DNA (9). In addition, the resulting supernatants are treated with DNase I to remove unencapsidated viral DNA that could have resulted from virally induced cytopathic effects (9). Samples are then processed for DNA extraction, and it is thus unlikely that drugs (especially DNA polymerase inhibitors) will still be present in sufficient concentrations to inhibit the PCR.…”

A method for evaluating antiviral drug susceptibility of Epstein-Barr virus

“…On day 2, 20-24 hours after TPA stimulation, the cells were repelleted, washed with phosphate-buffered saline, and resuspended in 10 mL of fresh medium containing the same antiviral drugs but without TPA. 5 On day 4, 10 mL of medium containing the same concentrations of antiviral drugs was added to the flasks. We demonstrated enhanced viability of the P3HR1 cells when TPA was removed on day 2 as compared with TPA being in contact with the cells for all 7 days.…”

Evaluation of Epstein-Barr Virus, Human Herpesvirus 6 (HHV-6), and HHV-8 Antiviral Drug Susceptibilities by Use of Real-Time-PCR-Based Assays

“…Due to the absence of virally induced plaque formation in cell culture, most susceptibility assays described so far for these viruses have measured the viral DNA load (7,8,9). For HHV-8 and EBV, the lytic viral replication must be first activated by treatment of latently infected B cells with 12-O-tetradecanoyl phorbol (TPA) and/or sodium butyrate (10,11).…”