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...