Cell cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere chamber. The virus strains used were: HSV-1 KOS and 29 R (Faculty of Pharmacy, University of Rennes, France), and HSV-2 333 (Department of Clinical Virology, Göteborg University, Sweden). Virus titers were determined Vemurafenib by plaque assay and expressed as plaque forming units (PFU/mL) (Burleson et al., 1992). The cytotoxicity of samples was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983). Briefly, confluent Vero cells were exposed to different sample concentrations for 72 h. The medium was then substituted by the MTT solution and incubated for 4 h. After dissolution

of formazan crystals, optical densities were read (540 nm) and the concentration of each sample that reduced cell viability by 50% (CC50) was calculated based on untreated controls. Subsequently, the potential antiherpetic activity was evaluated by the plaque reduction assay as previously described (Silva et al., 2010). Monolayers of Vero cells grown in 24-well plates were infected with 100 PFU per well of each virus for 1 h at 37 °C. Treatments were performed by adding samples either simultaneously with the virus (simultaneous treatment) or after the virus infection (post-infection treatment). Cells were subsequently covered with CMC medium (MEM containing 1.5% carboxymethylcellulose) and incubated

for 72 h. Cells were then fixed and stained with naphthol blue black and viral plaques was counted. The concentration of each sample required to reduce the

plaque number by 50% (IC50) was calculated by standard method (Burleson et al., selleck inhibitor 1992). Acyclovir (ACV), dextran sulfate (DEX-S), and heparin (HEP) were purchased from Sigma (St. Louis, MO) and used as positive controls. IC50 and CC50 values were estimated by linear regression of concentration–response curves generated from the data. The selectivity index (SI = CC50/IC50) was calculated for each sample. The virucidal assay was conducted as described by Ekblad et al. (2006), with minor modifications. Mixtures of equal sample volumes (20 μg/mL) and 4 × 105 PFU of HSV-1 (KOS and 29-R) or HSV-2 333 in serum-free MEM were co-incubated for Interleukin-2 receptor 20 min at 4 or 37 °C. Samples were then diluted to non-inhibitory concentrations (1:1000) to determine the residual infectivity by plaque reduction assay as described above. Ethanol 70% (v/v) served as a positive control. The attachment and penetration assays followed the procedures described by Silva et al. (2010). In the attachment assay, pre-chilled Vero cell monolayers were exposed to viruses (100 PFU per well), in the presence or absence of the samples. After incubation for 2 h at 4 °C, samples and unabsorbed viruses were removed by washing with cold phosphate-buffered saline (PBS) and cells were overlaid with CMC medium. Further procedures were the same as described above for the plaque reduction assay.