After that, to screen antiherpes activity, 5-Fluoracil datasheet a plaque reduction assay was performed following the general procedures described by Silva et al. (2010). Cell monolayers were infected with approximately 100 PFU of each virus for 1 h at 37 °C and then were overlaid with MEM containing 1.5% carboxymethylcellulose (CMC; Sigma) either with the presence or absence of different concentrations of the compounds. After 48 h (HSV-2) or 72 h (HSV-1) of incubation at 37 °C, cells were fixed and stained with naphthol blue–black (Sigma),

and plaques were counted. The IC50 of each compound was calculated as the concentration that inhibited 50% of viral plaque formation, when compared to untreated controls. Acyclovir was used as positive control. find more The selectivity index

(SI = CC50/IC50) was calculated for each tested compound. To investigate the potency of the detected antiherpes activity, an yield reduction assay was performed as previously described by Hussein et al. (2008). Vero cell monolayers were infected with HSV-1 at three different MOI (0.004, 0.04 and 0.4) for 1 h at 37 °C. Cells were washed, different concentrations of glucoevatromonoside were added, and the plates incubated during 72 h at 37 °C. After, culture supernatants were harvested and virus titers were calculated by plaque reduction assay as previously described. The virucidal assay was conducted as described by Ekblad et al. (2006), where the mixtures of serial two-fold dilutions of glucoevatromonoside and 4 × 104 PFU of HSV-1 in serum free MEM were co-incubated for 15 min at 37 °C prior to the dilution of these mixtures to non-inhibitory concentrations of this compound (1:100). The residual infectivity was Bay 11-7085 determined by viral plaque reduction assay as described above. The pretreatment (Bettega et al., 2004) was performed with Vero cell monolayers, which were pretreated with different concentrations of glucoevatromonoside

for 3 h at 37 °C prior virus infection. After washing, cells were infected with 100 PFU of HSV-1 for 1 h at 37 °C. The infected cells were washed, overlaid with MEM containing 1.5% CMC, incubated for 72 h, and treated as described earlier for plaque reduction assay. For the simultaneous treatment (Onozato et al., 2009), 100 PFU of HSV-1 and different concentrations of glucoevatromonoside were added concomitantly to Vero cells for 1 h at 37 °C. After washing, cells were overlaid with MEM containing 1.5% CMC, incubated for 72 h, and treated as described earlier for plaque reduction assay. The attachment and penetration assays followed the procedures also described by Silva et al. (2010).