Viability assay and Antiviral activity of NAAB-496 and NAAB-503 in Vero cells.

(A) The cytotoxicity effect of NAABs was evaluated on Vero cells in presence of different concentrations of NAABs compound (0.01, 0.05, 0.06, 0.08, 0.1, 0.15, 0.2, 0.4, 0.6, 0.8 and 1.6 mg/mL) separately. The cell proliferation index (%) was determined at 72 hours of treatment on the basis of ATP levels using the ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay Kit (Lonza Group Ltd., Basel, Switzerland). The CC50 was obtained from nonlinear regression analysis of concentration-effect curves by the GraphPad Prism 6 Demo program and expressed the means ± standard deviation of three independent experiments. (B) Antiviral activity of NAAB-496 and NAAB-503 was evaluated on the basis of plaques reduction assay in Vero cells infected with HSV-1 (F) and incubated in the presence of NAAB 496 and NAAB 503 at different concentrations (0.01, 0.05, 0.06, 0.08 and 0.1 mg/mL). The EC50 was obtained from nonlinear regression analysis of concentration-effect curves by the GraphPad Prism 6 program. (C) Antiviral activity of Acyclovir: Vero cells infected with HSV-1 (F) and incubated for 1 h at 37 °C. After the incubation time, the inoculum was removed, and the monolayers were overlaid with DMEM containing 0.8% methylcellulose in the presence of Acyclovir at 0.5, 1, 10, and 20 μM. The cells were stained with crystal violet for plaques detection. (D) The viral DNA was extracted from Vero cells 24h post HSV-1 infection and NAABs treatment (0.06mg/mL), as described in the Materials and Methods. Quantization of viral DNA was performed using real-time quantitative PCR and using the standard curve generated in parallel and expressed as a concentration of ng/μl. Results are the mean ± SD of three independent experiments (* p < 0.05, ** p < 0.01; *** p < 0.001).