Assays of antiviral activity and cytotoxicity were evaluated by the modified sulforhodamine B (SRB) assay previously described and recently reported by Choi et al. Ribavirin was purchased from DUCHEFA (Netherlands); azidothymidine, acyclovir, amantadine, and lamivudine were purchased from Sigma-Aldrich (St. Louis, MO, USA), as was SRB.
One day before infection, Vero cells were seeded onto a 96-well culture plate at a concentration of 2 × 104 cells per well. Next day, the medium was removed and then washed with 1 × phosphate buffered saline (PBS). Infectivity of the virus stock was determined by the SRB method and was determined as infectivity of the virus by SRB ID50 (50% infective dose). Following this, 0.09 mL of diluted virus suspension of ECV containing CCID50 (50% cell culture infective dose) of the virus stock to produce an appropriate cytopathic effects within 2 days after infection and 0.01 mL of medium containing an appropriate concentration of the compounds were added. The antiviral activity of each test material was determined with a 10-fold diluted concentration ranging from 0.1 to 100 μL/mL. Three wells were used as virus controls (virus-infected non-drug-treated cells) while three wells were used as cell controls (non-infected non-drug-treated cells). The culture plates were incubated at 37°C in 5% CO2 for 2 days. After washing once with 1 × PBS, 100 μL of cold (-20°C), 70% acetone was added to each well and left for 30 min at -20°C. 70% acetone was removed and 96-well plates were left at dry oven for 30 min. 100 μL of 0.4% (w/v) SRB in 1% acetic acid solution was added to each well and left at room temperature for 30 min. Unbound SRB was removed and the plates were washed 5 times with 1% acetic acid before oven drying and were then left in a dry oven for 1 day. Bound SRB was solubilized with 100 μL of 10 mM unbuffered tris-base solution and plates were left on a table for 30 min. The absorbance was read at 540 nm by using a VERSAmax microplate reader (Molecular Devices, Palo Alto, CA, USA) with a reference absorbance at 620 nm. To calculate the IC50 (50% inhibitory concentration) values, the results were transformed to percentage of controls and the IC50 values were graphically obtained from the dose-response curves.
2.4 Cytotoxicity assay
Vero cells were seeded into a 96-well culture plate at a concentration of 2 × 104 cells per well. Next day, the medium was removed and the 96-well plates were replaced with media containing the serially diluted compounds, and the cells were further incubated for 48 hrs. The culture medium was removed and washed with 1 × PBS. The next step was conducted by antiviral activity assay described above. To calculate the CC50 values, the results were transformed to percentage of controls and the CC50 values were graphically obtained from the dose-response curves.