SARS CoV-1 Antiviral Assays
All assays were performed in BSL3 containment. Vero 76 cells were plated at 10 000 cells per well using phenol red-free DMEM or IMEM (Gibco). After adhering for 2 h at 37 °C, various concentrations of compound (320, 100, 33, 10, 3.3, 1.0, 0.3, or 0.1 μM) were added and the cells were infected with 2.6 × 103 PFU/well SARS CoV Toronto-2 (provided as a gift from Dr. Heinz Feldman (NIAID, Hamilton, MT)) or mock-infected with the medium only. After 66 h, cell viability was determined using either the neutral red method or the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). For the neutral red method, the cells were washed twice with PBS, and 100 μL of DMEM pH 4.5 containing 0.066% neutral red was added to the cells for 2 h at 37 °C. The cells were again washed twice with PBS, 100 μL of buffer solution (50% EtOH, 1% acetic acid) was added, and the OD was read at 540 nM after a 10 min incubation at 37 °C with shaking. Data are expressed as the percent of neutral red or luminescent signal in wells of compound-treated cells compared to the signal in wells of uninfected, compound-free cells. The 50% effective concentration (EC50) is calculated as the concentration of the compound that increases the percent of the neutral red or luminescent signal in infected, compound-treated cells to 50% of that produced by uninfected, compound-free cells. The 50% cytotoxicity concentration (CC50) is calculated as the concentration of the compound that decreases the percent of the neutral red or luminescent signal in uninfected, compound-treated cells to 50% of that produced in uninfected, compound-free cells.51,52