SARS-CoV-2 cytopathic effect (CPE) assay

Vero-E6 cells previously selected for high ACE2 expression⁶⁴ (grown in EMEM, 10% FBS, and 1% Penicillin/Streptomycin) were cultured in T175 flasks and passaged at 95% confluency. Cells were washed once with PBS and dissociated from the flask using TrypLE. Cells were counted prior to seeding. A CPE assay previously used to measure antiviral effects against SARS-CoV⁶⁵ was adapted for performance in 384 well plates to measure CPE of SARS CoV-2 with the following modifications. Cells, harvested and suspended at 160,000 cells/ml in MEM/1% PSG/1% HEPES supplemented 2% HI FBS, were batch inoculated with SARS CoV-2 (USA_WA1/2020) at M.O.I. of approximately 0.002 which resulted in approximately 5% cell viability 72 h post infection. Compound solutions in DMSO were acoustically dispensed into assay ready plates (ARPs) at 3 point 1:5 titrations. ARPs were stored at -20 °C and shipped to BSL3 facility (Southern Research Institute, Birmingham, AL) for CPE assay. ARPs were brought to room temperature and 5 µl of assay media was dispensed to all wells. The plates were transported into the BSL-3 facility were a 25 μL aliquot of virus inoculated cells (4000 Vero E6 cells/well) was added to each well in columns 3–24. The wells in columns 23–24 contained virus infected cells only (no compound treatment). A 25 μL aliquot of uninfected cells was added to columns 1–2 of each plate for the cell only (no virus) controls. After incubating plates at 37 °C with 5% CO₂ and 90% humidity for 72 h, 30 μL of Cell Titer-Glo (Promega, Madison, WI) was added to each well. Following incubation at room temperature for 10 min the plates were sealed with a clear cover, surface decontaminated, and luminescence was read using a Perkin Elmer Envision (Waltham, MA) plate reader to measure cell viability.