Avicel rc 591

Viral titre was determined by plaque assay on Vero-C1008 cells (ATCC). Cells were plated at 650,000 cells per well and infected with 3 or 6 dilutions from a tenfold dilution series in 1% FBS/1× non-essential amino acids/MEM for 60 min at 37 °C, with rocking every 15 min. Overlay medium (2 ml) consisting of a 1:1 mix of 2× modified Eagle medium (Temin’s Modification) supplemented with 2  penicillin–streptomycin, 2× GlutaMAX, 10% FBS:2× Avicel RC-591 (Dupont) was added to cells. Cells were incubated for 3 days at 37 °C before the overlay medium was removed and cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature. The fixative was removed, and cells were stained for at least 5 min with crystal violet (0.2% crystal violet (Sigma-Aldrich), 20% ethanol), before removal and plaque enumeration.

SARS-CoV-2/CANADA/VIDO 01/2020 was a kind gift from Darryl Falzarano (University of Saskatchewan). Vero (Female green monkey kidney) E6 cells were infected with an MOI of 0.0001 pfu/cell in infection medium consisting of Dulbecco’s Modified Eagle’s medium supplemented with 1× non-essential amino acids (Gibco), 10 mM HEPES, 2% fetal bovine serum, 50 IU/mL penicillin, 50 IU/mL streptomycin different doses of GC373 or GC376. After 1 h, the infecting medium was removed and monolayers were overlaid with growth media (MEM supplemented with 10 mM HEPES) containing 1.2% Avicel RC-591 (DuPont), containing the relevant dose of either GC373 or GC376. After 48 h, cells were fixed in 10% formaldehyde, and stained using 0.5% (w/v) crystal violet. Plaques were counted and data were plotted as % inhibition vs the log₁₀ [drug] using Prism (GraphPad). EC₅₀ values were determined using a non-linear regression analysis. Experiments were done in triplicate. Error bars indicate standard deviation. To examine the reduction in secretion of viral RNA by Vero E6 cells caused by GC373 and GC376, cells were infected with SARS-CoV-2 MOI = 0.01 pfu/cell in the presence of varying concentrations of GC373 or GC376, for 1 h, the medium removed and replaced with growth media also containing GC373 and GC376 and supernatants harvested 48 h later and quantified by quantitative RT-PCR.