SARS-CoV-2 Antiviral Imaging Assay

An in vitro imaging assay using SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources) and Calu-3 cells was performed as previously described [23 (link)]. Briefly, Calu-3 cells were plated in 384-well plates. The following day antiviral activity was assessed in duplicates of eight serial dilutions of galidesivir, ranging from 0.023 to 50.0 µM. DMSO was included as a negative control, and remdesivir was included as a positive control. Two hours after incubation with galidesivir, cells were infected with SARS-CoV-2 under biosafety level 3 (BSL3) containment conditions at an MOI of 0.5. Forty-eight hours after viral infection, cells were fixed and incubated overnight at 4 °C with a primary antibody specific for dsRNA (anti-dsRNA J2). Following 1 h incubation with a secondary antibody at room temperature, the cells were processed for automated microscopy. Toxicity was measured by quantifying the number of cells per well. The percentage of infected cells was calculated using the formula: (dsRNA⁺ cells/cell number)/well.