HCoV 229E Antiviral Assay

HCoV 229E (a gift from Ralph Baric, UNC, Chapel Hill) was propagated on Huh-7 cells, and titers were determined by the TCID₅₀ assay on Huh-7 cells. Huh-7 cells were plated at a density of 25,000 cells per well in 96-well plates and incubated for 24 h at 37 °C and 5% CO₂. Cells were infected with HCoV 229E at an MOI of 0.1 in a volume of 50 μL of MEM 1 + 1 + 1 [modified Eagle’s medium, 1% FBS, 1% antibiotics, 1% N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer] for 1 h. The virus was removed, cells were rinsed once with PBS, and compounds were added at the indicated concentrations in a volume of 100 μL. Supernatants were harvested after 24 h, which were serially 10-fold diluted, and virus titer was determined by the TCID₅₀ assay on Huh-7 cells. CPE was monitored by visual inspection at 96 h post infection. TCID₅₀ titers were calculated using the Spearmann-Karber method.^105,106