| Assay Method Information | |
| | Cellular Antiviral Activity |
| Description: | Antiviral activity against SARS-CoV-2, SARS-CoV, MERS-CoV and HCoV-229E was assessed by monitoring cell viability; that against HCoV-OC43 was assessed by monitoring viral RNA in a cell suspension. EC50 values were determined by plotting compound concentration vs inhibition and fitting data with a 4-parameter logistical fit (Model 205, XLfit). EC90 values against HCoV-OC43 were determined from the resulting dose-response curves and calculated with the two-point method.Antiviral activities against SARS-CoV-2 were evaluated using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells (1.5 × 104/well) suspended in minimum essential medium (MEM) (Thermo Fisher Scientific) supplemented with heat-inactivated 2% FBS were seeded into 96-well plates with diluted compounds in each well. Cells were infected with each SARS-CoV-2 at 30 3000 TCID50/well and cultured at 37°C with 5% CO2 for 3 days or 4 days. Cell viability was assessed using a CellTiter-Glo 2.0 assay (Promega). The CC50 was assessed in the absence of viruses after being cultured for 3 days.Antiviral activities against SARS-CoV and MERS-CoV were evaluated at Hokkaido University using VeroE6/TMPRSS2 cells as previously reported23. VeroE6/TMPRSS2 cells (1.5 × 104/well) suspended in 2% FBS-containing MEM were seeded into 96-well plates with diluted compounds in each well. Cells were infected with each SARS-CoV at 1000 TCID50/well or MERS-CoV 2500 TCID50/well and cultured at 37°C with 5% CO2 for 3 days. Cell viability was assessed via (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Nacalai Tesque) as previously described26.Antiviral activity against HCoV-229E was evaluated using MRC-5 cells. MRC-5 cells (2.0 × 104/well) suspended in 2% FBS-containing MEM were seeded into 96-well plates and incubated at 37 °C with 5% CO2 overnight. The next day, the cells were infected with HCoV-229E at 1000 TCID50/well and incubated at 37 °C with 5% CO2 for 1 h, followed by removal of the inoculum and added 2% FBS-containing MEM with the diluted compounds. Cells infected with HCoV-229E were incubated at 37 °C with 5% CO2 for 3 days. Cell viability was assessed using a CellTiter-Glo 2.0 assay.Antiviral activity against HCoV-OC43 was evaluated using MRC-5 cells. MRC-5 cells (2.0 × 104/well) suspended in 2% FBS-containing MEM were seeded into 96-well plates and incubated at 37 °C with 5% CO2 overnight. The next day, the cells were infected with HCoV-OC43 at 100 TCID50/well and incubated at 37 °C with 5% CO2 for 1 h, followed by removal of the inoculum and added 2% FBS-containing MEM with the diluted compounds. Cells infected with HCoV-OC43 were incubated at 37 °C with 5% CO2 for 42 h, and viral RNA was extracted from the supernatants using a Quick-RNA Viral Kit (ZYMO RESEARCH, # R1041). Viral RNA was quantified via real-time PCR (Applied Biosystems, QuantStudio 3) with specific primers and probes for HCoV-OC43 detection27. |
| Affinity data for this assay | |
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