EV-A71 Antiviral Assay Protocol

The EV-A71 laboratory-adapted BrCr strain and clinical isolates representative of B genogroup (B2 subgenogroup: 11316; B5 subgenogroup: TW/70902/08) and C genogroup (C2 subgenogroup: H08300 461#812; C4 subgenogroup: TW/1956/05) were used at a low multiplicity of infection (MOI) in a standardized cell-based antiviral assay. Briefly, rhabdosarcoma (RD) cells were seeded in a 96-well plate. The day after, a serial dilution of the compounds and the virus inoculum were added to the cells. The assay plates were incubated at 37 °C, 5% CO₂ with virus inoculum and compounds until full virus-induced cell death was observed in the untreated, infected controls (3 days post-infection). Subsequently, the antiviral effect was quantified using a colorimetric readout with MTS/phenazine methosulfate (MTS/PMS method), and the compound concentration at which 50% inhibition of virus-induced cell death is observed (EC₅₀) was calculated from the antiviral dose–response curves. A similar assay setup was used to determine the adverse effect of the compound on uninfected, treated cells for calculation of the CC₅₀ (concentration of compound that reduces overall cell health by 50% as determined with the MTS/PMS method). The selectivity index (SI) was calculated as the ratio of CC₅₀ to EC₅₀.