Virus infection and plaque assay

Human RD cells were cultured in DMEM/F-12 (Gibco) supplemented with 10% FBS (GE Healthcare) and the infection medium used for all infections and compound treatment contained was supplemented with 2% FBS. For the compound treatment assays, monolayers of RD cells were first infected with EV71 at a multiplicity of infection (MOI) of 1 for 1 hour at 37 °C, 5% CO₂. The compounds were then introduced to the cells at different concentrations. Culture supernatant was collected at 12 hours post-treatment for determination of infectious virus titre by viral plaque assay. Each culture supernatant was 10-fold serially diluted and 100 μL was added, in triplicates, to a monolayer of RD cells in a 24-well format. The infection was allowed to proceed for 1 h at 37 °C, 5% CO₂ before the virus was removed. The cells were then washed to remove unbound virus particles with PBS (pH 7.4) and overlaid with infection medium containing 0.5% agarose. The cells were incubated for 48 hours at 37 °C before they were fixed with 4% paraformaldehyde and stained with crystal violet. Plaques that formed were counted visually and the infectious virus titre was calculated, expressed as the average number of PFU per milliliter (PFU/mL) of sample. The 50% effective concentration, EC₅₀, was determined for selected compounds by a non-linear regression graph fitting of the virus titre obtained at a minimum of 5 different compound concentrations and 0.05% DMSO was included as an untreated control.