EV-D68 Virus Plaque Assay Protocol

Plaque assay for EV-D68 US/MO/14-18947 was performed as previously described.^{38 (link), 39 (link)} RD cells were grown to more than 90% confluent and washed with PBS supplemented with magnesium and calcium after removing the growth medium. Cells were infected by EV-D68 virus and incubated at 33 °C incubator for 1-2 hours to allow virus attachment. After washing the unbound viruses, an overlay containing the indicated concentration of test compounds and 1.2% Avicel (FMC BioPolymer, Philadelphia, PA) in DMEM supplemented with 2% FBS and 30 mM MgCl₂ was added with 4 ml per well. The plates were incubated at 33 °C (5% CO₂) incubator for 3 days and the cells were stained with crystal violet after removing the Avicel overlay. Plaque areas were quantified by ImageJ and plotted with drug concentration for the calculation of EC₅₀ values.