Plaque assay CVB3 titer in cell supernatant was determined on monolayers of HeLa cells by an agar overlay plaque assay in triplicate as described previously [2] . Briefly, samples were serially diluted and overlaid on monolayer of HeLa cells. After 1 h incubation, medium was replaced with complete medium containing 0.75% agar. Cells were incubated for 72 h, then fixed with Carnoy's fixative (75% ethanol-25% acetic acid), and stained with 1% crystal violet. Plaques were counted and viral titer was calculated as plaque forming unit (PFU) per milliliter.