Lm 201 50

The soft agar assay was performed as previously described (19 (link)). The base layer of each well consisted of 1.5 mL of serum-free 2X DMEM (Welgene, LM 201-50) with a final concentration of 0.5% Noble agar (BD Biosciences). After bottom agar solidification, 1.5 mL of 0.35% agar containing NIH3T3 cells (20,000) was seeded on the bottom agar layer and incubated for 14 days. Medium was changed every 2 days for 2 weeks. Colonies were fixed with 4% paraformaldehyde and then stained with 0.05% crystal violet (Sigma-Aldrich, C6158).

The virus titer in the cell supernatant was determined as previously described (Wong et al. 2008 ). In brief, the supernatant from CVB3-infected cells was serially diluted 10-fold before being overlaid on a Vero cell monolayer that was 90–95% confluent. The Vero cells were rinsed in PBS after 1 h incubation, then overlayed with a 2 × DMEM (LM 20150; Welgene, Gyeongsan, Korea) / 2% agar (50100; Lonza, Walkersville, MD, USA) mixture. The cells were fixed with 3.75% formaldehyde (F8775; Sigma–Aldrich) solution and stained with 1% crystal violet (V5265; Sigma–Aldrich) after being cultured at 37°C for 72 h. The virus titer was determined as plaque-forming unit (PFU) per milliliter after the plaques were counted.