Plaque assay

Virus plaque morphologies were determined by plaque assay as described previously [39]. Briefly, approximately 2×10⁵ Vero cells per well were seeded in a 6-well plate 24 hours in advance. A series of 1:10 dilutions were made by mixing 25μl of virus sample with 225 μl of DMEM medium. 200 μl of dilutions of viral supernatant were inoculated to individual well of 6-well plate. The plates were incubated at 37°C with 5% CO₂ for 1 hour with being shaken every 15 minutes, and then the virus inoculum were replaced with 1 ml of DMEM medium containing 1% agarose. After 7 days of incubation at 37°C with 5% CO2, the agarose layer was covered with 1 ml of 1% agarose containing 0.01% neutral red to stain live cells at 37°C for4 hours in the absence of light. After staining, the number and the diameter of plaques were measured.