2.2. Viral Plaque Assay

The plaque-forming assay is described in our previous study [10]. In brief, the BHK-21 cells were seeded in 6-well plates at 4 × 10⁵ cells per well, followed by overnight incubation in RPMI 1640 medium containing 2% FBS to form a monolayer. Before infection, serial 10-fold dilutions of the supernatant of JEV-infected medium were prepared in serum-free RPMI medium, followed by incubation of the JEV-infected BHK-21 cells with serum-free RPMI 1640 medium for 1 h, and then, 0.5 mL of 10-fold dilutions and 0.5 mL of serum-free RPMI 1640 medium were added per BHK-21 monolayer for 1.5 h. The 6-well tissue culture (TC) plates were incubated at room temperature for 30 min to allow the 0.3% agarose overlay to solidify, followed by incubation at 37 °C for 3 days. The cells were then fixed with formaldehyde and stained with crystal violet stain solution for 2 min. Finally, plaque-forming units (pfu/mL) were calculated using a virus titer formula, where the virus titer equaled the number of plaques × (1 mL/0.5 mL) × the dilution factor.