2.5. Plaque assay and focus forming assay (FFA)

In the plaque assay, serial 10-fold dilutions of each sample were prepared, and 100 μL/well of the diluted virus was added into the 12-well plates. The cells were cultured in the mixture of 2× ​DMEM (Invitrogen) and 2% methylcellulose (Sigma) (v/v 1:1). Visible plaques were counted at 3–4 days post-infection (d.p.i.).

In the FFA, serial 10-fold dilutions of each sample were prepared, and 100 μL/well of diluted virus was added into the 96-well plates. The cells were cultured in the mixture of 2 ​× ​DMEM (Invitrogen) and 2% methylcellulose (Sigma) (v/v 1:1). Cells were washed with phosphate-buffered saline (PBS) at 48 ​h post-infection (d.p.i.), and then fixed with 1% paraformaldehyde (PFA). Cells were incubated with anti-WNV E60 MAb (a gift from Dr Michael S Diamond, Washington University School of Medicine) (Oliphant et al., 2005), followed by incubation with IRDye 680 RD conjugated anti-mouse IgG (LI-COR). The number of spots was determined by ImmunoSpot® S6 Ultra (CTL, USA).