After two washes with 16 PBS, cells were first fixed with BD cytofix solution (BD Biosciences) for 15 min and permeabilized by resuspending in permeabilization buffer (BD Cytoperm plus; BD Biosciences) and incubated at 25uC for at least 10 min. Cells were then washed twice in wash buffer (PBS containing 1% bovine serum albumin) then resuspended in wash buffer at 1610 6 cells per 100 mL. Primary antibody (JEV Nakayama strain; Chemicon, USA) were added in 1:100 dilutions and incubated for 30 min at 25uC. The cells were washed with wash buffer and pelleted by centrifugation followed by incubation with FITC conjugated secondary antibody for 30 min. After final wash with wash buffer, cells were resuspended in 400 mL FACS buffer and analyzed on a FACS Calibur. The percentage of population of JEV-positive cells was calculated after gating the populations on a Dot plot using Cell Quest Pro Software (BD Biosciences).