Vero cells (a kind gift from Dr. Guruprasad Medigeshi, Translational Health Science and Technology Institute, Gurgaon, India) and Neuro2A (obtained from National Centre for Cell Science, Pune, India) cells were grown in DMEM (Dulbecco's modified Eagles medium, supplemented with 10% fetal bovine serum (FBS) and antibiotics. The GP78 strain of JEV was propagated in suckling BALB/c mice and their brains were harvested when symptoms of sickness were observed. A 10% tissue suspension was made in MEM (minimum essential medium), followed by centrifugation at 10,0006g and finally filtered through a 0.22 m sterile filter [19] . JEV was titrated by plaque formation on Vero cell monolayer. Vero cells were seeded in six-well plates to form semi-confluent monolayer in about 18 h. Cell monolayer were inoculated with 10-fold serial dilutions of virus samples made in MEM containing 1% FBS and incubated for 1 h at 37uC with occasional shaking. The inoculum was removed by aspiration and the monolayers were overlaid with MEM containing 4% FBS, 1% low-melting-point agarose and a cocktail of antibiotic-antimycotic solution (Gibco, Invitrogen Corporation, Grassland, NY, USA) containing penicillin, streptomycin, and amphotericin B. Plates were incubated at 37uC for 72-96 h until plaques became visible. To allow counting of the plaques, the cell monolayer was stained with crystal violet after fixing the cells with 10% formaldehyde.