Virus isolation and titration.

Postnatal 3- to 4-day-old suckling BALB/c mice were infected with the P3 strain of JEV. Upon onset of clinical manifestations of JEV (poor brain response, limb paralysis, and whole-body tremor), animals were killed, and brains were excised. Brain homogenate prepared in Dulbecco's modified Eagle's medium (DMEM) was centrifuged at 10,000 × g to remove cellular debris. The resultant suspension was filtered through 0.22-μm-pore-size sterile filters to obtain a viral suspension. Immediately, aliquots of filtered virus suspension were stored at −80°C until further use. The virus titer was determined by plaque formation assay on baby hamster Syrian kidney (BHK-21) cells as described previously (45).