For immunohistochemical staining, brains from scarified animals were excised following repeated transcardial perfusion with ice-cold saline and fixed with 4% paraformaldehyde. Twenty micron thick cryosections were made with the help of Leica CM3050S cryostat and processed for immunohistochemical staining to detect presence of JEV antigen in the brain and to label activated microglia. Sections were incubated overnight at 4uC with mouse anti-JEV antigen (Nakayama, 1:250) (Chemicon, CA, USA) and rabbit anti-Iba-1 (1: 500; Wako, Osaka, Japan), respectively. After washes, slides were incubated with FITCconjugated anti-mouse or anti-rabbit secondary antibodies (Vector laboratories Inc. Burlingame, USA) and following final washes, sections were sections were cover slipped after mounting with 49-6diamidino-2-phenylindole (DAPI, Vector laboratories Inc.). The slides were observed under Zeiss Axioplan 2 fluorescence microscope and Zeiss Apotome microscope (Zeiss, Gottingen, Germany), respectively [21] .