FIG 7.
JEV NS4B induces encephalitis via PERK in mice. (A and B) Neuro-2a cells were transfected with NS4B-expressing plasmid, NS4BLIG-FHA/WD40mut-expressing plasmid, or empty vector and treated with GSK2606414 (0.8 nM) as indicated. At 2 days posttransfection, all groups were subjected to immunoblot analysis using NS4B, FLAG, or actin antibody (A) and apoptotic analysis by flow cytometry (B). (C to F) Mice were injected intracranially with NS4B- or NS4BLIG-FHA/WD40mut-expressing plasmid (200 μg/kg [body weight]) and injected intracranially GSK2606414 (800 μg/kg [body weight]) as indicated. At day 5 after injection, brain tissues were collected and subjected to immunoblot analysis using NS4B, FLAG, or actin antibody (C), TUNEL assays using the TUNEL assay kit (D), and H&E staining (E and F). Meningitis (E) and perivascular cuffing (F) that appeared in the JEV-infected groups are indicated by arrows. Left panels (B, D to F) show representative images; right panels (B, D to F) show quantitation data. Error bars indicate the SD of the mean (n = 3). Scale bars, 200 μm. **, P < 0.01; ***, P < 0.001.