Fig. 3. JEV targets oRGCs in cortical organoids during development.

Brain organoids at different stages were inoculated with JEV (SA14, 10⁵ PFU) or mock treated for 24 h and analyzed at day 8. a Immunostaining of oRGC markers FAM107A (green) and JEV NS1 protein (JN1, red) in cortical organoids. White dashed box point to JN1⁺ FAM107A⁺ oRGC-like cells in the oSVZ region. Scale bars: 20 μm. b Statistics for JN1⁺ FAM107A⁺ cells/FAM107A⁺ cells in the oSVZ region demonstrates that JEV can targets oRGCs at days 55 and 90, respectively. Values represent mean ± SEM; n = 5. c Immunostaining of astrocyte marker GFAP (green) and JN1 (red) in Hoechst-stained (HO, gray) telencephalon cortical organoids at day 91. White arrows point to the existence of JN1⁺ GFAP⁺ astrocytes. Scale bars: 100 μm. d Immunostaining of mature neurons marker PSD95 (postsynaptic density-95 kDa, green) and JN1 (red) in cortical organoids at day 91. The results illustrate that mature neurons, which almost lack JN1 expression, generally are resistant to JEV infection. Scale bars: 100 μm