Fig. 3

TBEV damages human neurons. HNPCs were differentiated for 13 days and infected with TBEV-Hypr at MOI 10⁻². a Cells in non-infected (NI) and infected (TBEV) co-cultures were immunostained with an antibody directed against HuC/HuD (neurons, green) at 14 dpi. Nuclei were counterstained with DAPI. Scale bars = 100 μm. b Enumeration of HuC/HuD-positive cells using an ArrayScan Cellomics instrument. Normalization to non-infected HuC/HuD-positive cells at 14 hpi. c Cells in non-infected and infected cultures were immunostained with an antibody against βIII-tubulin (neurons, red) at 7 dpi. Note the paucity of neurites in TBEV-infected co-cultures. Scale bars = 100 μm. d Quantification of neurite network density (neurite length per square millimeter) using an ArrayScan Cellomics instrument. e Cells in non-infected and TBEV-infected cultures were TUNEL stained at 72 hpi. Note that TUNEL staining matches with βIII-tubulin immunostaining (arrows). Right panel show an apoptotic neuron (co-localization of βIII-tubulin and TUNEL staining). Scale bars = 20 μm. (f) Percentage of apoptotic cells based on TUNEL staining at 24 and 72 hpi. Results in b, d, and f are expressed as the mean ± SD and are representative of four (b) and two (d, f) independent experiments performed in triplicate, respectively. Statistical analysis was performed using a two-tailed unpaired t test with Graphpad Prism V6.0.1. ns, non-significant (p > 0.05); *p < 0.05, **p < 0.01, ***p < 0.001