Fig 1: TBEV infection dynamics assessment in in vitro differentiated human neurons and astrocytes reveals higher neuronal susceptibility. Astrocytes were differentiated for 22 days, neurons for 14–17 days prior to infection with TBEV strains Hypr and Neudoerfl at MOI of 5. (A) Immunofluorescent labelling of selected differentiation markers in astrocytes (GFAP – red and S100B – green) – left and neurons (HuD – red and MAP2 -green) – right. Nuclei were stained with DAPI. Controls are labelled with secondary antibodies only. The scale bar represents 20 µm. Representative images from two independent experiments are shown. (B) TBEV growth curve of strains Hypr and Neudoerfl, virus shedding into cultivation media was quantified by plaque assay using A549 cells. Data summarise three independent experiments and values in graphs are expressed as mean ± SD. Significant difference from control was calculated by unpaired Student’s t-test (p < 0.05 (*), p < 0.01(**)). (C) Relative viability of neurons and astrocytes upon TBEV infection measured by metabolic conversion of alamarBlue and related to the viability of mock-treated cells (1.0 = 100 %, dotted line). Data summarise four biological and two technical replicates experiments, and values in graphs are expressed as mean ± SD. Welch´s t-test showed a significant difference from control with the multiple testing correction by FDR (Benjamini, Krieger, and Yekutieli, q = 0.05), p = 0.05 (*), p = 0.01 (**), p = 0.001 (***). (D) TBEV replication in astrocytes and neurons was assessed by the amount of genomic RNA quantified by qRT-PCR and related to mock with the ??-ct method. Results are represented as means ± SD of three biological and technical replicates. (E) TBEV C (capsid protein) and GAPDH (loading control) levels in infected astrocytes and neurons at 24 and 72 h p.i. were determined by immunoblotting. Representative data from three independent experiments are shown. N – Neudoerfl strain, H – Hypr strain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)