Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects

Lindqvist, Richard; Mundt, Filip; Gilthorpe, Jonathan D.; Wölfel, Silke; Gekara, Nelson O.; Kröger, Andrea; Överby, Anna K.

Cells were grown on 96-well plates (Greiner CELLSTAR® ) , fixed in 4 % formaldehyde and permeabilized in PBS containing 0.5 % Triton X-100 and 20 mM glycine. The cells were stained with primary antibodies; a mouse monoclonal TBEV anti-E antibody (Hypr, 1493 1:1000 [44] , Sofjin and Aina, 1786 1:1000 [44] ), flavivirus anti-E antibody (JEV, WNV, and ZIKV, HB112 1:1000, ATCC D1-4G2-4-15 [45] ), and rabbit polyclonal anti-GFAP antibody (1:1000 Abcam, ab7260 [46] ). Secondary antibodies were as follows (Thermo Fisher Scientific); donkey anti-rabbit Alexa Fluor 488 (A21206), donkey anti-mouse Alexa Fluor 555 (A31570), and goat anti-mouse Alexa Fluor 647 (A21236) which were diluted 1:500. Nuclear counterstaining was performed using DAPI (Life Technologies, D1306) 1 μg/ mL. Viral spread was quantified using a TROPHOS Plate RUNNER HD® (TROPHOS SA, Marseille, France). Images of immunofluorescence staining were acquired under a Zeiss Axiovert 25 microscope using an infinity3 luminera® camera.