Fig. 1
Isolation and characterization of extracellular vesicles. WNV titres (a) and detection of viral RNA by RT-PCR (b) in EV preparations before and after heat-inactivation and RNase A treatment. Culture fluids from WNV-infected were centrifuged to remove cell debris and incubated for 30min at 56oC with or without 1U/ml of RNaseA. EVs were then isolated from treated and untreated culture fluids using and analysed for the presence of infectious viral particles by plaque-assay on BHK-21 cells. b RT-PCR for viral RNA on sample of culture fluids and EV preparations c Transmission electron microscopy of EV preparations. d Western-blot analysis of EV lysates for the presence of exosomal markers. e Confocal microscopy analysis of A549 cells treated with Mock or WNV EVs containing fluorescently stained RNA cargo. Bright green fluorescence corresponds to SYTO RNASelect-stained EV RNA, blue fluorescence corresponds to DAPI-stained DNA. e Values in a represent the means of 4 biological replicates ±SD