*2.5. Immunofluorescence Assay*

A549 and HEK cells grown, infected and treated on glass coverslips were fixed with 3.7% formaldehyde at room temperature for 10 min. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 4 min. Coverslips were incubated with primary antibodies (1:1000 dilution) in 1% PBS BSA. Antigen staining was visualized with Alexa Fluor-conjugated secondary antibodies (1:1000, Invitrogen). Nucleus morphology was revealed by DAPI staining. The coverslips were mounted with VECTASHIELD®(Clinisciences, Nanterre, France) and fluorescence was observed using a Nikon Eclipse E2000-U microscope. Images were captured and processed using a Hamamatsu ORCA2 ER camera and the imaging software NIS-Element AR (Nikon, Tokyo, Japan). From the immunofluorescence imaging, percentage of ZIKV-E-positive cells with a stained active mitochondrial BAX or cytosolic cytochrome-c or cleaved caspase 3 was estimated after counting a minimum of six microscopic fields i.e., about 1000 cells examined for each condition.
