ZIKV induces cell death in virus-positive cells. (a) Confocal micrographs of A549 cells stably expressing the ZIKV-NLS-GFP reporter (green) that were infected with ZIKV-PR or ZIKV-DAK (MOI 10), or mock for 48 h and then immunostained for ZIKV (anti-Envelope (Env); red) and cleaved Caspase-3 (CC3; magenta). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. (b) Schematic of the live cell imaging analysis performed to quantify cell death in ZIKV-uninfected and infected cells. (c,f) Fluorescence microscopy shows sequential images of the same field of A549 cells stably expressing ZIKV-NLS-GFP after infection (MOI 10) with ZIKV-PR (c) or ZIKV-DAK (f) at the indicated hours post infection (hpi) between corresponding DIC (top) and GFP (bottom) images. Arrows mark cells of interest and asterisks denote cells that have died. Scale bar, 10 µm. (d,e,g,h) Quantification of the time of death of cells in ZIKV-infected cultures from live cell imaging experiments (represented by ). Individual fluorescent cells were tracked and identified as positive for ZIKV by determining the incidence and time point of nuclear GFP translocation. The time point of cell death was determined by assessing cellular morphological changes as seen in the DIC channel, and the time between GFP translocation and cell death was calculated. For uninfected cells that did not undergo nuclear translocation, the time between apoptosis and the first nuclear translocation event in the entire field was calculated and plotted. Values represent the mean ± SEM (n = 10 fields) from three independent experiments, with 10 cells counted per field. Cell death in infected (Nuc GFP) and uninfected (Cyto GFP) cells is directly compared at 40 h post-nuclear GFP translocation in (e,h). Asterisks denote significance; ** p = 0.0002 (e) and *** p < 0.0001 (h), as determined by an unpaired t-test.