Fig 1: ZIKV does not replicate in human neutrophils. (A) Neutrophils morphological features after isolation shown by flow cytometry and microscopy (bright-field, bar = 25 µm, magnification = 100x.). (B) The cells were stimulated in vitro for 2 hours with ZIKV BR 2015/15261, ZIKV PE243, ZIKV MR766 (1 MOI), or mock (C6/36 cells-conditioned media in an equivalent volume of ZIKV strains). Then, neutrophils were washed to remove the stimuli (indicated by ?) and evaluated directly after (2 hours) and at 6, 12, and 24 hours after stimulation. When indicated, neutrophils were treated with trypsin after the wash step at 2 hours of stimulation with ZIKV strains. (C) Immunostaining of neutrophils with 4G2 (E protein; green) at 24 hours after ZIKV PE243 (a-d) or mock (e-h) stimulation. Nuclei (blue) were stained with Vybrant DyeCycle Violet. Merge of these stains and the bright-field colocalization are also shown. C6/36 cells (l-p) were used as a positive infection control (bar = 50 µm, magnification = 40x.) (D) Histograms showing 4G2 intensity of fluorescence in neutrophils and C6/36 cells at 24 hours after mock and ZIKV PE243 stimulation, as measured through flow cytometry. Infected-unstained cells were used as a negative fluorescence control. (E) Histograms showing AXL receptor intensity of fluorescence in neutrophils and A549 cells. An isotype control antibody was used as a negative fluorescence control. One result that is representative of three independent experiments is shown. (F) RNA levels of ZIKV strains in neutrophils at 2, 6, 12, and 24 hours after stimulation. (G) ZIKV loads on neutrophil culture supernatant at the same time after stimulation with ZIKV strains. The dashed line named input represents the mean of the titers of the three ZIKV strains that were still detected on the supernatant after stimuli removal after 2 hours of stimulation. (H) RNA levels of ZIKV PE243 detected in neutrophils treated or not with trypsin after 2 hours of stimulation. Bars indicate the standard error of the mean (SEM). Three-four independent experiments are shown (n = 9-15). The asterisk (*) denotes the statistical difference between 6 and 12 hours of the ZIKV PE243 stimulation.