Fig. 5

Seminal extracellular vesicles inhibit ZIKV infection. a 50% SP was filtered through a 0.2 µm syringe filter. b 0.2 µm filtered SP was applied to a 300 kDa molecular weight filter and the retentate and filtrate diluted with PBS to the originally applied volume. c SP was separated into extracellular vesicles (EVs) larger and smaller than 220 nm by centrifugal size filtration. d EV samples generated in c were boiled at 99 °C for 20 min, centrifuged, and denatured protein discarded. All samples (a–d) were added to Vero E6 cells at the indicated concentrations and incubated for 10 min before cells were inoculated with ZIKV MR766. After 2 days, infection was determined by cell-based ZIKV immunodetection assay that enzymatically quantifies the flavivirus protein E. Average infection rates are normalized to the corresponding infection averages in the absence of SP. Data represent average values obtained from triplicate infections ± standard deviations. e ZIKV virions were allowed to attach to Vero E6 cells in the presence of the indicated concentrations of SP-derived EVs (<220 nm) for 2 h at 37 °C or f 4 °C. Cells were then washed and stained for ZIKV protein E and cell nuclei. A z-stack of 14 confocal microscopic images were taken and combined to a maximum intensity projection. Protein E fluorescence was quantified and normalized to the absence of EVs in three z-stacks ± standard deviation (see Supplementary Fig. 12). *P < 0.01, **P < 0.001, ***P < 0.0001 (by one-way ANOVA with Bonferroni post-test)