Figure 4.

Lipid content of SEV blocks ZIKV infection. (A) The protein content of SEV was denatured by heating at 95° for 8 min. Nanoparticle tracking analysis (NTA) profile comparing heat-treated and untreated SEV showing that the size profiles do not change. Size profiles of liposomes (see part C) are also plotted. (B) Equal amounts of heat-treated or untreated SEV were incubated with ZIKV virions at a 10⁶ SEV:PFU, then added to epithelial cell lines for 1.5 h before washing and assessing ZIKV binding by ddPCR. As a control, ZIKV virions were also heated at 95° for 8 min. Each line is a separate cell line, points are mean of two replicate wells and error bars are standard deviation. Significance by one-way ANOVA with Tukey’s multiple comparisons test. ^**p > 0.01. (C) ZIKV was pre-incubated with 10⁶ SEV or 10⁶ liposomes for 1 h prior to infecting genital epithelial cells. Seventy-two hours post-infection cells were lysed and ZIKV genomes quantified by ddPCR. Each line is the average of duplicate wells from a separate experiment. Reduction in ZIKV genomes was significant by one-way ANOVA with Dunnett’s multiple comparisons test. ^*p < 0.05, and ^**p < 0.005. (D) The data from C, and additional conditions with 10⁵ SEV or liposomes, are plotted as percent reduction from ZIKV alone. Each symbol represents the average of two technical wells from a different cell line. The horizontal lines are the mean for each condition. (E) Comparison of pre-incubation of either ZIKV or epithelial cells with liposomes on viral infection. Liposomes were pre-incubated with ZIKV (ratio:10⁶ liposome per PFU) or with cells (same amount of liposomes) for 1 h at 37°, then virus was added to cells for 1.5 h. Cells were washed and cell-associated ZIKV genomes quantified by ddPCR. Each symbol represents the average of two technical replicates from different epithelial cell lines. Significance by one-way ANOVA with Dunnett’s multiple comparisons test. ^*p < 0.05.