Supplementary Materials1: Movie S1, Related to Figure 2. ACC were calculated by Kruskal-Wallis test with Dunns multiple comparisons test: *, 0.05; **, 0.01; ***, 0.001; ****, 0.0001. Statistical significance in E was determined by an unpaired two-tailed Students t-test: *, 0.05. Dots indicate data from individual fetuses or placentas, and bars and dashed lines indicate median values and limits of detection, respectively. D. fetal viability at the time of Ataluren supplier harvest was assessed 5 dpi at the timepoints shown (0% for E6CE11, 90% for E9CE14, and 96% for E12CE17, 0.0001 by the Chi-Square test; = 44 fetuses for E6CE11; = 70 for E9CE14; and = 68 for E12CE17). Data in this figure is pooled from at least three independent experiments. See also Figure S1. To corroborate these findings, we used a second established model of ZIKV-induced maternal-fetal disease in which dams were mated with WT sires so that fetal-derived placentas were heterozygous for Ifnar1 expression (Miner et al., 2016). Pregnant dams were inoculated with ZIKV at E6, E9, or E12, and placentas later were analyzed five times, at E11, E14, and E17, respectively (Fig 1B). placentas also suffered higher degrees of ZIKV replication when dams had been contaminated at E6 in comparison to E9 or E12 (10-collapse for E6 versus E9, 0.0001; 65-collapse for E6 versus E12, 0.0001). These results suggest that Ataluren supplier even though the gestational stage from the fetus effects the degree of ZIKV replication in the placenta, with highest amounts observed at previously stages, vertical transmitting and suffered ZIKV replication in the placenta may appear throughout pregnacy. Additionally, the integrity of the sort I IFN signaling pathway in the mom does not look like an integral determinant of the time-dependent phenotype. Because differential disease from the placenta at different gestational instances could reflect variant in maternal disease, we evaluated the degrees of ZIKV in cells from Erg the pregnant dams five times after disease using the 0.0001) or seven days (250-fold higher, 0.0001) after disease (Fig 1C). Disease at E9 led to intermediate degrees of viral disease at both timepoints. When the related test was performed in pregnant dams mated to WT sires, the medical phenotype was even more pronounced; ZIKV inoculation at E6 uniformly led to fetal demise (Fig 1D), as noticed previously (Miner et al., 2016; Yockey et al., 2016). A lower life expectancy rate of recurrence of fetal demise was noticed when ZIKV inoculation was performed at E9 ( 0.0001), no additional fetal demise occurred with inoculation in E12 beyond that observed in WT uninfected mice. Although viral quantification cannot become performed after disease at E6 because of fetal resorption, ZIKV RNA amounts in the fetal mind 5 times after inoculation at E9 had been somewhat higher (2-collapse, 0.05) than corresponding ones from E12 inoculated dams (Fig 1E). Therefore, in mice, previously gestational age during ZIKV disease results in higher levels of viral replication in the placenta (Fig 1ACB), which faciliates more viral replication in the fetus and severe clinical outcomes. Ultrasound analysis of ZIKV-infected pregnant dams reveals gestational age-dependent placental and fetal pathology Several fetal and neonatal pathological sequelae of ZIKV infection have been reported in humans, including microcephaly, intrauterine growth retardation, defects in neuronal development of hearing and vision, and cerebral cortical thinning (de Paula Freitas et al., 2016; Driggers et al., 2016; Honein Ataluren supplier et al., 2017; Leal et al., 2016; Martines et al., 2016). To gain insight into the basis for these phenotypes, we examined fetuses by ultrasound analysis after (Mu et al., 2008). Open in a Ataluren supplier separate window Figure 2 Ultrasound assessment of fetuses from mock- and ZIKV-infected pregnant micevalues were determined using a one-way ANOVA: *, 0.05; **, 0.01; ****, 0.0001. Bars indicate median values. Data is pooled from independent experiments with placentas and fetuses from 2 mock-infected and 4 ZIKV-infected dams at E9CE15 and 2 mock-infected and 3 ZIKV-infected dams at E12CE17. See also Movie S1. Ultrasound images were captured and feto-placental dimensions recorded from multiple fetuses in ZIKV- and mock-infected pregnant dams (Fig 2). As noted previously (Miner et al., 2016), infection at E6 in this model resulted in universal fetal.