Zika Virus Infection during Pregnancy in Mice Causes Placental Damage and Fetal Demise

Abstract

Zika virus (ZIKV) infection in pregnant women causes intrauterine growth restriction, spontaneous abortion, and microcephaly. Here, we describe two mouse models of placental and fetal disease associated with in utero transmission of ZIKV. Female mice lacking type I interferon signaling (Ifnar1(-/-)) crossed to wild-type (WT) males produced heterozygous fetuses resembling the immune status of human fetuses. Maternal inoculation at embryonic day 6.5 (E6.5) or E7.5 resulted in fetal demise that was associated with ZIKV infection of the placenta and fetal brain. We identified ZIKV within trophoblasts of the maternal and fetal placenta, consistent with a trans-placental infection route. Antibody blockade of Ifnar1 signaling in WT pregnant mice enhanced ZIKV trans-placental infection although it did not result in fetal death. These models will facilitate the study of ZIKV pathogenesis, in utero transmission, and testing of therapies and vaccines to prevent congenital malformations.

Figure 1. Mortality, viral burden, and size of mouse fetuses after maternal infection with ZIKV

A. Schematic depiction of two models of infection during pregnancy. Model 1: WT males were crossed with Ifnar1^−/− dams. Pregnant dams were infected subcutaneously with ZIKV (10³ FFU) on E6.5 or E7.5 followed by harvest on E13.5, or 15.5, respectively. Model 2: WT males were crossed with WT dams. Pregnant dams treated with 1 mg of an anti-IFNAR antibody on days −1, +1, and +3 relative to ZIKV (10³ FFU) or DENV-3 (10³ FFU) infection. Mice were sacrificed on E13.5 or E15.5 and fetuses and placentas were harvested for measurements of fetal size by crown-rump length (CRL) and occipito-frontal (OF) diameter. B. E13.5 uteri from ZIKV-infected WT and Ifnar1^−/− dams. Most Ifnar1^+/− fetuses carried by Ifnar1^−/− dams died in utero and had undergone resorption, leaving only the residual placenta. In the lower three panels are representative images of fetuses carried by ZIKV-infected WT and mock-infected Ifnar1^−/− dams, the latter of which exhibited growth restriction at E15.5. C. Fetus survival on E13.5 after infection with ZIKV on E6.5. Mice were either treated with three 1 mg doses of control or anti-Ifnar antibody (left two bars) or untreated mock- or ZIKV-infected Ifnar1^−/− dams (right two bars). Data are representative of at least 3 independent experiments with 1 pregnant female dam per experiment. The n for each group is indicated above each bar. ****, P < 0.0001. D. Fetus size as assessed by CRL × OF diameter in E13.5 fetuses following E6.5 infection of the indicated pregnant dams with either ZIKV or DENV-3. Bars indicate the mean size of 8-20 fetuses from 2 or 3 independent experiments from fetuses carried by 2 to 3 pregnant dams. ***, P < 0.0005; ****, P < 0.0001. E and F. Viral burden was measured by qRT-PCR assay from the fetal head and placenta on E13.5 after infection at E6.5. Symbols represent individual fetuses pooled from several independent experiments with the exception of 4 intact Ifnar1^+/− fetal heads that were carried by a single dam. Bars indicate the mean of 4 to 17 mice per group. Dotted lines represent the limit of sensitivity of the assay. *, P < 0.05 **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001. G. Fetus survival on E15.5 after infection with ZIKV on E7.5. Data are representative of at least 2 independent experiments with 1 pregnant female dam per experiment. The n for each group is indicated above each bar. ****, P < 0.0001. H. Fetus size as assessed by CRL × OF diameter in E15.5 fetuses following E7.5 infection of the indicated pregnant dams with ZIKV. ****, P < 0.0001. I-K. Viral burden was measured by qRT-PCR assay from maternal serum, spleen, and brain at E13.5. Symbols are derived from individual animals and pooled from 2 or 3 independent experiments. Bars indicate the mean of 4 to 5 mice per group. Dotted lines represent the limit of sensitivity of the assay. See also Figure S1.

Figure 2. ZIKV infects maternal and fetal cells within the placenta

Pregnant Ifnar1^−/− dams were infected on E7.5 with 10³ FFU of ZIKV via a subcutaneous route and placentas were harvested on E15.5 for histological analysis. A. Schematic representation of the mouse placental structure. B. Representative RNA FISH images in uninfected and infected Ifnar1^−/− placentas. Images in each column correspond to the same field of view generated under bright-field or confocal microscopy. Higher magnification of images is displayed as inserts. Scale bar, 25 μm. C. Transmission electron microscopy images of ZIKV infected Ifnar1^−/− placentas. ZIKV particles were identified within the endoplasmic reticulum in the maternal sinus (left panel), and in the fetal endothelium (right panel) lining fetal capillaries in the labyrinth layer. MT = Mononuclear trophoblast; MS = Maternal sinus; FE = Fetal endothelial cell; FC = Fetal capillary; MRBC = Maternal erythrocyte; and ER = Endoplasmic reticulum. See also Figure S2.

Figure 3. ZIKV infection triggers apoptosis and vascular damage in the placenta

Pregnant dams were infected on E7.5 with 10³ FFU of ZIKV via a subcutaneous route and placentas were harvested on E15.5 for histological analysis. A. Representative hematoxylin and eosin staining showed pathological features of placentas at E15.5. Labyrinth layers were marked with a solid line on the cross section of mouse placentas. Black arrows indicate apoptotic trophoblasts. Blue arrows indicate increased number of nucleated fetal erythrocytes in fetal capillaries. B. Immunofluorescence staining of cytokeratin (CK) and vimentin in mouse placentas. CK, a marker for trophoblasts; vimentin, a marker for the endothelium in fetal capillaries. See also Figure S2.

Figure 4. ZIKV infection is associated with evidence of apoptosis in the fetal brain

Pregnant Infar1^−/− dams were infected with 10³ FFU of ZIKV via a subcutaneous route. Infected (left) or uninfected (right) Ifnar1^+/− E13.5 fetuses were stained with the apoptotic marker activated caspase-3 (AC3; red) and the proliferative marker Ki-67 (green). Sagittal images of representative infected (A) and uninfected (E) fetal heads showing high expression of Ki-67 along brain regions adjacent to ventricles indicative of proliferating neural progenitor cells in the neuroepithelium. Lettered box regions (B-D and F-H) in these images are magnified in corresponding panels below. Higher levels of apoptosis can be seen in the midbrain (Panels B-C) and hindbrain (Panel D) of the infected Ifnar1^+/− fetus. Alternatively, low levels of physiological apoptosis are seen in the absence of infection (Panels F-H). I. Diagram depicting the developing E13.5 fetal brain in sagittal view including the forebrain (green), midbrain (blue), and hindbrain (red). Images are representative of 4-5 sections per fetus from 2 fetuses.