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.

Given the urgent need to understand the basis for in utero transmission of ZIKV and its pathological consequences, we developed two models of ZIKV infection during pregnancy using Ifnar1 −/− females crossed to WT males as well as pregnant WT females treated with an anti-ifnar-blocking antibody. We found that ZIKV infects pregnant dams and the placenta, and this resulted in damage to the placental barrier and infection of the developing fetus, as well as placental insufficiency and IUGR. In severe cases, ZIKV infection of Ifnar1 −/− females led to fetal demise. When dams were treated with an anti-ifnar antibody, infection of the developing fetus occurred but was less severe and did not cause fetal death. These findings establish models for studying mechanisms of in utero transmission and testing of candidate therapies for preventing congenital malformations. They also highlight the concern that ZIKV infection can occur in fetuses of otherwise healthy-appearing dams with uncertain neurodevelopmental consequences.

Recently, we and others have developed models of ZIKV pathogenesis in adult mice that recapitulated several features of human disease (). Whereas 4- to 6-week-old wild-type (WT) mice did not develop overt clinical illness after infection with a contemporary clinical strain of ZIKV, mice lacking the ability to produce or respond to type I interferon (IFN) (e.g., Ifnar1mice) developed severe neurological disease that was associated with high viral loads in the brain and spinal cord and substantial lethality. In a complementary approach using WT mice treated with a blocking anti-ifnar antibody (MAR1-5A3), we reported a less severe model of ZIKV pathogenesis that also resulted in replication of ZIKV in several organs (). These animals, however, survived infection and did not develop neurological signs or neuroinvasive disease.

In 2015, Brazil experienced a sharp rise in the number of cases of pregnancy-associated microcephaly, and this was linked to a concurrent epidemic of ZIKV infection. Mounting evidence suggests that ZIKV infection in pregnant women causes congenital abnormalities and fetal demise (). Initial case descriptions of microcephaly and spontaneous abortion have been bolstered by evidence of viral RNA and antigen in the brains of congenitally infected fetuses and newborns (). These findings were substantiated by a prospective study of a cohort of symptomatic, ZIKV-infected pregnant women in which 29% of fetuses exhibited developmental abnormalities including microcephaly and IUGR, which in a subset of cases resulted in fetal demise or stillbirth (). Preliminary reports suggest that ZIKV-induced fetal abnormalities can occur in all trimesters of pregnancy although the most severe manifestations are associated with infections in the first and second trimesters (). Congenital abnormalities associated with ZIKV infection also have been described in French Polynesia (by retrospective analysis) and other Latin American countries (). These findings suggest that ZIKV strains in French Polynesia and Latin America share the potential to cause disease during pregnancy.

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that was first isolated from a febrile rhesus macaque in Uganda in 1947 and is related to other globally relevant arthropod-transmitted human pathogens including dengue (DENV), yellow fever (YFV), West Nile (WNV), Japanese encephalitis (JEV), and tick-borne encephalitis viruses (). Over the last decade, ZIKV has emerged from a relatively obscure status to causing large epidemics in Micronesia, French Polynesia, and South and Central America. Although in most instances ZIKV infection results in a self-limiting febrile illness associated with rash and conjunctivitis, severe neurological phenotypes can occur, including Guillain-Barre syndrome and meningoencephalitis (). Infection in pregnant women is of major concern, as it is linked to catastrophic fetal abnormalities including microcephaly, spontaneous abortion, and intrauterine growth restriction (IUGR) due to placental insufficiency (). Because of the growing public health concern, there is an urgent need to establish animal models of intrauterine ZIKV infection that define mechanisms of fetal transmission and facilitate testing of therapeutics and vaccines. Furthermore, an in utero animal model of ZIKV infection would establish causality and satisfy the criteria for proof of teratogenicity ().

Histopathological assessment of ZIKV-infected Ifnar1fetal brains demonstrated abundant apoptotic cells within multiple regions at E13.5 ( Figures 4 A–4D). Activated caspase-3 staining showed low levels of physiological apoptosis in uninfected fetuses ( Figures 4 E–4H), whereas infected animals had apoptotic cells throughout the midbrain and hindbrain ( Figures 4 B–4D and 4I). Although we could localize viral RNA in infected placentas, multiple attempts at RNA FISH staining of ZIKV-infected fetal brains did not yield a clear pattern of viral RNA expression (data not shown), despite the recovery of infectious virus ( Figures S1 A and S1B). Accordingly, we cannot state with certainty whether the enhanced apoptosis within ZIKV-infected fetuses results from infection-induced apoptosis or another process, including ischemia due to placental insufficiency. The presence of numerous apoptotic cells within the developing central nervous system (CNS) coupled with the established neurotropism of ZIKV (), however, suggests direct infection may contribute.

(B-H) 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 (B-C) and hindbrain (D) of the infected Ifnar1 +/− fetus. Alternatively, low levels of physiological apoptosis are seen in the absence of infection (F-H).

Pregnant Infar1 −/− dams were infected with 10 3 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).

Pathological analysis of ZIKV-infected Ifnar1(maternal) and Ifnar1(fetal) placentas showed severe vascular injury characterized by irregularly shaped, reduced fetal capillaries and destruction of the placental microvasculature ( Figures 3 A and 3B , Ifnar het severe). Infected Ifnar1placentas were smaller, mostly because the labyrinth zone was markedly thinned. In addition, apoptotic trophoblasts were evident in ZIKV-infected placentas ( Figure 3 A, black arrows). Immunofluorescence staining of pan-cytokeratin, a pan-trophoblast marker, was diminished in infected Ifnar1placentas, consistent with evidence of apoptotic trophoblasts ( Figure 3 B). Apoptosis in trophoblasts can cause disruption of the placental barrier, which compromises protection against pathogens (). Indeed, ZIKV-infected Ifnar1placentas contained large numbers of nucleated fetal erythrocytes ( Figure 3 A, blue arrows), key indicators of fetal stress. Evidence of vascular damage and fewer blood vessels also was reflected by diminished staining of vimentin, a marker of fetal blood vessels in mouse placentas ( Figure 3 B).

(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.

We evaluated ZIKV localization in the placenta to define whether transmission occurred by a trans-placental route. The mouse placenta is comprised of the maternal decidua and the fetal embryo-derived compartments, including the junctional and labyrinth zones ( Figure 2 A). Different types of trophoblasts with distinct functions reside within all three layers, including trophoblast giant cells, glycogen trophoblasts, and spongiotrophoblasts. Within the labyrinth zone, fetal capillaries are lined by fetal blood vessel endothelium, which are separated from maternal sinusoids by a layer of mononuclear trophoblasts and a syncytiotrophoblast bilayer ( Figure 2 A) (). We performed RNA fluorescence in situ hybridization (FISH) coupled with histopathological analysis in ZIKV-infected Ifnar1placentas and confirmed the presence of ZIKV RNA in different trophoblast cells, including glycogen trophoblasts and spongiotrophoblasts ( Figure 2 B) and to a lesser extent in mononuclear trophoblasts and syncytiotrophoblasts (data not shown). These findings are consistent with cell culture studies demonstrating ZIKV infection of human trophoblast cell lines () and suggest that the mouse model of infection during pregnancy recapitulates features of human disease including placental tropism of ZIKV. We independently confirmed ZIKV infection and replication in two of three human trophoblast cell lines ( Figure S2 ). Transmission electron microscopy of placentas revealed multiple 50 nm dense bodies within the endoplasmic reticulum of the mononuclear trophoblasts ( Figure 2 C, left), consistent with ZIKV infection of the maternal placenta. As these bodies resemble flavivirus virions () and were not present in uninfected animals, they are suggestive for the presence of virus. Proximity to non-nucleated maternal erythrocytes ( Figure 2 C, left) confirmed the location as within the maternal face of the placenta. Consistent with a trans-placental route of infection, we also observed bodies resembling virions within the endoplasmic reticulum of fetal endothelial cells that lined damaged fetal capillaries ( Figure 2 C, right). The cellular and ultrastructural evidence of ZIKV infection in trophoblasts and fetal endothelium suggests that maternal viremia leads to compromise of the placental barrier by infecting fetal trophoblasts and entering the fetal circulation.

(C) Viral titer in the supernatants of ZIKV-infected trophoblasts at the indicated time points were assessed by infectious focus-forming assay on Vero cells. Results represent the mean ± SEM of two independent experiments.

(A) Flow cytometry showing the efficiency of ZIKV infection in the indicated trophoblast cell lines. The indicated trophoblast cell lines were infected with ZIKV strain H/PF/2013 at MOI 0.1 for 2 hr, washed with PBS to remove the input virus, and collected at the indicated time points.

(C) Transmission electron microscopy images of ZIKV infected Ifnar1 −/− placentas. ZIKV particles were identified within the endoplasmic reticulum in the maternal sinus (left), and in the fetal endothelium (right) lining fetal capillaries in the labyrinth layer.

(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.

The levels of ZIKV RNA detected in WT fetuses were affected by the dose of anti-ifnar mAb administered, with the greatest amounts of ZIKV RNA present in fetuses receiving 2 or 3 mgs of anti-ifnar mAb ( Figure 1 E). ZIKV RNA persisted in the anti-ifnar mAb-treated fetal heads and bodies at least through E16.5 ( Figures S1 C and S1D), a critical time in early development of the mouse brain. The placentas in both the Ifnar1and anti-ifnar antibody models exhibited higher levels of infection than the fetal tissues, and ZIKV RNA accumulation in the placenta was independent of the anti-ifnar mAb dose above 0.5 mg ( Figure 1 F). In comparison, mice treated with the isotype control antibody sustained low levels or no detectable ZIKV infection in the placenta, fetal heads or maternal tissues ( Figure 1 E, 1F, and 1I–1K). Collectively, these data suggest that the mouse placenta is vulnerable to infection with ZIKV, and that high-grade infection may cause placental insufficiency, IUGR, and fetal demise, at least in Ifnar1animals. Anti-ifnar mAb-treated animals sustained less infection and no enhanced lethality although a mild IUGR phenotype was observed.

In our second model of ZIKV infection during pregnancy, WT mice were treated with MAR1-5A3, a blocking anti-ifnar monoclonal antibody (), on E5.5, inoculated with ZIKV on E6.5 or E7.5, and fetuses were analyzed on E13.5 or E15.5, respectively ( Figure 1 A). Although demise was not observed, fetuses exhibited evidence of IUGR compared to control mAb-treated and mock-infected animals (62.3 mmversus 50.2 mm, p < 0.005), albeit to a lesser extent than seen in Ifnar1animals ( Figure 1 D). In contrast, anti-ifnar mAb-treated mice inoculated subcutaneously with 10FFU of a clinical DENV serotype 3 (DENV-3) isolate that replicates in mice () did not exhibit evidence of placental or fetal infection by qRT-PCR or signs of IUGR ( Figure 1 D and data not shown). These results suggest that ZIKV may have greater tropism for placental cells than other flaviviruses.

To determine whether direct infection of the placenta and fetus occurred, we measured ZIKV RNA levels by quantitative real-time RT-PCR (qRT-PCR) as well as infectious virus by plaque assay. High levels of viral RNA and infectious virus were detected within the placenta and also within the fetus head by E13.5 ( Figures 1 E and 1F and Figures S1 A and S1B). As seen with the fetuses from dams infected on E6.5, ZIKV inoculation on E7.5 also resulted in fetal demise and resorption by E15.5 as well as growth restriction (141.8 mmversus 79.5 mm, p < 0.0001, Figure 1 H) and pallor ( Figure 1 B) of intact fetuses. As expected from prior studies with Ifnar1males (), high levels of ZIKV were present in the blood, spleen, and brain of Ifnar1dams at day 7 after infection ( Figures 1 I–1K). Of note, the amount of ZIKV RNA within the placenta was ∼1,000-fold greater than in maternal serum ( Figures 1 F and 1I), suggesting that ZIKV replicates preferentially within this tissue.

(C-G) Viral burden was measured by qRT-PCR assay from the fetal head (C), placenta (D), and in the indicated maternal tissues (E-G) on E16.5 after infection at E6.5 after dams were treated with anti-ifnar or control antibody. In this Figure, symbols represent individual fetuses or tissues pooled from 2 independent experiments with the exception of 4 intact Ifnar1 +/− fetal heads that were carried by a single dam.

Pregnant dams were infected with 10 3 FFU of ZIKV or via a subcutaneous route. A and B. Infectious virus detected by plaque assay in fetal head (A) and the placenta (B) on E13.5 after infection of Ifnar1 −/− dams.

In the Ifnar1model, pregnant dams mated with WT mice were inoculated on embryonic days 6.5 (E6.5) and E7.5 and sacrificed on E13.5 and E15.5, respectively ( Figure 1 A). To minimize confounding effects of maternal illness on fetal viability, we evaluated pregnant Ifnar1mice prior to the onset of disease, which is characterized by hunched posture, fur ruffling, or hind-limb paralysis (). Individual fetuses were evaluated morphologically for size and appearance by measuring the crown-rump length and the occipito-frontal diameter of the fetal head, the latter of which establishes microcephaly in human fetuses (). By E13.5, the majority of ZIKV-infected Ifnar1heterozygous fetuses had undergone fetal demise and been resorbed, leaving only a placental remnant ( Figures 1 B, upper, and 1 C). The remaining intact Ifnar1fetuses exhibited significant IUGR (60.2 mmversus 48.7 mm, p < 0.0001, Figure 1 D). In ZIKV-infected pregnant women, multiple phenotypes have been described including fetal demise, IUGR, and microcephaly (). Although we did not observe isolated microcephaly in this in utero model of ZIKV infection, several other abnormalities were visible in ZIKV-infected Ifnar1fetuses, including pallor and foci of necrotic tissue in the placenta ( Figure 1 B).

Since the type I interferon (IFN) response prevents efficient replication of ZIKV in peripheral organs of WT mice (), we initially used Ifnar1mice to facilitate high levels of ZIKV replication during pregnancy. Ifnar1female mice were bred with WT males so that resulting fetuses would be heterozygous (Ifnar1) and thus exhibit a largely intact type I IFN signaling response. In parallel, we developed a second model of ZIKV infection during pregnancy by treating WT pregnant dams with an anti-ifnar-blocking antibody 1 day prior to infection ( Figure 1 A). Both sets of pregnant mice were inoculated via a subcutaneous route in the footpad with 10focus forming units (FFU) of a clinical isolate from French Polynesia (H/PF/2013) that was passaged in Vero cells. This ZIKV strain is at least 97% identical at the nucleotide level to the sequence of an epidemic strain of ZIKV in Brazil (). We confirmed the sequence of our ZIKV H/PF/2013 stock by next-generation sequencing (data not shown), which also allowed us to exclude the presence of adventitious pathogens.

(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

(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.

(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.

(D) Fetus size as assessed by CRL x OF diameter in E13.5 fetuses following E6.5 infection of the indicated pregnant dams with either ZIKV or DENV-3. For these experiments, a total dose of 3 mg of anti-ifnar antibody was used. 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.

(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.

(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.

(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 (10 3 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 (10 3 FFU) or DENV-3 (10 3 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.

Discussion

Epidemiological studies have found that ZIKV infection during pregnancy causes catastrophic neurodevelopmental outcomes in human fetuses, but there currently is no effective treatment or prevention of ZIKV infection other than avoidance of its mosquito vectors. Given the devastating effects of this rapidly emerging infectious disease, small animal models of ZIKV infection during pregnancy are urgently needed to test candidate therapeutics and vaccines that could prevent or mitigate intrauterine infection with ZIKV. We developed two mouse models that support ZIKV replication and trans-placental transmission in pregnant dams: (1) a model of severe disease in pregnant Ifnar1−/− dams that resulted in fetal demise; and (2) a less severe model of ZIKV pathogenesis in utero using pregnant WT dams that were given anti-ifnar antibody prior to and during infection, which resulted in mild IUGR and viral infection within the fetal head during a key period in neurodevelopment.

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et al. Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth. Several aspects of our models of ZIKV infection during pregnancy resemble intrauterine infection by ZIKV in humans. Common features included tropism of ZIKV for the placenta, evidence of intrauterine infection, and fetal demise. However, infection during pregnancy in mice did not recapitulate all aspects of human disease, as we did not detect microcephaly, brain calcifications, or absence of individual brain structures, such as the corpus callosum. There are several reasons why ZIKV may not have induced these pathological manifestations in our models. In mice, brain neurogenesis begins around E10 (), and the brain of a newborn pup is relatively immature at postnatal day 1, akin to the developmental stage of the human brain at mid-gestation (). As the development of the mouse brain includes a major postnatal component, examination of the neurodevelopmental effects of ZIKV infection in mice may require infection later during pregnancy. Since ZIKV infects mature neurons as well as neural cell progenitors and limits their growth (), the morphological effects of ZIKV infection on brain development may be more apparent in species with larger cerebral cortices.

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et al. Interim Guidelines for the Evaluation and Testing of Infants with Possible Congenital Zika Virus Infection - United States, 2016. Although ZIKV-associated microcephaly has drawn major public health and media attention, ZIKV infection of human fetuses does not always cause this manifestation, and the sequelae of intrauterine infection in the absence of gross morphological abnormalities remain to be defined. In pregnant WT dams that were treated with an anti-ifnar antibody, we observed only mild growth restriction in the developing fetus although ZIKV RNA was detectable in the fetal head at both E13.5 and E16.5 after infection. Future behavioral studies may define whether intrauterine infection by ZIKV has long-term neurological effects in mice and serves as a model for evaluation of disease in humans ().

−/− dams led to severe placental damage and destruction of the microvasculature, which most likely limited blood flow to the developing fetus and caused severe IUGR, ischemia, and fetal demise. In vivo infection of the mouse placenta with ZIKV may provide a model for defining host factors required for or that restrict infection, which could suggest a path for developing therapies to limit placental and intrauterine infection. For example, since IFN-λ restricts ZIKV replication within human trophoblasts from term placentas ( Bayer et al., 2016 Bayer A.

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Coyne C.B. Type III Interferons Produced by Human Placental Trophoblasts Confer Protection against Zika Virus Infection. We found that the murine placentas can be infected by ZIKV. Infection of Ifnar1dams led to severe placental damage and destruction of the microvasculature, which most likely limited blood flow to the developing fetus and caused severe IUGR, ischemia, and fetal demise. In vivo infection of the mouse placenta with ZIKV may provide a model for defining host factors required for or that restrict infection, which could suggest a path for developing therapies to limit placental and intrauterine infection. For example, since IFN-λ restricts ZIKV replication within human trophoblasts from term placentas (), studies are planned to test the effects of exogenously administered IFN-λ on in utero transmission in mice.

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Hanson R.P. Intrauterine infection of mice with St. Louis encephalitis virus: immunological, physiological, neurological, and behavioral effects on progeny. Intrauterine infection with flaviviruses may be an underappreciated phenomenon. Prior to the ZIKV epidemic, there were isolated descriptions of trans-placental infection in humans with other flaviviruses including WNV and JEV (). These reports suggest that sporadic cases of flavivirus-induced miscarriage or fetal demise might have been unrecognized, although this remains speculative. We are currently testing whether additional variations in ZIKV infection (e.g., dose, route of administration, virus strain, time of infection during pregnancy, and time of analysis) in our mouse model of in utero transmission can recapitulate other morphological abnormalities in the CNS that are described in human disease. Of note, intrauterine infection with Saint Louis encephalitis virus, a less well studied flavivirus, caused severe neurological outcomes in mice. In that model, disease depended on the gestational date of infection (); mice infected early in gestation survived, whereas those inoculated later developed neurological malformations and died as neonates (). In our studies with DENV and an anti-ifnar-blocking antibody, inoculation of mice did not result in placental infection, although it is possible that this was due to a diminished ability of DENV to replicate in mice compared to ZIKV. Experiments that test infection of pregnant animals with additional related viruses may clarify whether placental infection is an underappreciated clinical manifestation of flavivirus pathogenesis.

Treatment and prevention of ZIKV infection will likely require small animal models for testing of vaccines and potential therapies. The mouse models described in our study may be relevant to studying mechanisms of pathogenesis and determining whether vaccines given prior to pregnancy can prevent infection in the developing fetus. Mouse models of ZIKV infection during pregnancy also may provide fundamental insights into how the placental barrier prevents viral infection from the developing fetus, and why this process fails in the context of specific pathogens. Finally, our animal model of in utero transmission establishes causality of a fetal syndrome associated with ZIKV infection in mice.