Zika in Mice Causes Placental Damage & Fetal Demise - Cell

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.

niman · May 12, 2016

Highlights

niman · May 12, 2016

Introduction

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 (Lazear and Diamond, 2016). 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 (Carteaux et al., 2016, Oehler et al., 2014). 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 (Brasil et al., 2016). 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 (Rasmussen et al., 2016).

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 (Brasil et al., 2016, Sarno et al., 2016, Ventura et al., 2016). 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 (Martines et al., 2016, Mlakar et al., 2016). 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 (Brasil et al., 2016). 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 (Brasil et al., 2016). Congenital abnormalities associated with ZIKV infection also have been described in French Polynesia (by retrospective analysis) and other Latin American countries (Cauchemez et al., 2016). These findings suggest that ZIKV strains in French Polynesia and Latin America share the potential to cause disease during pregnancy.

Recently, we and others have developed models of ZIKV pathogenesis in adult mice that recapitulated several features of human disease (Aliota et al., 2016,Lazear et al., 2016, Rossi et al., 2016). 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., Ifnar1−/− mice) 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 (Lazear et al., 2016). These animals, however, survived infection and did not develop neurological signs or neuroinvasive disease.

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.

niman · May 12, 2016

Results

Since the type I interferon (IFN) response prevents efficient replication of ZIKV in peripheral organs of WT mice (Lazear et al., 2016), we initially used Ifnar1−/−mice to facilitate high levels of ZIKV replication during pregnancy. Ifnar1−/−female 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 1A). Both sets of pregnant mice were inoculated via a subcutaneous route in the footpad with 103 focus 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 (Calvet et al., 2016, Faria et al., 2016). 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.

In the Ifnar1−/− model, 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 1A). To minimize confounding effects of maternal illness on fetal viability, we evaluated pregnant Ifnar1−/− mice prior to the onset of disease, which is characterized by hunched posture, fur ruffling, or hind-limb paralysis (Lazear et al., 2016). 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 (Chervenak et al., 1987, Staples et al., 2016). By E13.5, the majority of ZIKV-infected Ifnar1+/− heterozygous fetuses had undergone fetal demise and been resorbed, leaving only a placental remnant (Figures 1B, upper, and 1C). The remaining intact Ifnar1+/− fetuses exhibited significant IUGR (60.2 mm2versus 48.7 mm2, p < 0.0001, Figure 1D). In ZIKV-infected pregnant women, multiple phenotypes have been described including fetal demise, IUGR, and microcephaly (Brasil et al., 2016, Sarno et al., 2016). Although we did not observe isolated microcephaly in this in utero model of ZIKV infection, several other abnormalities were visible in ZIKV-infected Ifnar1+/− fetuses, including pallor and foci of necrotic tissue in the placenta (Figure 1B).

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 1E and 1F and Figures S1A 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 mm2 versus 79.5 mm2, p < 0.0001, Figure 1H) and pallor (Figure 1B) of intact fetuses. As expected from prior studies with Ifnar1−/− males (Lazear et al., 2016, Rossi et al., 2016), high levels of ZIKV were present in the blood, spleen, and brain of Ifnar1−/−dams at day 7 after infection (Figures 1I–1K). Of note, the amount of ZIKV RNA within the placenta was ∼1,000-fold greater than in maternal serum (Figures 1F and 1I), suggesting that ZIKV replicates preferentially within this tissue.

In our second model of ZIKV infection during pregnancy, WT mice were treated with MAR1-5A3, a blocking anti-ifnar monoclonal antibody (Sheehan et al., 2006), on E5.5, inoculated with ZIKV on E6.5 or E7.5, and fetuses were analyzed on E13.5 or E15.5, respectively (Figure 1A). Although demise was not observed, fetuses exhibited evidence of IUGR compared to control mAb-treated and mock-infected animals (62.3 mm2 versus 50.2 mm2, p < 0.005), albeit to a lesser extent than seen in Ifnar1+/− animals (Figure 1D). In contrast, anti-ifnar mAb-treated mice inoculated subcutaneously with 103 FFU of a clinical DENV serotype 3 (DENV-3) isolate that replicates in mice (Pinto et al., 2015, Sarathy et al., 2015) did not exhibit evidence of placental or fetal infection by qRT-PCR or signs of IUGR (Figure 1D and data not shown). These results suggest that ZIKV may have greater tropism for placental cells than other flaviviruses.

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 1E). ZIKV RNA persisted in the anti-ifnar mAb-treated fetal heads and bodies at least through E16.5 (Figures S1C and S1D), a critical time in early development of the mouse brain. The placentas in both the Ifnar1−/− and 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 1F). 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 1E, 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 inIfnar1+/− animals. Anti-ifnar mAb-treated animals sustained less infection and no enhanced lethality although a mild IUGR phenotype was observed.

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 2A). 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 2A) (Simmons and Cross, 2005, Watson and Cross, 2005). We performed RNA fluorescence in situ hybridization (FISH) coupled with histopathological analysis in ZIKV-infected Ifnar1+/− placentas and confirmed the presence of ZIKV RNA in different trophoblast cells, including glycogen trophoblasts and spongiotrophoblasts (Figure 2B) 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 (Bayer et al., 2016) 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 2C, left), consistent with ZIKV infection of the maternal placenta. As these bodies resemble flavivirus virions (Allison et al., 2003) and were not present in uninfected animals, they are suggestive for the presence of virus. Proximity to non-nucleated maternal erythrocytes (Figure 2C, 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 2C, 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.

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 3A and 3B , Ifnar het severe). Infected Ifnar1−/− placentas were smaller, mostly because the labyrinth zone was markedly thinned. In addition, apoptotic trophoblasts were evident in ZIKV-infected placentas (Figure 3A, black arrows). Immunofluorescence staining of pan-cytokeratin, a pan-trophoblast marker, was diminished in infected Ifnar1−/− placentas, consistent with evidence of apoptotic trophoblasts (Figure 3B). Apoptosis in trophoblasts can cause disruption of the placental barrier, which compromises protection against pathogens (Robbins and Bakardjiev, 2012). Indeed, ZIKV-infected Ifnar1+/− placentas contained large numbers of nucleated fetal erythrocytes (Figure 3A, 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 3B).

Histopathological assessment of ZIKV-infected Ifnar1+/− fetal brains demonstrated abundant apoptotic cells within multiple regions at E13.5 (Figures 4A–4D). Activated caspase-3 staining showed low levels of physiological apoptosis in uninfected fetuses (Figures 4E–4H), whereas infected animals had apoptotic cells throughout the midbrain and hindbrain (Figures 4B–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 S1A 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 (Lazear et al., 2016), however, suggests direct infection may contribute.

niman · May 12, 2016

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.

The placenta acts as a barrier against infections, due to multiple unique structural, cellular, and immune properties. The detrimental effects of congenital viruses on pregnancy and fetal outcomes occur in part because of impaired trophoblast function (Arechavaleta-Velasco et al., 2002). Defective placentas can lead to severe maternal and fetal morbidity and mortality during pregnancy, including spontaneous abortion, stillbirth, preterm birth, IUGR, and other complications. We observed profound pathological changes in ZIKV-infected placentas, including trophoblast apoptosis, abnormal fetal capillary features, and increased fetal nucleated erythrocytes, indicating malfunction of mouse placentas caused by ZIKV infection.

We observed variability in susceptibility to ZIKV infection of different human trophoblast cell lines. Trophoblast cell lines (JEG-3 and HTR-8) originally cultured from choriocarcinoma explants and first trimester human villous explants, respectively, which exhibit features of extravillous trophoblasts (EVTs) including high invasive capacity and expression of HLA-G, a MHC class II molecule, were susceptible to ZIKV infection. In contrast, a relatively undifferentiated cytotrophoblast cell line (BeWo) was not. Previous studies have shown that EVTs are most susceptible to bacterial infections, particularly during the first and second trimesters (Cao and Mysorekar, 2014, Robbins and Bakardjiev, 2012,Zeldovich and Bakardjiev, 2012). Thus, it is possible that in early pregnancy, ZIKV infects EVTs and enters the fetal circulation. Placentas nearer to term, which have reduced EVTs on the tips of anchoring villi and a more fully developed placental barrier, in general exhibit greater resistance to infection. Indeed, human primary trophoblasts of the villous syncytiotrophoblast phenotype from term placentas were resistant to ZIKV infection due to the production of IFN-λ in paracrine manner (Bayer et al., 2016).

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 (Finlay and Darlington, 1995), 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 (Semple et al., 2013). 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 (Tang et al., 2016), the morphological effects of ZIKV infection on brain development may be more apparent in species with larger cerebral cortices.

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 (Staples et al., 2016).

We found that the murine placentas can be infected by ZIKV. Infection ofIfnar1−/− 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), studies are planned to test the effects of exogenously administered IFN-λ on in utero transmission in mice.

Maternal-fetal transmission of pathogens can be mediated by diverse pathways, with the most common being via an ascending route and hemochorial transmission. Viruses from the urogenital tract can disseminate into intrauterine space and colonize the fetal membrane or the placenta in an ascending manner (Edwards et al., 2015). In contrast, viruses from the maternal blood circulation can be transported to the feto-maternal space and infect trophoblasts lining the maternal-fetal interface. These trophoblasts include the EVTs, which embed in maternal decidua, endovascular extravillous cytotrophoblasts, and villous trophoblasts, which are bathed in maternal blood (Delorme-Axford et al., 2014). Our studies suggest that ZIKV can infect the placenta through blood-placental transmission and bypass the placental barrier to infect the fetus. Nonetheless, ascending infection routes might be important in sexual transmission during pregnancy. ZIKV has been found in human semen (Musso et al., 2015) and mouse testes (Lazear et al., 2016, Rossi et al., 2016) and can be transmitted sexually from male to female in humans (Musso et al., 2015).

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 (Chaturvedi et al., 1980, Nguyen et al., 2002). 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 (Andersen and Hanson, 1970); mice infected early in gestation survived, whereas those inoculated later developed neurological malformations and died as neonates (Andersen and Hanson, 1970, Andersen and Hanson, 1975). 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.

Author Contributions

J.J.M., J.G., B.C., K.K.N., R.S.K., I.U.M., and M.S.D. designed experiments. J.J.M., B.C., J.G, A.M.S, O.H.C., E.F., C.G., M.N., and K.K.N. performed the experiments. J.J.M, B.C, I.U.M., and M.S.D. analyzed the data. J.J.M., B.C., I.U.M., and M.S.D. wrote the first draft of the paper; all authors edited the manuscript.

Acknowledgments

This work was supported by grants from the NIH (R01 AI073755 and R01 AI104972 to M.S.D., R01 HD052664 to K.K.N., and R01 NS052632 to R.S.K.) and the Intellectual and Developmental Disabilities Research Center at Washington University (NIH/NICHD U54 HD087011). J.J.M., E.F., and D.J.P. were supported by a Rheumatology Research Foundation Scientist Development Award, NIH Pre-doctoral training grant award (T32 AI007163), and the NIH Research Education Program (R25 HG006687), respectively. The work also was supported by a Preventing Prematurity Initiative grant from the Burroughs Wellcome Fund and a Prematurity Research Initiative Investigator award from the March of Dimes (to I.U.M). We thank Wandy Betty for her TEM assistance, Fredrik Kraus for his expertise in placental pathology, and Justin Richner for advice with establishing the RNA FISH assays. Finally, we acknowledge Xavier de Lamballerie (Emergence des Pathologies Virales, Aix-Marseille Université, Marseille, France) and the European Virus Archive goes Global (EVAg) for consenting to the use of H/PF/2013 ZIKV strain for this study under a material transfer agreement with the EVAg parter, Aix-Marseille Université. M.S.D. is a consultant for Inbios, Visterra, and Takeda Pharmaceuticals and on the Scientific Advisory Boards of Moderna and OraGene.

Supplemental Information

Document S1. Article plus Supplemental Information

niman · May 12, 2016

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