Rodents, including laboratory mice, are not a natural host of ZIKV infection because their IFN-driven immune responses can inhibit ZIKV replication11,26,35. As a result, murine models of ZIKV infection require IFN manipulation and are thus inherently limited in their potential for both translational relevance and mechanisms of congenital pathogenesis11,26,35,36. Here we show that ZIKV inoculation of pregnant marmosets causes a maternal asymptomatic infection and seroconversion similar to that seen in humans. This maternal infection is measured as prolonged viremia and viruria, and is accompanied by active viral replication in the placenta, with resultant fetal infection, a predominant neuropathology of the developing fetal brain and eye, and early demise. Although atypical for non-pregnant human cases, persistence of virus in serum and urine (i.e. viremia and viruria) beyond 14 days and as long as 8 months has been reported in ZIKV-infected pregnant women33,37, and is felt to be due to permissive replication in the placenta and/or fetus functioning as a viral reservoir3,29. In both inoculated dams, spontaneous abortion was observed 16–18 days post infection, with extensive productive viral infection in the placenta and fetal tissues documented by qRT-PCR, immunohistochemistry, and ISH for (+) and (−) ssRNA strands. We also visualized direct infection of neural progenitor cells associated with disorganized migration. The most straightforward explanation for the measured 50% expected biparietal diameter of the fetuses from dam 1 is cranial collapse following intrauterine demise; however, we also cannot rule out this finding as an early predictor of microcephaly since it is temporally and spatially accompanied by aberrant migration patterns which are hallmarks of secondary microcephaly. In 1 of the 2 inoculations, a spontaneous de novo mutation, F252L, shared in common with a ZIKV genome from a human case of microcephaly, was identified. Finally, maternal interferon-associated host responses and increased activity of proinflammatory cytokines were demonstrated at early as day 2 post-inoculation. Collectively, these findings reveal several intriguing and novel mechanistic insights pertaining to the potential roles of placental replication, mutation-driven viral adaptation, and maternal responses in ZIKV-mediated fetal pathogenesis.

The first trimester of pregnancy in both humans and marmosets is critical for appropriate development of the major organ systems, especially the brain and its associated neuroprogenitor cells. Gestational ages of 79–83 days (dam 1) and 68–72 days (dam 2) at the time of inoculation correspond to approximately 6.6–8.6 weeks in humans (Fig. 2), and is a time of crucial placental development in the marmoset. Indeed, the marmoset spends a third of its entire 143-day gestation invested in placentation. When compared to the human, which completes organogenesis by day 56 of a 266-day gestation, the marmoset will not complete organogenesis until day 80 (Fig. 2). Spontaneous abortion rates in the absence of congenital viral infections at this stage are hard to document, given the small sizes of the placental and fetal material being expelled; however, the available data indicate that two abortions in this gestational age range is an unexpected, spontaneous finding. For 2016–17, the SNPRC non-infected marmoset colony documented two abortions with placental/fetal material indicating this gestational age range out of 103 pregnancies (1.9%). The most comprehensive data documenting loss rates at this stage of marmoset pregnancy come from hormonal verification of pregnancy and pregnancy loss in a large (n = 596) number of marmoset pregnancies in a research colony in Berlin38. The investigators documented a pregnancy loss rate during days 80–142 of 4.4%; our finding of 2 of 2 ZIKV-infected pregnancies resulting in spontaneous abortion is thus statistically significant (p = 0.011 by Fisher’s Exact Test). In comparison, the overall loss rate in experimentally ZIKV-infected macaques at a comparable stage of embryonic development is 38%, as compared to expected non-infected loss rates of approximately 4–10% (Dudley et al., under review). In humans, documented rates of pregnancy loss due to ZIKV across all 3 stages at time of infection are estimated to be ~3%, with 8% of completed pregnancies following infection during the first trimester resulting in ZIKV-associated birth defects4. However, due to inherent limitations in the studies of miscarriages, early and mid-gestation ZIKV-induced pregnancy loss has been likely underreported and largely limited to case reports and limited retrospective series25,39. We speculate that our observation of uniform pregnancy loss in our marmoset model is a highly significant novel finding of potential translational significance and worthy of prioritized further human studies and re-analysis of existing epidemiologic data40.

Only rodents rendered deficient of type I interferon through experimental manipulations are highly susceptible to maternal-fetal transmission and resultant congenital disease26,35. It is thus important to note that in ZIKV-infected pregnant marmoset dams, maximal RNA-seq differential expression between the infected and uninfected state and significant up-regulation of interferon-associated genes in peripheral blood were observed at day 2. This early IFN systemic burst in pregnant dams is in contrast to day 9 in a previous study of ZIKV-infected male marmosets22. This may reflect the enhanced susceptibility of pregnant versus non-pregnant individuals to ZIKV infection, driving a more rapid host response and resulting in higher viral loads as well as persistence in body fluids and tissues33,37. In addition, it may provide crucial insight into the role of the placenta as both reservoir and conduit. We found early (day 2) increases in the concentrations of several proinflammatory molecules, including IFN-γ, which drives the type II interferon pathway. Interestingly, an antiviral response characterized by increased interferon and proinflammatory cytokine activity has been previously reported in association with ZIKV infection of primary human placental macrophages (Hofbauer) cells and cytotrophoblasts41. Here we observed extensive ZIKV infection of the placenta and fetus in the absence of overt inflammation and despite induction of a robust host antiviral response. This is consistent with ours and others recently described human placental pathogenesis39,42,43,44,45,46,47.

Among the 31 candidate DEGs at day 7 post-infection, the decreased expression of TRPV6 and LYPD6 relative to baseline is especially intriguing. TRPV6 is highly expressed in placenta48, and has been shown to be critical for maternal-fetal Ca2+ transport and fetal Ca2+ homeostasis in an aromatase knockout pregnant mouse model49. In this mouse model, decreased expression of TRPV6 was associated with estrogen deficiency, which increases risk of miscarriage50. Like TRPV6, LYP6 is highly expressed in placenta51, and was critical for normal neuroectodermal development by way of Wnt/β-catenin signaling in zebrafish embryos52. Further studies are needed to determine whether viral suppression of these highly expressed maternal placental genes plays a role in the spontaneous abortions and disrupted fetal neurodevelopment seen here in association with first-trimester ZIKV infection.

An important point of consideration for applying our current findings to human congenital disease is an appreciation of the timeline of embryonic and placental development in marmoset monkeys versus humans. Marmosets have a delayed period of embryogenesis relative to humans (Fig. 2), resulting in the marmoset undergoing critical stages of neural development in association with a relatively far more mature placenta (both structurally and in terms of duration of the placenta as a functioning organ). Our findings further suggest that marmosets may be particularly sensitive to fetal Zika infection with respect to fetal loss, perhaps related to the initial infection of a relatively mature placenta when considered against their human counterparts (Fig. 2).

Our findings herein summarily show that the marmoset model faithfully recapitulates key characteristics of CZS in humans that are not observed in other rodent nor NHP models. Notably, ZIKV-infected rhesus macaques exhibited no fetal CNS abnormalities with maternal infection15. Pigtail macaques exhibited neural injury with severe hypoplasia and asymmetry within the occipital-parietal lobes9, but did not manifest the diffuse effects on neuroprogenitor cells observed in both the marmoset and human cases. In contrast, neuronal disorganization in ZIKV-infected pregnant marmosets was observed during early gestation at the posterior and crown in what will later comprise the occipital–parietal lobes (Fig. 8). We speculate that the capacity for the marmoset to better recapitulate human infection is related to ongoing placental development and trophoblast renewal, whereby the virus is permissive to replication in both placental trophoblasts42 and placental macrophages (Hofbauer cells)39 without evidence of inflammation39,42,43,44,45,46,47. As is likely the case in humans, the placenta may be viewed as an effective “shuttle” to the fetus, functioning first as a reservoir and later as a source of fetal infection of susceptible cell types and tissues, including neuroprogenitor cells43. Nevertheless, in fetuses from both dam 1 and dam 2, the virus appears to have caused injury to the neocortex documented by histopathological examination and neuronal disorganization in the absence of localized inflammation, as also seen in mouse models11 and human cases44.

For both ZIKV-infected marmosets and humans, fetal infection appears to be established in the absence of localized fetal and placental inflammation39,42,43,44,45,46,47. However, there is clear evidence of injury to the fetal neocortex as visualized by histopathological examination showing neuronal organization, mimicking findings in mice models of ZIKV vertical transmission as well as human cases11,44. This notion of the significance of placental mechanics and placental cell infection in the pathogenesis in CZS has been previously brought forward by a number of investigators36,41,42,43,45,46,47,53. It has long been supposed that shuttling of bloodborne ZIKV to the developing human fetus requires establishment of maternal blood in the intervillous space, which occurs approximately at 6–8 weeks post-conception (mean of roughly 42 days) (Fig. 2), or 8–10 weeks of pregnancy, corresponding to the late first trimester. However, epidemiologic observations reveal that both first and second trimester are highly vulnerable windows of CZS susceptibility4,40. It has thus been unclear whether CZS susceptibility in the first trimester may relate to leaks in placental architecture or shifting antiviral capacity of the maturing trophoblast36.

Our results here document a definitive role for early infection of a maturing placenta in the response to ZIKV infection during the critical period of fetal neurodevelopment (Fig. 2). We observed (1) active and robust ZIKV replication in the absence of local inflammation, (2) a maternal host response dominated by interferon type I and II-associated genes and proinflammatory cytokines, and (3) evidence of a functional placenta (normal amniotic fluid volume) and viable fetus despite viral replication and disruption of neuronal migration in developing brain and eye demise. Taken together, these findings suggest that contemporary ZIKV strains are capable of both replicating in the placenta and migrating to the developing fetus despite robust antiviral maternal responses. Our findings are consistent with previous work showing that multiple placental cell types, including macrophages, cytotrophoblasts, and primary human trophoblasts, are permissive to ZIKV infection39,41,42,43,44,45,46,47. Thus, our studies in both humans and marmoset reveal that the placenta likely serves both as a conduit for fetal infection and a reservoir for ongoing replication41,42.

Contemporary ZIKV strains may have evolved to acquire the capacity for placental replication and fetal neuropathogenesis36,41,42. Notably, viral genome sequencing showed both viruses retained the S139N mutation in the pre-membane (prM) protein (associated with increased ZIKV infectivity of neural progenitor cells and more severe microcephaly in a mouse model54). Of note, the African strains used in the work of Bayer et al.45, where they failed to see robust ZIKV replication in primary human trophoblasts, did not demonstrate the S139N mutation. The virus from dam 2 had a further de novo F252L coding mutation shared in only 1 of 6 available microcephalic ZIKV genomes in humans33. The serendipitous finding of a F252L coding mutation is unlikely to be coincidental, as the mutation was not seen in any of the other 603 ZIKV genomes sequenced to date not associated with microcephaly (p = 0.0135). Further investigation of the potential functional significance of the F252L mutation is warranted.

We are enthused by both the potential relevance and significance of our findings employing pregnant marmosets. First, they show that the marmoset faithfully recapitulates all crucial aspects of human CZS and does not require the exogenous manipulation of interferons, necessary in murine models11,26,35,36. Given the capacity for the marmoset to complete multifetal gestations in 143 days and undergo repeat pregnancy within 10 days, this is an efficient model whose reproductive capacity over short time intervals rivals most rodent models. Second, these studies underscore crucial mechanistic insights pertaining to the role of the placenta in modulating (or failing to modulate) congenital infection and maternal-to-fetal transmission. Understanding the role of placental permissiveness in this process is of utmost crucial importance and reinforces the emerging notion of the placenta not as a de facto barrier, but as an active conduit for maternal and fetal communication during gestation. Thus, the identification of drugs not only to treat or prevent ZIKV infection of the fetus but also to prevent placental infection and viral transport may be critical to prevent the irreversible neuronal damage associated with CZS. Third and of potential crucial clinical importance, our findings demonstrate a high rate of pregnancy loss with maternal ZIKV infection and placental and fetal transmission. These observations should prompt reexamination of human epidemiologic data and provide additional endpoints in prospective studies as ZIKV continues to emerge in newly endemic communities in Texas, Florida, and other at-risk regions of the Americas.