Common Pathways Targeted by Viral Hemorrhagic Fever Viruses to Infect the Placenta and Increase the Risk of Stillbirth

Abstract

Viral hemorrhagic fevers (VHF) are endemic to Africa, South America and Asia and contribute to significant maternal and fetal morbidity and mortality. Viruses causing VHFs are typically zoonotic, spreading to humans through livestock, wildlife, or mosquito vectors. Some of the most lethal VHF viruses also impart a high-risk of stillbirth including ebolaviruses, Marburg virus (MARV), Lassa virus (LASV), and Rift Valley Fever Virus (RVFV). Large outbreaks and epidemics are common, though the impact on the mother, fetus and placenta is understudied from a public health, clinical and basic science perspective. Notably, these viruses utilize ubiquitous cellular surface entry receptors critical for normal placental function to enable viral invasion into multiple key cell types of the placenta and set the stage for maternal-fetal transmission and stillbirth. We employ insights from molecular virology and viral immunology to discuss how trophoblast expression of viral entry receptors for VHF viruses may increase the risk for viral transmission to the fetus and stillbirth. As the frequency of VHF outbreaks is expected to increase with worsening climate change, understanding the pathogenesis of VHF-related diseases in the placenta is paramount to predicting the impact of emerging viruses on the placenta and perinatal outcomes.

Graphical Abstract

Condensation:

We review how viral hemorrhagic fever viruses use commonly expressed placental cellular receptors to invade the placenta to mediate maternal-fetal transmission and stillbirth.

Introduction

Viral hemorrhagic fevers (VHF) are viral diseases characterized by severe fever, vasculopathy, organ damage, and potentially death. Large outbreaks and epidemics of these viruses occur frequently across vast regions of Africa, Asia and South America, and are expected to increase in frequency as climate change worsens. Our knowledge of VHF pathogenesis in pregnancy and how these viruses impact maternal, placental and fetal health is currently insufficient. The consequence of not addressing these research gaps for maternal-fetal health is a poor knowledge base to predict the outcomes of new therapeutics and vaccines in pregnancy for these lethal diseases. Pandemic preparedness must include a thorough investigation of the pathogenesis of viral disease in pregnancy, which begins with understanding the impact of infection on the placenta. The placenta is both the largest of fetal organs and the gateway to the fetus. Knowledge of viral tropism for the placenta can inform risk for maternal-fetal transmission as well as perinatal morbidity and mortality.

We lack a conceptual model for predicting the potential impact a newly emerging virus has on the fetus. In this manuscript, we present a hypothesis to explain why select VHF viruses are associated with high rates of stillbirth. We focus on the VHF viruses characterized by maternal-fetal transmission and a high risk for stillbirth: ebolaviruses, Marburg virus (MARV), Lassa virus (LASV), and Rift Valley Fever (RVF). Although stillbirth can be caused indirectly by severe maternal illness (e.g., influenza A viruses[1–3]), we propose the primary reason for the high rates of fetal death after VHF infection is the ubiquitous expression of viral entry receptors that enables widespread direct viral infection of the placenta (Table 1, Fig. 1). To support this hypothesis, we begin by describing why the placenta is an ideal virus “factory” and elucidate the general mechanisms used by VHF viruses to target placental and immune cells at the maternal-fetal interface. Next, we review four significant VHF viruses (i.e., ebolaviruses, MARV, LASV, RVF) and describe their known placental receptors for viral entry and perinatal outcomes. We further support our hypothesis by providing evidence from transgenic murine studies demonstrating amelioration of disease after viral infection when these viral entry receptors were knocked out. Finally, we contrast this discussion with the distribution of viral entry receptors in the placenta for other viruses for which maternal-fetal transmission and stillbirth is comparatively rare.

Table 1.

Viral Hemorrhagic Fever Viruses that Increase Risk for Stillbirth

Virus (Abbreviation, Virus Family)	Viral Structure	Examples of Viral Entry Receptors Expressed in STB and CTB	Adverse Maternal and Pregnancy Outcomes	Adverse Fetal Outcomes
Ebolaviruses (filovirus) and Marburg Virus (MARV, filovirus)	Enveloped, helical virus with a non-segmented, negative-sense single strand RNA genome	Apoptotic mimicry (PtdSer receptors) C-type lectin receptors	Ebolaviruses: Increased maternal disease severity, death. hemorrhagic fever, and maternal death. MARV: Few case reports. Maternal death in 100% (3/3) case reports.	Both genera of viruses result in maternal-fetal transmission. Ebolaviruses: Spontaneous abortion and stillbirth occur in nearly 100% of cases. Rare case reports of fetal/neonatal survivors after ebolavirus disease during pregnancy. MARV: Few case reports. Spontaneous abortion (67%, 2 out of 3 cases) and neonatal death (33%, one of 3 cases).
Lassa Virus (LASV, arenavirus)	Enveloped, helical virus with a bisegmented, negative-sense single strand RNA genome	Apoptotic mimicry (PtdSer receptors, e.g., TIM-1) α-dystroglycan modified with matriglycan, Gas6	Increased maternal mortality, hemorrhage, convulsions, and oliguria.	Maternal-fetal transmission, stillbirth, neonatal death, and preterm birth.
Rift Valley Fever Virus (RFV, phenuivirus)	Enveloped, tri-segmented, negative sense single strand RNA genome	Entry by acid-based cleavage of membrane fusion protein C-type lectin receptor (e.g., DC-SIGN)	Unknown	Maternal-fetal transmission, spontaneous abortion, and stillbirth.
Dengue Fever Virus (DENV, flavivirus)	Enveloped, positive sense single strand RNA genome	Wide variety of viral entry receptors used such as DC-SIGN (C-type lectin receptor) and heat shock proteins (e.g., HSP90/HSP70)	Preterm labor, preterm birth, maternal hemorrhage during labor, and maternal mortality.	Maternal-fetal transmission, stillbirth, neonatal death, low birth weight, and perinatal infection.

This table illustrates that VHF viruses imparting a considerable risk for stillbirth typically have very broad mechanisms for cellular entry that target nearly all cell types in the placenta. Note that the ebolavirus genus consists of five species, which include the Zaire ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus, Tai Forest (Cote d’Ivoire) ebolavirus, and the Reston ebolavirus. Abbreviations: CTB, cytotrophoblast; DC-SIGN, DC-specific intercellular adhesion molecule-3 grabbing nonintegrin; PtdSer, phosphatidylserine; and STB, syncytiotrophoblast.

Figure 1.

This figure illustrates the anatomy of a chorionic villous and the viral entry receptor distribution for several VHF viruses in syncytiotrophoblast and cytotrophoblast cells.

Exploitation of Placental Biology by VHF Viruses for Invasion

The placenta as an ideal virus factory

From a viral perspective, the placenta is an ideal organ to infect and exploit to achieve rapid replication and proliferation. The placenta is highly perfused by maternal vasculature and has an extremely high metabolic rate, consuming a disproportionately high percentage of oxygen (40%) compared to its relative mass (20%) to ensure adequate oxygen delivery to the fetus.[4] Growth of the human placenta is continuous throughout most of pregnancy, gradually generating a complex “tree-like” structure of chorionic villi that creates a large surface area across which gas and nutrient exchange can occur. The chorionic villi are covered by an outer layer of syncytialized cells, termed the syncytiotrophoblast (STB) cells; immediately under the STB layer are the cytotrophoblast (CTB) cells, which continuously renew the STB cells through fusion (Fig. 1).[5] Viruses typically induce aerobic glycolysis and fatty acid synthesis to increase available energy that supports their replication.[6] However, aerobic glycolysis and synthesis of fatty acids is already ongoing in the CTB and STB, meaning that an invading virus will find an energy-rich and metabolically active environment ideal for rapidly producing new virions.[7, 8]

Immunohistochemical analyses of infected human placentas or placental explants have revealed that ebolaviruses, LASV, and RVFV can invade the STB and/or CTB (Fig. 2).[9–13] Many VHF viruses can also infect immune and other cell types within the maternal decidua as well as placental chorioamniotic membranes which may also contribute to maternal-fetal transmission through infection of the amniotic fluid. Maternal macrophages are abundant in the extraplacental membranes and decidua and express cellular surface receptors (i.e., C-type lectins) used by many VHF viruses for viral entry.[14] The contribution of other cell types beyond STB and CTB to VHF maternal-fetal transmission and subsequent stillbirth are unknown. Ultimately, the lower the threshold for viral entry, the easier maternal-fetal transmission becomes.

Figure 2.

This figure illustrates immunohistochemical staining for ebolavirus in the syncytiotrophoblast from a placenta obtained from an infected pregnant individual.

As one of the main functions of the placenta is to facilitate nutrient transport across the STB and CTB, VHF viruses can take advantage of these cellular properties for viral replication and trafficking. Many viruses exploit a trophoblast transport mechanism for movement of macromolecular cargo (i.e., nutrients, waste products, growth factors), called transcytosis, to establish viral replication.[15] Viruses also use tightly packed lipid molecules within lipid rafts of the STB apical membrane to facilitate viral replication.[16] These lipid rafts serve as platforms for sorting and segregating proteins into cellular compartments, which is helpful for the STB in facilitating nutrient uptake from the maternal blood. To a certain extent, the normal anatomical, functional and cellular properties of the STB and CTB naturally facilitate viral replication.

The unique immunologic milieu at the maternal-fetal interface may also be exploited by VHF viruses to infect the placenta and fetus. Although a discussion of maternal-placental-fetal immunity is beyond the scope of this manuscript, we have highlighted a few mechanisms that may contribute to viral infection of the placenta. At the maternal-fetal interface, maternal T cell function is regulated by enzymatic depletion of tryptophan (indoleamine 2,3-dioxygenase; IDO), chemokine silencing, and the presence of Fas ligand (FasL) and B7 family molecules on placental trophoblast.[17] Whether the maternal decidua might represent an immunologically privileged sanctuary site is unknown, but there is data that indicate other viruses (i.e., Zika virus) may take advantage of this tolerogenic niche to replicate.[18, 19] Further, maternal T cells are known to acquire a state of tolerance for fetal alloantigens during pregnancy, in addition to being actively suppressed through several mechanisms.[17] The pregnancy-associated expansion of maternal T regulatory cells (T_REG) cells, which suppress antigen-specific immune responses, may also play a role in creating a more permissive milieu for viral infection.[20] Although viruses may broadly exploit alterations of maternal immunity to facilitate their propagation and vertical transmission, stillbirth is an uncommon outcome for most viral infections in pregnancy. Therefore, we propose that modifications to maternal immunity in the periphery or at the maternal-fetal interface are typically insufficient by themselves to predispose to viral-induced stillbirth.

Viral Entry Receptors Commonly Used by VHF Viruses

VHF viruses imparting a high risk for stillbirth typically have remarkably broad mechanisms for cellular entry that target nearly all cell types in the placenta (Fig. 1). For example, viral protein expression of phosphatidylserine (PtdSer) moieties disguises the virus as an apoptotic body, which is engulfed by the cell in a process termed apoptotic mimicry.[21] This process leads to viral entry and immune evasion across many cell types. Due to the ubiquity of PtdSer receptor expression, viruses that express PtdSer have expanded tropism, including to placenta tissues.[22, 23] Highly virulent VHF viruses, including ebolaviruses, MARV, and LASV, employ high viral tropism to take advantage of apoptotic mimicry. These viruses express PtdSer and engage widely with PtdSer receptors such as TIM-1 and Axl, leading to viral distribution throughout many tissue types including the placenta. [24–27]

The use of C-type lectin receptors (CLRs) as viral entry receptors is also a useful strategy, as CLRs are ubiquitously expressed on host cells and function as pattern recognition receptors (PRRs). Exploiting CLRs for viral entry allows the virus to both infect the cell and evade this path of immune recognition and viral restriction. CLRs that VHF viruses exploit include DC-SIGN and L-SIGN, both of which are widely expressed in the placenta. Interestingly, a potential mechanism for DC-SIGN mediated viral maternal-fetal transmission has been described in studies of HIV and could be similarly implicated in VHF that use CLRs.[28] In summary, we hypothesize that many VHF viruses increase the risk of fetal demise because they exploit highly expressed placental cellular surface receptors in addition to their high virulence (Table 1). For several highly virulent VHF viruses, we describe their mechanisms for viral entry and perinatal outcomes accordingly.

Ebolaviruses and Marburg Virus (Filoviridae)

The ebolavirus genus comprises 5 species of single stranded positive RNA viruses that belong to the Filoviridae family and are highly lethal to pregnant women and their fetuses.[29] The placenta is highly permissible to ebolaviruses and high viral loads have been detected in placental and fetal tissues as well as amniotic fluid.[30] Ebolaviruses are suspected to be transmitted by fruit bats to humans and subsequently spread from human-to-human through direct contact with infected bodily fluids (i.e., blood, sweat, saliva).[31] Ebolaviruses are endemic to Central Africa with large outbreaks reported in the Democratic Republic of the Congo in 2021 and Western Africa (i.e., Guinea, Sierra Leone, Liberia) in 2014–2016, the latter claiming 11,000 lives with more than 5,000 cases reported in reproductive-aged women.[29] In pregnancy, ebolaviruses are associated with maternal hemorrhage, preterm labor, miscarriage, stillbirth, neonatal death and high mortality rates. The obstetrical impact of Marburg (MARV) virus, a related filovirus, appears similar; there are very few case reports of MARV infection in pregnancy.[32] A viral outbreak in the Democratic Republic of the Congo was reported to have caused the deaths of 3 pregnant women and their fetuses through spontaneous abortion in 2 cases and neonatal death shortly after delivery in the third case.[32, 33]

Nearly all pregnancies in which a mother was infected with an ebolavirus have resulted in a spontaneous abortion or stillbirth; rare case reports of pregnant women infected with ebolaviruses have described infected mothers surviving the initial infection and clearing ebolaviruses from their bloodstream without fetal loss during the acute infection. These mothers, however, went on to have a stillbirth later in the pregnancy with increased ebolavirus RNA levels in both the placenta and fetus.[9, 34] There are rare cases of fetal survival; one neonate was born at 36 weeks and survived after treatment with ebolavirus therapeutics including monoclonal antibodies, buffy coat transfusions from an ebolavirus survivor, and a broad spectrum anti-viral.[35] To our knowledge, all untreated cases of ebolaviruses in pregnancy have resulted in spontaneous abortion, stillbirth or neonatal demise and further research is needed to understand how these experimental treatments have prevented neonatal demise.[36] Moreover, limited fetal autopsy research and availability of placental histopathology inhibits further elucidation of how ebolaviruses and MARV contribute to congenital disease.[29]

Most placental cells express the cell surface receptors that ebolaviruses and MARV use for cellular attachment. Cellular entry of ebolaviruses and MARV occurs via a viral spike glycoprotein (GP) which latches the virus to the cell surface and is subsequently cleaved by a protease, Cathepsin L (CatL), to fuse with the cell membrane and subsequently allow for communication with endosomes to engage in macropinocytosis or endocytosis.[37] This clathrin-mediated process is facilitated by the Niemann-Pick C1 protein (NPC1) - a late-endosomal membrane entry receptor. The npc1 gene has been shown to be expressed on placental STBs; cells with defective NPC1 function have been shown to be resistant to infection by these filoviruses, lending credence to its important role in ebolaviruses or MARV entry into the placenta.[37] [38] Limited evidence from placental studies of pregnant women infected with ebolaviruses suggests that STB and CTB are the primary cells permissible to ebolaviruses infection.

Cellular attachment of ebolaviruses can also occur via a broad spectrum of non-canonical cell surface receptors including CLRs and PtdSer receptors via apoptotic mimicry.[22, 26, 39, 40] CLRs are strongly expressed by multiple cell types in the placenta, including STB and CTB cells.[28, 41–43] After cell attachment, filoviruses enter the endosomal pathway through a macropinocytosis-type uptake mechanism, where they are cleaved by cysteine proteases to expose the receptor-binding domain of the filovirus glycoprotein. Exposure of the ebolaviruses receptor binding domain allows GP to interact with the luminal C-domain of the NPC1 receptor.[44]

Once the virus establishes its replication cycle in trophoblast cells, the placenta can become a viral reservoir that is difficult to eradicate.[33] Ebolavirus antigens have been detected in villous STB cells from infected pregnant women, with placental histopathology and immunohistochemical analyses revealing mild subchorionitis with fibrin deposits, eosinophilic cytoplasmic granules, and atypical macrophage infiltration of the intervillous space (Fig. 2).[9] IHC analyses of decidua, fetal placental villous stroma, amnion, and umbilical cord have not detected placental injury.[9]

Initial clinical trials of ebolavirus drugs and vaccines excluded pregnant women, which was highly criticized as “protecting pregnant women to death”.[45–47] In response, the rVSV-ZEBOV vaccine was offered to pregnant women at risk in the Democratic Republic of the Congo after a revision to an earlier exclusionary protocol.[46, 47] Pregnant women inadvertently enrolled in a Sierra Leone study of the rVSV-ZEBOV vaccine showed no significant difference in pregnancy loss compared with those who were unvaccinated, and no congenital abnormalities were reported. The sample size was only 84 women; more data will be required to establish safety and efficacy of the ebolavirus vaccine in pregnancy.[48] There remains a significant need for studying the impact of ebolavirus vaccines and therapeutics on perinatal outcomes.

Lassa Virus (Arenaviridae)

LASV is an enveloped, single-stranded RNA virus of the Arenaviridae family. LASV is zoonotically spread through exposure to urine or fecal excretions of infected Mastomys natalensis rats or to the infected bodily fluids of other people.[49] LASV is found throughout West Africa and is endemic in Benin, Ghana, Guinea, Liberia, Mali, Sierra Leone, and Nigeria. The Sierra Leone Civil War of 1991–2002 created profound political instability, mass displacement, and contributed to significant epidemics of Lassa fever.[50] In 2018, Nigeria experienced one of the largest recorded LASV outbreaks with more than 600 confirmed cases and 170 deaths.[51] There are an estimated 300,000–500,000 cases of Lassa fever annually, with a reported case fatality rate of 15–20% that can climb to 50% during epidemics.[52, 53] Lassa fever has a maternal mortality rate of 7% in the first and second trimester and 87% in the third trimester.[54, 55] Spontaneous abortion rates among women infected with LASV in pregnancy are approximately 95%.[52]

The high maternal and fetal mortality rates associated with LASV result from its affinity for highly vascular tissues including the placenta; clinical case studies have identified elevated rates of viral replication in STB suggesting high placental viral loads may correlate with worse pregnancy outcomes.[12, 56] One study also observed a higher viral load in pregnant versus non-pregnant people.[57] LASV engages a viral GP in a pH-dependent process to attach LASV viral particles to surface membrane protein alpha-dystroglycan (alpha-DG) present on lysosomes and endosomes, enabling viral entry.[58] Alpha-DG is expressed in the STB and variably in the chorioamniotic membranes.[59] Further study has revealed the LASV GP can also target a lysosome-associated membrane protein 1 (LAMP1) - a cell-surface receptor normally utilized for cholesterol absorption – and markedly enhance its capacity for membrane fusion.[58] LAMP1 has been detected in placental tissues; LAMP1 murine cell line knock-out studies have demonstrated significantly decreased viral replication. [58, 60]

High viral loads in 4 of 5 placentas were documented at autopsy after deaths from virologically documented cases of LASV.[61] Significant pathology was not observed in the two available placental samples; however, the extent to which these placentas were sampled for histopathology is unknown.[61] High viral loads may be due to LASV suppression of innate immune signaling through nucleoproteins that digest double-stranded host RNA. Histopathologic placental staining suggests LASV interacts with STB during pregnancy; maternal-fetal transmission of LASV is supported by immunohistochemistry findings in maternal placental, endometrial, and cervical tissues as well as fetal endothelial and mononuclear cells.[12] In a retrospective cohort study in Nigeria, 17 of 30 pregnant women infected with LASV had a stillbirth or spontaneous abortion.[62] Positive maternal and fetal outcomes have occurred in cases where ribavirin, an antiviral drug, was administered early. Maternal outcomes generally improved after uterine evacuation, spontaneous abortion or normal delivery.[57]

Rift Valley Fever Virus (Bunyaviridae)

RVFV, like CCHFV and other bunyaviruses, is an enveloped, single-stranded RNA virus. It is a zoonotic arbovirus, transmitted by Aedes and Culex spp. mosquito vectors to livestock (e.g., cows and sheep) as well as humans. Outbreaks of RVFV have occurred in the Arabian Peninsula and across the African continent. RVFV is known to threaten healthy pregnancies in RVFV-infected mammals; RVFV infection in utero correlates with teratogenesis and pregnancy loss in livestock.[63–65] Of concern, vaccines employed in various endemic areas to prevent RVFV outbreaks among livestock induce birth defects in sheep, a finding that delayed development of vaccine use in humans or in livestock from non-endemic regions.[66–68] In humans, case studies have shown that maternal-fetal transmission occurs during RVFV infections and is associated with spontaneous abortion.[69–71]

Ex vivo experiments with human trophoblast have identified that RVFV can reliably infect both CTB and STB in the placenta, supporting the hypothesis that transplacental infection may have precipitated maternal-fetal transmission in the clinical studies.[10, 72] Low-density lipoprotein receptor-related protein 1 (Lrp1), a highly expressed protein in the placenta and other organs, has been identified as host entry factor for RVFV.[73] Lrp1 is a key component of heme transport in the placenta and is highly conserved across animal species in the RVFV epizootic cycle.[74, 75] DC-SIGN has also been identified as a mediator of viral endocytosis for RVFV and is expressed by Hofbauer cells in the human and nonhuman primate STB/CTB and human trophoblast cell lines.[10, 43, 76–79] This C-type lectin is expressed on dendritic cells and macrophages and has been previously characterized as an important mediator of HIV immune-cell entry.[80] Studies evaluating DC-SIGN protein expression in the chorionic villi have detected high levels on the Hofbauer cells (fetal macrophages).[28, 43] In summary, RVFV uses at least two abundantly expressed cellular surface receptors on the placenta for viral entry.

An epidemiological challenge in this field of research is the paucity of studies assessing the impact of RVFV on pregnant women during outbreaks. Most existing studies have focused on livestock and their predominantly male herders, but animal models have demonstrated the ability of RVFV to infect the mammalian placenta. In rats, infection with RVFV led to placental injury, higher mortality rate, and fetal death.[72] Immunohistochemical analysis of ovine and murine placentas revealed hemorrhaging and placental necrosis accompanying RVFV infection of the placental tissue.[10, 72, 81] Animal models are needed to better understand the pathogenesis of placental injury and to test efficacy of therapeutics and vaccines.

Placental Distribution of Viral Entry Receptors for Other Viruses with a Lower Rate of Maternal-Fetal Transmission

Many viruses have the potential for maternal-fetal transmission and the placenta expresses many commonly used viral entry receptors. Nonetheless, the placental distribution of viral entry receptors for VHF viruses with high rates of stillbirth can be compared with that for viruses that are thought to have a lesser impact on the fetus. For example, respiratory syncytial virus (RSV) is estimated to annually cause more than 33 million cases in children (globally) and 1.5 million cases in older adults (industrialized countries) [82, 83]; adults can be repeatedly infected with RSV, sometimes in the same year. Despite the common nature of this infection, there exists only a single case report of a neonate with maternal-fetal transmission.[84] RSV uses ICAM-1 as its viral entry receptor, which is not expressed by either decidual or villous trophoblast cells (STB, CTB), potentially explaining its lack of placental virulence; however, ICAM-1 can be upregulated in cases of placental villitis.[85–87] Though LDL and cadherin receptors are also involved,[86] 90% of human rhinoviruses use ICAM-1 as their viral entry receptor.[88]

Some viruses are known to rarely transmit to the fetus; for many of these viruses, the viral entry receptors have low- to medium- expression in the STB and CTB. For example, hepatitis C Virus (HCV) is a pathogen that is rarely passed from mother to fetus. Estimates of transmission rates vary, but are lower than in VHF; one study demonstrated a rate of 4–7% transmission and 3.4% fetal death in mothers with HCV viremia.[89] HCV exploits CD81 receptor proteins like calpain-5 and CBLB, which have low to medium trophoblastic expression, respectively.[60, 90] Similarly, maternal-fetal transmission and stillbirth due to the severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) are uncommon[91]. Notably, co-expression of the SARS-CoV-2 canonical viral entry receptor ACE-2 and the enzyme TMPRSS2 is rare in STB/CTB from healthy placentas.[92] However, ACE2 can also be expressed in immune cells (macrophage and neutrophils) that infiltrate the placenta during chorioamnionitis; whether maternal-fetal transmission of SARS-CoV-2 can occur due to maternal immune cell infiltration or by other mechanisms is unknown.[92, 93]

Effect of Knockout of VHF Viral Entry Receptors on Disease

Evidence to support the key role of placental entry receptors in VHF transmission and disease can be found in studies that demonstrate reduced viral cellular invasion or amelioration of disease in transgenic murine models knocked out for viral entry receptors. Although few studies have evaluated viral entry receptor knockouts for VHF viruses, and none having assessed receptor knockout in placental cells or pregnancy models, the broader principle is well-established that inhibiting cellular receptor gene expression or blocking receptor activity can confer reduced viral entry and replication. For example, studies using a transgenic mouse model knocked out for the Npc1 viral entry receptor, revealed a strikingly limited replication of ebolavirus replication and complete prevention of ebolavirus disease (EVD).[94] This finding was supported by an additional study in which mutations disrupting NPC1 function resulted in resistance to ebolavirus infection among human cells, which remained otherwise susceptible to a suite of viruses with alternative viral entry receptors.[37] A similar concept was demonstrated regarding LASV and knockout studies involving two of its predominant entry receptors, alpha-DG[95] and LAMP1[96]. Beyond VHF, studies of hepatitis A virus [97] and tick-borne encephalitis virus [98] have shown that knockout of the PtdSer receptor, TIM1, produces markedly reduced infection rates by preventing receptor-binding interactions in the early stages of infection. Knockout studies in mice have also been performed to determine the impact of PtdSer knockout on Zika virus infection and disease.[99] These studies provide evidence to support the concept that receptor distribution of viral entry receptors on placental tissues may be the most critical factor for susceptibility of the fetus to congenital infection and stillbirth.

Clinical Implications for the Treatment of Infected Pregnant People

Whether blocking viral entry receptors on the placenta for VHF or other viruses might prevent congenital infection or ameliorate fetal disease is unknown but represents an interesting concept. Recently, a binding reagent (sTIM1dMLDR801) was developed capable of specifically blocking the PtdSer-dependent virus entry mechanism used by several enveloped viruses.[100] Whether such a therapeutic might help prevent stillbirth due to ebolavirus, LASV or MARV infections that use PtdSer for viral entry is unknown, but could be tested in vitro and in animal models. Current therapies for ebolavirus infections are insufficient to protect the fetus. There is only a single case reported in the literature of fetal survival after a pregnant individual contracted EVD; in this case, the mother was treated with multiple therapies (i.e., monoclonal antibodies, antiviral).[37] Use of a PtdSer receptor blocker may increase fetal survival in the context of EVD and should be tested to determine if there are detrimental effects of the therapeutic on placental or fetal health.

Discussion

We anticipate that VHF as well as new, emerging viruses will pose an increasing threat to pregnant individuals and their fetuses due to the synergistic actions of climate change and globalization of the world through air travel.The ability of these viruses to use ubiquitously expressed receptors on placental trophoblast cells sets the stage for maternal-fetal transmission and stillbirth. In a rapidly unfolding public health crisis with a new virus, we might predict the outcome of an infection during pregnancy on the fetus through an analysis of receptor expression by cells in the placenta. Based on our hypothesis, new therapeutics blocking PtdSer receptors might impart a benefit for both maternal and fetal survival in the context of EBOV, MARV or LASV infections and should be tested in pregnant animal models before a large outbreak. Learning how VHF viruses use placental cellular machinery to their biological advantage may yield important lessons for pandemic preparedness for maternal and fetal health.

Highlights.

• Viral hemorrhagic fever viruses can cause severe infections in pregnancy

• Entry receptors for viral hemorrhagic fever viruses are highly expressed in placenta

• High placental expression of viral entry receptors may increase stillbirth risk

• Climate change may increase global infections from viral hemorrhagic fever viruses