**1. Introduction**

The placental syncytiotrophoblast (STB) layer that covers the floating chorionic villi in direct contact with maternal blood in the intervillous space constitutes a major barrier to vertical transmission of parasites and microorganisms. The consequences of STB damage have been studied in first

trimester placental explants where the subsyncytial cytotrophoblasts (CTB) becomes infected by *Listeria monocytogenes* if the STB layer is damaged with collagenase [1]. With *Toxoplasma gondii*, the situation is more complex, as multiparasite vacuoles in chorionic villi are present not only immediately below or besides syncytial interruptions, but also at sites of no visible syncytial interruptions, suggesting the possibility of hard to detect breaks in the STB layer or of earlier breaks that had subsequently healed [2]. These observations highlight the importance of considering syncytial permeability upon studying placental vulnerability to infection.

Several viruses cross the uterine-placental interface, infecting the fetus and causing birth defects, including rubella, varicella-zoster, parvovirus B19, human cytomegalovirus, hepatitis E type 1 and Zika (ZIKV) (see [3]). ZIKV is a flavivirus which is transmitted by *Aedes* mosquitoes; it was first identified in 1947 in an African forest, and triggered epidemics in the South Pacific in 2007 and in the Americas in 2015–2017 [4]. ZIKV infection during pregnancy is associated to an array of devastating birth defects known as congenital Zika syndrome, which include microcephaly, brain calcifications, neurological impairment, and retinal damage.

We are interested in exploring the mechanisms that allow ZIKV transplacental transmission in humans. During the development of human placenta, CTB epithelial cells may be di fferentiated in two ways. Firstly, they aggregate into cellular columns that invade the uterine interstitium and colonize the spiral arterioles, allowing the anchorage of the fetus to the mother and the flow of blood to the placenta. Secondly, CTB form a bilayer in which the cells of the external sheet fuse to form the multinucleated STB that covers the chorionic villi. The STB layer is crucial for the interchange of ions, nutrients, gases, and waste between the fetus and the mother. During pregnancy, as a result of syncytial ruptures or focal degeneration of STB, lateral cell membranes subdivide segments of STB from the surrounding STB continuum. This appears to be a dynamic process where the disconnected parts of the STB eventually fuse after the disintegration of the lateral separating membranes [5].

Tight junctions (TJs) regulate transit through the paracellular pathway of epithelial cells. In the STB, these cell-cell adhesion structures located at the uppermost portion of the lateral membranes that subdivide the STB layer constitute a cornerstone of the blood-placental barrier (BPB) that protects the fetus from toxins and pathogens. TJs in the STB of human placental chorionic villi have been observed by freeze fracture, and their function as paracellular seals has been demonstrated by electron microscopy in thin sections, with the blockade of the transit of electron-dense markers through the paracellular pathway [6–9]. The apical surface of the STB of chorionic villi expresses several TJ proteins, including the integral proteins occludin, claudins -1, -3 and -16, and the adaptor protein ZO-1 [9,10], while claudin-4 is strongly expressed during all trimesters of pregnancy [11], but localizes at the basal membrane of the STB [9].

ZIKV infects cells that strongly express TIM-1 [12], a cell surface phosphatidylserine and phosphatidylethanolamine receptor [13]. ZIKV in humans replicates in the glandular epithelium of the decidua and in decidual cells and infects invasive CTB, the CTB of cell columns, as well as fetal fibroblasts and macrophages known as Hofbauer cells which are present in the parenchyma of floating chorionic villi [12,14,15]. These observations led to the proposal of a ZIKV transmission route that goes from the cells of the basal decidua in the mother to the fetal invading CTB, followed by the infection of cell columns of CTB and Hofbauer cells present in the parenchyma of chorionic villi [12]. Moreover, since the envelope (E) proteins between the dengue virus (DENV) and ZIKV are very similar structurally [16], cross-reactive antibodies are generated that may enhance ZIKV infection [17]. In this respect, it has been observed that ZIKV infection in human placental explants are enhanced by the presence of dengue virus antibodies, suggesting that ZIKV immune complexes could use the neonatal Fc receptor for IgG as a transport system to transcytose across the STB layer to infect Hofbauer cells in the chorionic villi [18]. However, since the clinical severity of maternal ZIKV infection has not being associated with the existence of prior dengue antibodies [19], and not all pregnan<sup>t</sup> women infected with ZIKV have been previously infected with DENV, here, we explore another complementary route of vertical ZIKV transmission. Taking into account that STB cells are not infected by ZIKV [12], we explore

the possibility of ZIKV reaching the chorionic mesenchyme via transit through the paracellular route of the STB.

Virus passage through the paracellular route of epithelial and endothelial cells has previously been reported. Thus, after human airway epithelia infection with adenovirus, the viruses are first released to the basolateral surface and then escape to the apical surface. This process involves the binding of the fiber proteins of the adenovirus to its receptor CAR located within TJs, which triggers a disruption of junctional integrity that allows the virus to escape through the paracellular pathway between the cells to reach the apical surface [20]. Another case is that of human immunodeficiency virus (HIV), whose disruption of TJs and adherens junctions (AJs) in oral epithelial cells facilitates the paracellular spread of the herpes simplex virus 1 (HSV1), emerging as a mechanism to explain the rapid development of HSV-associated oral lesions in HIV infected individuals [21]. Likewise, sealing the blood-brain barrier (BBB) is compromised by HIV-1 induced inflammatory cytokines; in this respect, TNFα has been shown to open the paracellular route for HIV-1 invasion across the BBB [22]. With regards to *Flaviviruses*, the West Nile virus disrupts the BBB in mice inducing an increase in BBB permeability and a reduced expression of TJ and AJ proteins [23]; moreover, in vitro infection of mouse brain endothelia with DENV delocalizes TJ proteins from the membrane to the cytoplasm, reduces the transendothelial electrical resistance, and increases the macromolecule permeability and the paracellular passing of free virus particles [24].

Here, we analyzed the permeability of the paracellular pathway and the molecular composition of TJs in the STB cell layer of placentae derived from ZIKV-infected women. In addition, in the trophoblast cell line BeWo, we observed that basolateral incubation with ZIKV reduces the transepithelial electrical resistance (TER).

Our results indicate that ZIKV infection alters the composition of placental TJs and increases paracellular permeability.
