Cytotrophoblasts Aid in Zika Transmission from Mother-to-Fetus

One of the biggest reasons that Zika virus is such a public health concern is the danger that it poses for women who are pregnant and their unborn fetuses. Women who become infected with the Zika virus when pregnant are known to have “prolonged viremia” and a higher risk of passing the virus on to the fetus, a transmission that can result in a number of serious birth defects, such as microcephaly.

In a presentation on February 24, 2017 at The First International Zika Conference in Washington, DC, Lenore Pereira, PhD, professor of Cell and Tissue Biology at the University of California, San Francisco, highlighted her research, which is dedicated to answering the question of how the Zika virus is able to spread from maternal blood to the placenta to reach the fetus.

Dr. Pereira opened up her talk with an image illustrating the surface of the placenta in the maternal blood space, that depicted how maternal blood infuses around the villi in the placenta. Dr. Pereira explained how the branching structure of chorionic villi, “increases [the] exchange of nutrients, gages, and IgG for passive immunity.” She continued, “If you look closer, you can see the surface of the villi branch creating a huge surface area for this exchange between maternal blood and the villi themselves.”

Underneath the surface of the villi are the cytotrophoblasts, which are “the building blocks of the placenta.” These are the cells that will proliferate at the tips of the villi and then invade the decidua of the mother. Dr. Pereira explained, “they create a new vasculature, so they move the maternal blood vessel components and they create a low-resistance blood vessel, so that by mid-gestation, a liter a minute of maternal blood is pouring on the surface of the placenta.” She added that this is the time when the baby starts to grow and all “that material and nutrients” are needed in order to do that.

Dr. Pereira stressed that the biggest takeaway from her presentation involves a model that her and her colleagues have developed. In the laboratory, the team used dissected pieces from first trimester placentas placed in matrigel and found that “at the tips of the villi, the cytotrophoblasts proliferated and then they began to invade the matrigel.”

Dr. Pereira explained, “We [took] the villus and we [fixed] it and we put it in a matrix, an embedding medium, and then as an intact tissue, it [was] sectioned.” She continued, “We [could] see at different levels the various cells. So, at any one section you won’t see everything, but you will see some portion of what the villus is doing as it grows, what the functional differences are.” The model allows you to see the mitotic cells that form a cell column as well as the invasive cytotrophoblasts and researchers are then able to measure the distance that the cells invade. Dr. Pereira explained, “So, we [were able to] see how far they go in 3 days, cell-by-cell.”

In another slide Dr. Pereira shared a picture of one cross-section of an entire villus, while she stressed that there is a lot of work that goes into any one section that is analyzed. She added, “This is the model that we felt confident in by studying isolated primary cells from the placenta and also we had already begun to characterize their pattern of infection at the uterine placental interface.”

Seeing this, Dr. Pereira and her colleagues sought to find the answer to another question: What is the real target for the Zika virus? The researchers compared a Ugandan prototype strain (MR766) of the virus (from their studies) with an epidemic Nicaraguan strain of Zika virus. After further exploration, she found that the patterns pertaining to the cytotrophoblasts and the Hofbauer cells “are consistently reproducible in all of the tissues.” She added that there are functional differences manifested in cytotrophoblasts infected by the Nicaraguan strain of the virus and those infected by the phototype strain. “We see that Zika-infected cytotrophoblasts downregulate a marker of cell proliferation [Ki67] and we think that the [villus] explant model is useful for testing drugs to [fight] Zika virus that are suitable for pregnancy,” she said.

Taking a closer look at the Ugandan prototype strain versus the Nicaraguan strain, the researchers began to see the differences “which we had hints at but didn’t really know until we counted all of the migrating cells and measured everything.” Dr. Pereira showed pictures illustrating the Ugandan prototype strain and how only a few cells had migrated out at 3 days’ post-infection. Another picture illustrated how the cells in the Nicaraguan strain actually traveled four times the distance than what was seen in the prototype strain; they both expressed ZIKV E and NS3 proteins that produce infectious virus.

In order to prove it, the researchers took a closer look at the other cell type that was infected: Hofbauer cells in the villus core. She explained that the Hofbauer cells “express E [and] NS3 [proteins]. They are in the villus core [that border proximal cell columns]. There are no infected cytotrophoblasts around them, so they are independently infected from the cytotrophoblasts. They have to be close to an area of cell columns and somehow prepare the area for Hofbauer cells to become infected. We don’t know what that is, but that connection happens.”

As the villi branch, first they sprout, and then make a new villus; this actually works to prepare the villus core to “allow the virus to reach the Hofbauer cells inside.” The cytotrophoblasts outside are not infected. Dr. Pereira also stressed that the Hofbauer cells surround the villus blood vessel so closely that, at first, the researchers couldn’t discern if they were actually a part of the blood vessel edge. They also found that, “A Hofbauer cell being infected could bring virus close to this blood vessel and infect the fetal bloodstream.”

Dr. Pereira summarized that through the analysis of 355 villus explant villi that were infected with the Ugandan porotype strain and the Nicaraguan strain, they were able to “determine that the sites of infection [most vulnerable to Zika infection ex vivo] are reproducibly in proximal cell columns.” In fact, around 20% of the villi had proximal cell columns and 50% of the villi had invasive cytotrophoblasts. They also found that Hofbauer cells were also infected, and that 25% of them were in the villi. Thus, their findings suggest that “the maternal virus infects the placenta in the intervillous blood space and undermine development in early gestation.”

In an exclusive interview with Contagion® Dr. Pereira explained another potential route of transmission. She said, “Of course, the third possibility, which we didn’t mention in our paper was the possibility that the virus could come from the sexual route, and, again, cross the fetal membranes, because they then about the access from the vagina— which of course there’s a plug in-between, but still virus might get through. The possibility then, is crossing from the maternal bloodstream, either in the first trimester from the basal decidua, or, at late-gestation through the parietal decidua to the fetal compartment.”

Dr. Pereira concluded, “What I do is show you the model that we now refined to limit the cells of infection is the mitotic cytotrophoblasts which also can infect the Hofbauer cells and pass the virus into the villus core, basically the bloodstream of the baby.”

DISCLOSURES

None

SOURCE

First International Conference on Zika Virus

PRESENTATION

Session 3: Virology

Zika Virus Infects Cytotrophoblasts and Hofbauer Cells in Chorionic Villi From First-Trimester Human Placentas and Downregulates Cellular Markers

Exclusive interview with Contagion®