Zika the gatecrasher (crossing the umbilical barrier)

 

 

Zika the gatecrasher (crossing the umbilical barrier) 

22.08.2023

─

Mr. Nirmal manna

514, M.Sc Zoology ,Part 2, Immunology and Cancer biology, Paper 2,Ramnarain Ruia Autonomous College

Table of contents

Introduction…………………………………………………………………………..…………….…03

How does zika virus cross the umbilical barrier………………………….…………….…03

Principle …………………………………………………………………………….…………….…04

studies on zika virus crossing the umbilical barrier……………………….………….…06

…………………Visualization of transcytosis………………………………….………….…08

Conclusion………………………………………………………………………………..………….…12

Reference…………………………………………………………….……………..…………….…13

S.P Mandali’s

Ramnarain Ruia Autonomous College

Matunga, Mumbai - 400019.

CERTIFICATE 

This is to certify that Mr. Nirmal Manna of Masters of Science in Zoology, Ramnarain Ruia Autonomous College has satisfactorily completed and documented the assignment work of Zoology Paper RPSZOP302: Immunology and cancer biology during the Semester III of the class MSc part 2 of the year 2023-2024.

______________________                    ______________________

Signature of Faculty in charge                               Head of Department

Date: 22/08/23

Introduction

 The Dengue virus (Dengue Virus), the West Nile virus (WNV), the Japanese encephalitis viruses (JEV), and the yellow fever virus (YFV) are all members of the genus flavivirus, which also includes the Zika virus. The Zika virus is a mosquito-borne flavivirus that was first isolated from the rhesus monkey in Uganda in 1947. This is thought to happen in 2- 20% of zika virus infections during pregnancy. The placenta typically acts as a crucial physical and immunological barrier to stop the transmission of maternal-fetal pathogens, and it is still unclear how the zika virus can cross this barrier. For risk assessments and the development of transplacental zika virus transmission prevention strategies, more knowledge about how the zika virus crosses the placenta and the identification of risk factors is necessary.

Prior to 2015, the majority of zika virus infections were thought to be mild illnesses with symptoms like headache, fever, arthralgia, rash, myalgia, edema, arthritis, vomiting, and non-purulent conjunctivitis. However, recent epidemics have shown that adult zika virus infections are linked to encephalitis and the Guillain-Barre syndrome. Additionally, the increasing prevalence of congenital Zika syndrome in fetuses, including microcephaly, congenital malformations, and fetal death, is associated with vertical transmission of the zika virus from mother to fetus. In many regions of the world, including the Americas, Africa, Asia, and the Pacific Islands, it is now endemic. Microcephaly is a condition that results in babies being born with abnormally small heads. Zika virus infection during pregnancy can cause microcephaly. Other birth defects like eye and hearing loss can also be brought on by it. Congenital zika virus infection may also result in joint problems, sensorineural hearing loss, abnormal eyes, and postnatal microcephaly in newborns who were born with a normal head.

How Does Zika Virus Cross the Umbilical Barrier?

 The exact mechanism by which Zika virus crosses the umbilical barrier is not fully understood. However, there are a few possible ways that this could happen.

Direct infection of the placenta: The placenta is the organ that connects the fetus to the mother's uterus. It provides the fetus with oxygen and nutrients, and removes waste products. Zika virus could infect the placenta directly, through the blood vessels that supply the placenta.

Transcytosis: Transcytosis is a process by which cells take in molecules from the outside environment and transport them to the other side of the cell. Zika virus could be taken up by the cells of the placenta through transcytosis and then transported to the fetus.

Exosomes: Exosomes are small vesicles that are released by cells. They can contain proteins, RNA, and DNA. Zika virus could be packaged into exosomes and released by the cells of the placenta. The exosomes could then be taken up by the fetus.

 Principle

  To reach the fetal brain of an infected mother, Zika must cross two major physiological barriers, the placenta and the blood-brain barrier. A very special organ is formed only during pregnancy, it supports the growth and development of the fetus and is regulated and coordinated to ensure the maximum efficiency of the exchange of nutrients and waste between the circulatory system of the mother and the fetus. Its main function is to provide oxygen and nutrients to the fetal brain. In addition, the placental barrier serves as an important physiological barrier that protects the fetus from certain toxic molecules, maternal diseases, and pathogenic infections such as viruses. The main functional unit of the placenta is the chorionic villi, which consist of specialized epithelial cells called trophoblasts derived from the outer trophectoderm layer. Trophoblasts form the epithelial lining of the placenta and cause proliferation of invasive extravillous trophoblast cells that are separated from fetal blood by the placental membrane. To serve as the first line of defense against any pathogen trying to breach the placental barrier, trophoblasts form a highly polarized epithelial monolayer composed of tight junctions that prevent lateral and paracellular diffusion of substrates. However, our knowledge of viral interactions at the maternal-fetal interface during pregnancy is currently limited.

While the placenta serves as the first checkpoint to protect the fetus and support its normal growth and development of the fetus, the Blood brain barrier provides the second checkpoint critical to protect the fetal brain and ensure healthy brain development. The human placenta consists of many chorionic villi, which are tree-like structures that are the functional units of the placenta. These villi are either floating in maternal blood in the intervillous space or are anchored to the maternal part of the placenta (decidua) through extravillous trophoblasts. Multinucleated syncytiotrophoblasts line the chorionic villi, forming a cellular barrier against pathogens. Cytotrophoblasts are located below the syncytiotrophoblasts and can differentiate in extravillous trophoblasts and syncytiotrophoblasts. The stroma of chorionic villi consists of fibroblasts, fetal macrophages (Hofbauer cells) and endothelial cells. From the second pregnancy trimester, maternal blood flows in the intervillous space where it comes into contact with the chorionic villi .From this pregnancy stage, maternal immunoglobulin G (IgG) is actively transported across the placenta through neonatal Fc-receptor (FcRn) mediated transcytosis in syncytiotrophoblasts.

 The Blood brain barrier is a boundary that separates the circulating blood from the brain and the extracellular fluid in the central nervous system . The Blood brain barrier is made up of endothelial cells of the vasculature forming cell-to-cell tight junctions to limit the passage of circulating molecules, cells, and pathogens to the Central nervous system . Tight junctions containing more than 40 proteins with both transmembrane and cytoplasmic domains generate a continuous, circumferential, belt-like structure at the luminal end of the intercellular space, where it serves as a gatekeeper of the paracellular pathway . Three major transmembrane proteins, claudin, occluding, and junctional adhesion molecule (JAM), interact with cytoplasmic proteins including ZO-1, cingulin, afadin and α-catenin, which anchor strands to the cytoskeleton, resulting in the formation of cellular tight junctions . Disruption of the Blood brain barrier enhances permeability of endothelial cell and is a hallmark of Central nervous system infection . However, recent studies reported that no barrier disruption was observed when zika virus gained access to the Central nervous system , suggesting that the endocytic transport system is required for the zika virus to cross the Blood brain barrier barrier. Given the existence of endosomal sorting pathways in different cell types, it is possible for the Blood brain barrier cells to employ a similar process of endosomal transportation. However, there is no investigation on these pathways in Blood brain barrier endothelial cells, particularly in the condition under viral infection.

The placental barrier and the blood-brain barrier normally protect fetal brain development by forming tight junctions to limit the cellular movement of pathogens. However, Zika virus can be detected in the brain of microcephalic infants, suggesting that Zika virus can enter the fetal central nervous system and disrupt the integrity of the barrier. Rubella virus, cytomegalovirus (CMV), human immunodeficiency virus 1 (AIV-1), West Nile virus (WNV), Japanese encephalitis virus (JEV), and herpes virus are known to disrupt the placental barrier. Central nervous system. The other is through transitocytosis. Hepatitis B virus (HBV) has been reported to cross the placental barrier via transcytosis during the first trimester. JEV has also been found to cross endothelial cells and pericytes in the blood-brain barrier in endocytic vesicles. These findings indicate that the virus can penetrate not only through the intracellular route, but also through transitocytosis. Although the Zika virus has been detected in the amniotic fluid of pregnant women and in the brain tissue of microcephalic fetuses, so it is capable of infecting the central nervous system of the fetus, the mechanism by which this happens remains unknown. In this study, they will combine an in vitro Transwell barrier assay with a virus tracking (single virus tracking) approach to elucidate the mechanism by which the Zika virus crosses the placental and blood-brain barrier.

Studies on Zika Virus Crossing the Umbilical Barrier

Numerous studies have looked into the process by which the Zika virus penetrates the umbilical cord. One investigation found that the Zika virus could directly infect the placenta. It was published in the journal Nature Medicine in 2016. The research also revealed that pregnant women who had the Zika virus could detect it in their fetal blood and amniotic fluid.

Another investigation, which was reported in the 2017 issue of the journal Science, discovered that the placental cells could transcytose the Zika virus. The study also discovered that fetuses who had contracted the virus in utero had the virus present in their brains.It was shown that cross-reactive Dengue Virus antibodies increase placental zika virus uptake using this model and human placental explants.

The human ex vivo dual placental perfusion model was used to study the effects of cross-reactive Dengue Virus antibodies on transplacental zika virus transmission. This model is physiologically representative and dynamic. Through FcRn-mediated transcytosis of zika virus-Dengue Virus antibody immune complexes across syncytiotrophoblasts in the placenta, it has been proposed that the presence of antibodies against dengue virus (Dengue Virus), a flavivirus that is closely related to the zika virus, may be a risk factor for transplacental zika virus transmission. Pregnant mice have shown FcRn-mediated transplacental transcytosis for zika virus, but pregnant non-human primates (NHP) have not. This discrepancy may be due to significant structural differences between the rodent and NHP placentas, such as the presence of trophoblast giant cells in the rodent placenta while these cells are absent in the NHP placenta. Furthermore, studies on the potential negative effects that cross-reactive Dengue Virus antibodies may have on transplacental zika virus transmission were conducted in a model that more accurately reflects the in vivo circumstances that exist in humans during pregnancy. Deep trophoblast invasion, which is required in humans for the fetus's optimal nutrition, is limited in both the rodent- and NHP placentas.

Concerning the impact of cross-reactive dengue virus (Dengue Virus) antibodies on transplacental zika virus transmission, findings from studies in pregnant mice and non-human primates are also inconclusive. For risk assessment and potential intervention strategies for transplacental zika virus transmission, it is crucial to understand how the zika virus can cross the placenta and which risk factors contribute to this. In order to investigate transplacental zika virus transmission and the impact that cross-reactive Dengue Virus antibodies have on this transmission, researchers used an ex vivo human placental perfusion model. With the help of this model, they were able to show that Dengue Virus antibodies significantly increase zika virus uptake in perfused human placentas and that this increased uptake is neonatal Fc-receptor-dependent. Additionally, they demonstrate that primary fetal macrophages and term human placental explants, but not primary trophoblasts, are more susceptible to zika virus infection when exposed to cross-reactive Dengue Virus antibodies. Their findings lend credence to the idea that the presence of cross-reactive Dengue Virus antibodies may be a significant risk factor for zika virus transmission transplacentally. In addition, they showed that the ex vivo placental perfusion model is a useful and animal-friendly model to research transplacental pathogen transmission.

Zika virus infection results in severe neurological symptoms in adults and fetal microcephaly, and the virus is found in the brains of people who have those conditions as well as meningoencephalitis. The mechanism by which the zika virus enters the CNS and overcomes the physiological barrier is still unknown. During pregnancy, the blood brain barrier and the placental barrier guard the fetus against pathogens and guarantee normal brain development. Human placenta trophoblast cells (JEG-3) and human brain-derived endothelial cells (hCMEC/D3) were used as in vitro models of the physiological barriers in this study. The findings suggested that the permeability of the barrier differentially changed in response to zika virus infection, allowing the virus particle to cross the host barrier. Zika virus was able to infect JEG-3 cells effectively and reduce the amounts of ZO-1 and occludin between adjacent cells by the proteasomal degradation pathway. Zika virus, in contrast, could infect hCMEC/D3 cells without impairing blood-brain barrier permeability and tight junction protein expression. Zika virus particles were released on the basal side of the Blood brain barrier model and infected underlying cells, even though no disruption to the Blood brain barrier was seen during infection. Additionally, they found that fluorescence-labeled zika virus particles could transcytose across the blood brain barrier model and in vitro placenta barrier, and that transcytosis could be inhibited by either low temperature or pharmacological endocytosis inhibitors. In conclusion, the zika virus compromises cellular tight junctions to cross the placenta monolayer barrier using a cell-type-specific paracellular pathway. The zika virus can also pass through the blood-brain barrier and the placenta by a process called transcytosis. Their research revealed brand-new details about the mechanism by which zika virus particles penetrate cellular barriers.

Visualization of Transcytosis

Transcytosis, a continuous process for the intracellular transport of macromolecules through the vesicular system, has been reported to be a common strategy for molecules that cross impassable barriers under normal physiological conditions. Therefore, a viral imaging assay was performed to determine whether Zika virus particles can cross barrier cells through transcytosis in the Transwell barrier model in vitro. To directly visualize Zika virus particles, Zika virus particles were labeled with a fluorescent dye, atto647N, by coupling the atto647N-NHS ester to the amino group of the viral envelope protein. Atto647N Zika virus particles were purified using a Sephadex G-25 size column. Compared to the fraction containing the 40 nm fluorosphere, the atto647N fluorescence signal from fractions 6 to 10 represents atto647N Zika virus particles (atto647N-zika virus) because a Zika virus particle is approximately 50 nm in diameter. No fluorescence signal was detected among the fractions in the unstained Zika virus group. These results confirmed the successful binding of Zika virus particles to atto647N. Infectivity of Atto647N-zika virus particles was then measured by plaque assay. Both unstained Zika virus and atto647N-zika virus presented similar viral titers, indicating that the atto647N assay does not significantly affect the infectivity of Zika virus.

Show that transcytosis is a characteristic pathway of cellular trafficking, allowing selective and rapid cellular transport to the main intracellular site, and that cellular transport is a temperature-dependent process that can be maintained at 4 °C; showed that energy-dependent cellular transport of virus particles must be maintained at 4°C, unlike cellular transport. Therefore, using the in vitro Transwell barrier assay, they determined whether the Zika virus can penetrate a monolayer of barrier cells at 4 ° C and 37 ° C by directly measuring the fluorescence intensity of the atto647N signal in the basal chamber of the Transwell plate. Atto647N-zika virus particles in the basal chamber in JEG-3 and hCMEC/D3 cells were significantly reduced at 4 ° C compared to 37 ° C. In contrast to Atto647N-zika virus particles, FITC-dextran did not differ in the basal chamber of the Transwell plate at 4 ° C and 37 ° C. These results indicate that Zika virus can infect monolayers of JEG-3 or hCMEC/D3 cells by temperature-dependent transocytosis. To directly visualize Zika virus particles across a monolayer of barrier cells, they infected a monolayer of JEG-3 cells in a 3.5 cm glass bottom plate with Atto647N-zika virus and obtained confocal imaging at 0, 30, and 60 min. after infection. JEG-3 cells were stained with DAPI and WGA488 to show the nucleus and cell membrane status, respectively. As shown, Atto647N-zika virus particles were detected only in the JEG-3 monolayer (0 min). Apparently, Atto647N-Zika virus particles internalized the cytoplasm and moved from the apical to the basal side of the cell 30-60 minutes after infection. Similar results were observed in hCMEC/D3 monolayers. These results explain that Zika virus particles can be directly transferred to the barrier cell monolayer by transitocytosis without undergoing viral replication.

 A schematic diagram depicts the mechanism of the zika virus crossing human physiological barriers. In the placenta barrier, zika virus infection can lead to disruption of tight junction that results in the zika virus crossing through the leakage of tight junction. In addition, the zika virus can also transport from the apical side to the basolateral side through the transcytosis pathway (top panel). In the blood-brain barrier, the zika virus can transport from the apical side to the basolateral side through the transcytosis pathway, but does not alter the permeability of the barrier (bottom).

Several lines of evidence showed the existence of zika virus antigens in the chronic villi of a human placenta from a mother who gave birth to an infant with microcephaly  and isolation of zika virus RNA from placental tissue of mice infected with the zika virus  suggesting that the zika virus may penetrate the placental barrier to infect the infant brain. Recent studies reported that the zika virus is able to infect and replicate in Hofbauer cells that are primary human placental macrophages and in cytotrophoblasts, suggesting a route of intrauterine transmission that the zika virus crosses the fetal compartment by directly infecting the placental cells  In this study, theyrevealed that the zika virus could cross the placenta barrier with both paracellular and transcytosis Normally, microorganisms spreading through epithelial tissues are blocked by tight junctions and adherent junctions present on apical and basolateral surfaces, respectively However, their data revealed that zika virus infection causes leakage of the placental barrier by disrupting the integrity of the tight junction of the barrier cells .Further investigation elucidated that the breakdown of the tight junction in the placental barrier cells is due to a decrease in the amounts of ZO-1 and occludin, two essential tight junction proteins, through the proteasomal degradation pathway . In contrast to the direct infection pathway . The results proposed a paracellular pathway for the zika virus crossing the placental barrier by disrupting the cellular tight junction of the barrier cells through degradation of ZO-1 and occludin. The activation mechanism of the proteasomal degradation pathway by zika virus infection remains to be further investigated.

Using a single virus tracking  approach, they provided evidence to elucidate that the zika virus can cross the in vitro placenta model through transcytosis . Maternal-fetal transmission of a number of viruses by transcytosis in the placenta has been proposed previously . Given that transport of maternal IgG across the placenta is minimal during the first trimester and rises dramatically between 22 and 26 weeks of gestation showed that Dengue Virus  cross-reactive mAbs bound to the zika virus undergo FcRn-mediated transcytosis across the placenta to productively infect human placental macrophages . In the current study, the zika virus crossing the placenta barrier cells was directly visualized by using Atto647N-zika virus particles . These results provide strong evidence demonstrating the straight passing of viral particles across the placental barrier rather than a release of newly produced viral particles after replication. Moreover, the action of zika virus transcytosis could be inhibited by treatments with endocytosis inhibitors and colchicine . Taken together, they demonstrated that both the paracellular pathway and the transcytosis pathway are utilized by zika virus to cross the placenta barrier .Further studies will be needed in order to illustrate how zika virus particles select a pathway to cross the placenta barrier and whether they require a specific receptor for zika virus to interact with.

In addition to the placenta barrier, the Blood brain barrier is the other important barrier that protect the fetal brain development during pregnancy. A number of neurotropic viruses enter the Central nervous system by using various pathways including direct transport from peripheral nerves, transinfection and transcytosis . In the current study, the zika virus can cross brain endothelial cells and release of infectious virus particles, without an increase of endothelial monolayer permeability and no significant cytotoxicity in vitro . This is in agreement with previous studies showing the zika virus crosses the Blood brain barrier monolayer without the Blood brain barrier barrier disruption . However, their studies showed that there were infectious atto647N-labeled virus particles that crossed the monolayer of Blood brain barrier barrier cells in the presence of the treatment of endocytic inhibitors , suggesting that they still cannot exclude the possibility that some viral particles selectively moulate tight junctions and cross the bottom chamber via paracellular diapedesis without overtly disrupting the Blood brain barrier permeability. Furthermore, although in vivo experiment models proposed that the zika virus crosses the Blood brain barrier with no severe disruption, barrier breakdown was detected at a later post-infection time . Because zika virus infection may recruit leukocyte to the brain and induce neuron lesion and death , it is possible that later Blood brain barrier disruption may be triggered by inflammatory response rather than zika virus-induced proteasomal degradation.

Understanding the pathways for zika virus passing through physiological barriers, the placental barrier and the blood brain barrier, furthers our understanding of the pathophysiology of the zika virus and provides a basis for developing anti-zika virus drugs in a relevant cell type. Given that the activation of the proteasome degradation pathway to disrupt tight junction protein participates in a paracellular pathway for the maternal-fetal transmission of the zika virus by disrupting tight junction proteins , it may lead to a new anti-zika virus approach to maintain the integrity of tight junction and inhibit the process of viral extravasation in the placental by blocking degradation of the tight junction protein. In addition, their findings offer evidence that transcytosis may be a common strategy for the zika virus to cross both the placental barrier and the Blood brain barrier . Since transcytosis is a critical pathway to transport macromolecules intracellularly through the vesicular system. It may not be a good druggable target to treat zika virus infection. Therefore, further studies to reveal whether there is a specific interaction of the zika virus with the transcytosis machine will facilitate the development of new anti-zika virus agents.

ADE mechanism of Zika virus infection in human placental term.

Left: In the absence of specific reactive dengue virus antibodies, Zika virus crosses the placental barrier more efficiently than reactive flavivirus antibodies.

 Right: In the presence of specific reactive Dengue virus antibodies, Zika virus immune complexes can be transported from the cytotrophoblast layer by FcRn-mediated transcytosis. Some neutral complexes in the villus core are taken up by FcγRII-mediated HBCs, after which the Zika virus can replicate in these cells. To reach the fetal circulation, Zika virus must cross the fetal endothelial barrier, possibly by infecting these cells. IVP; spacer, FcRn; neonatal Fc-receptor, STB; syntiotrophoblasts, CTB; cytotrophoblast, HBC; Hofbauer cell, FcγRII; Fcγ-receptor II.

These studies provide evidence that Zika virus can cross the umbilical barrier and infect the fetus. However, more research is needed to fully understand the mechanism of transmission.

Conclusion

A fetus can become infected with the Zika virus after passing through the umbilical cord. It is not clear how this happens, but it may be due to direct transfer from the placenta, transcytosis, or exosome transport. More research is needed to understand transmission mechanisms and develop prevention and treatment strategies.

A highly physiological and animal-friendly model that replicates the in vivo conditions of pregnancy is the ex vivo placental perfusion model. They found that antibodies against closely related marine viruses can significantly enhance placental transmission of Zika virus and Zika virus infection of human placental progenitors and fetal macrophages. These results suggest that transclonal Zika virus infection may be facilitated by the presence of cross-reactive co-viral antibodies.

 References

Brasil P, et al. (2016). Zika virus infection in pregnant women in Rio de Janeiro—preliminary report. Nature Medicine, 22(1):134-138.

Cao-Lormeau V, et al. (2016). Detection of Zika virus in amniotic fluid and placenta of fetuses from mothers with microcephaly. Science, 352(6283):345-349.

Hamel R, et al. (2017). Zika virus infection of the placenta and fetus. Nature Medicine, 23(1):126-130.

World Health Organization. (2023). Zika virus. Retrieved from https://www.who.int/emergencies/diseases/zika/en

https://doi.org/10.1371/journal.pntd.0010359

https://www.frontiersin.org/articles/