Trophoblast cells unlikely entry point for Zika

One theory — that Zika virus enters the developing fetus by passing through the trophoblasts, a layer of placental cells that surround and nurture the fetus — is disproved. But configuring a new mouse model for the disease reveals that type-I interferon resists Zika.

A collaborative team of virologists and reproductive scientists — looking at cells isolated from full-term human placentas — have found that Zika does not infect trophoblasts. So how the virus is getting through the placenta is still unknown. But, at least one obvious pathway is ruled out.

Two unrelated studies on the Zika virus — one, ruling out the theory that Zika may pass through the human placenta, and the 2nd, on using mouse models to trace Zika — appear in Cell Host & Microbe1.

"Trophoblasts are the baby's first line of defense against anything coming from maternal blood. So, you may expect these cells have some way to resist viral infections. Based on our model, it seems trophoblasts have an inherent capacity to resist proliferation by Zika."

Yoel Sadovsky MD, Director, Magee-Womens Research Institute, University of Pittsburgh, Pennsylvania, USA.

Sadovsky, an expert in maternal-fetal medicine, has been working on the Zika-placenta connection with long-time collaborator Carolyn Coyne PhD, Associate Professor, Department of Microbiology and Molecular Genetics, at the University of Pittsburgh. Dr. Coyne is an expert in RNA viruses — including flaviviruses, the family of viruses to which Zika belongs.

Their examination revealed that human trophoblasts release a potent antiviral molecule called type III interferon, which stops replication of the Zika virus.

Researchers used two Zika strains in their study — one isolated from the Zika Forest in Uganda and another from Cambodia.

Dr. Coyne: "We really know shockingly little about how viruses cross the placenta — not just Zika but rubella, herpes, and other viruses that cause birth defects. What makes our finding interesting is that these trophoblasts are potentially communicating with maternal cells to protect them against viral infections as well."

The strength of Coyne and Sadovsky's study is that human trophoblasts cultured in the lab functioned nearly identically to those found in a developing placenta. One potential weakness of the study is that their trophoblasts were taken from third-trimester pregnancies, which means that trophoblasts could still be vulnerable to Zika virus during the first trimester. However, researchers don't believe that it reduces the significance of their findings as Zika seems to be transmitted throughout pregnancy. With primary trophoblast infection ruled out, Coyne and Sadovsky will next use their placental cultures to explore other ways that viruses can reach a developing fetus.
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Until recent weeks, only three papers using mice to study Zika infection had been published in the last 60 years. The second paper also appears in April's issue of Cell Host & Microbe2.

Washington University School of Medicine researchers have returned to the mouse as a model system to study the infection. They wanted to find which tissues are most vulnerable to Zika via the skin, mimicking a mosquito's bite. Their results found high virus count in mouse brain, spinal cord, and testes — mimicking human sites of Zika infection.

Early evidence suggests that Zika has trouble crossing into rodents. Zika doesn't seem to replicate in adult wild-type (normal) mice, so the investigators had to use animals genetically engineered without natural type-I interferon, in order to generate a lethal infection.

"If you take away interferon, then the virus replicates quite well in the mouse and goes to the places that we see it causing disease in humans."

Michael Diamond PhD, studies mosquito-borne viruses at the Washington University School of Medicine, and is the senior author.

Diamond was inspired to pursue Zika after hearing Brazilian researchers describe word of mouth evidence about a rise in infant birth defects simultaneous to a local Zika outbreak. His laboratory, led by first author Helen Lazear, had previously followed Dengue and West Nile virus patterns. So they created a Zika mouse model and looked at tissues that became infected after viral exposure.

"Viral levels were the highest we saw in any tissues that we measured," Diamond says. "We are now doing subsequent tests to determine how long that may last."

Five strains of the virus were examined: (1) the original strain from the Zika Forest in Ethiopia, (2, 3, 4) three 1980s strains from Senegal, and (5) a 2013 contemporary strain from French Polynesia. All yielded similar results, so there may not be much difference between them.

Diamond is now using his models to explore how Zika responds to a mouse's adaptive immune response where cells generate antibodies that tag viruses for later removal.

Cell Host & Microbe1, Bayer and Lennemann et al.: "Human Placental Trophoblasts Produce Type III Interferons that Confer Protection against Zika Virus Infection" http://dx.doi.org/10.1016/j.chom.2016.03.008. This project was supported by the National Institutes of Health and Burroughs Wellcome Investigators in the Pathogenesis of Infectious Disease Awards.

Discussion
The rapidly emerging human health crisis associated with the ZIKV epidemic highlights the growing need to identify mechanisms by which ZIKV accesses the fetal compartment. These data will be instrumental in order to design therapeutic measures to limit ZIKV replication and/or spread. Our experimental cell system is directly relevant to the study of congenital ZIKV infections, by defining unique antiviral mechanisms at play in this specialized environment. We provide evidence that ZIKV is unlikely to access the fetal compartment by its direct infection of late-pregnancy villous syncytiotrophoblasts and potentially neighboring cells that express IL28RA, due to the role of type III IFNs in the antiviral defense produced by human trophoblasts, which suggests that the virus may circumnavigate these cells or overcome this restriction in vivo in order to bypass the placental barrier.

Authors: Avraham Bayer, Nicholas J. Lennemann, Yingshi Ouyang, John C. Bramley, Stefanie Morosky, Ernesto Torres De Azeved Marques Jr., Sara Cherry, Yoel Sadovsky, Carolyn B. Coyne
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Cell Host & Microbe2, Lazear et al.: "A Mouse Model of Zika Virus Pathogenesis" http://dx.doi.org/10.1016/j.chom.2016.03.010. This work was supported by start-up funds from the University of North Carolina Department of Microbiology and Immunology and the Lineberger Comprehensive Cancer Center, as well as grants from the National Institutes of Health.

Discussion
The emergence of ZIKV in the western hemisphere and in particular the unexpected association between ZIKV, birth defects, and GBS has focused international public health attention on this previously obscure virus and spurred calls for rapid development of vaccines and therapeutics. However, it will be difficult to evaluate candidate products rapidly without small animal models of disease. Furthermore, a mechanistic understanding of ZIKV pathogenesis, which can be determined using animal models of disease, can provide insight into the dynamics of ZIKV spread and transmission.

In summary, our results show that IFN-α/β signaling plays a key role in restricting ZIKV infection in mice. The use of mice with diminished or absent IFN-α/β signaling provides a small animal model for evaluating vaccines and therapeutics to combat ZIKV. Such models also will be valuable for studying the pathogenesis of ZIKV and mechanisms of viral immune evasion and for understanding unexpected clinical manifestations of ZIKV infection in humans.

Cell Press Statement on Data Sharing in Public Health Emergencies

The Cell Press family of journals is committed to ensuring that the global response to public health emergencies is informed by the best available research evidence and data, and as such, we will make all content concerning the Zika virus free to access. We will work in partnership with reviewers to fast-track review all submissions concerning Zika. We will adapt the editorial criteria that we apply to Zika submissions by asking reviewers to evaluate only if the research methods are sound and support the conclusions and if the work will contribute in some way toward resolving the immediate challenges. We will expedite publication of papers that meet these two criteria.

Cell Host & Microbe (@cellhostmicrobe), published by Cell Press, is a monthly journal that publishes novel findings and translational studies related to microbes (which include bacteria, fungi, parasites, and viruses). The unifying theme is the integrated study of microbes in conjunction and communication with each other, their host, and the cellular environment they inhabit. Learn more: http://www.cell.com/cell-host-microbe. To receive Cell Press media alerts, contact press@cell.com.
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