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Cellular Studies of Zika Virus Offer Clues to a Potential Mechanism for Microcephaly — and a Therapeutic Target

Talan, Jamie

doi: 10.1097/01.NT.0000482740.29873.68
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ARTICLE IN BRIEF

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Researchers observed that in cellular models Zika virus was able to infect neural stem cells, causing dysfunction or death under laboratory conditions. The investigators contend that the cellular studies serve as a potential model for understanding the link between Zika and microcephaly.

Scientists have discovered that the Zika virus infects human cortical neural progenitor cells, triggering cell damage and death. The finding, they said, provides a model for future studies and a clue about the mechanism that may be contributing to an associated link between the virus and microcephaly.

Studies of fetuses and babies with microcephaly in Zika-affected areas have found abnormalities in the cortex, the researchers noted, and Zika virus has been found in the fetal tissue. [See “More Research on the Association Between Zika and Microcephaly.”]

The study, published online first on March 4 in Cell Stem Cell just two months after the inception of the experimental study, reflects the frenzied pace of collaborative research to resolve what has become a major public health concern in at least 31 countries, to date.

Scientists have been scrambling to determine which cells Zika infects in the brain and whether the virus has a direct or indirect effect, or if there is another player involved. Ultimately such studies are critically important to creating strategies to stop the virus from infecting the developing brains of fetuses in pregnant women exposed to the virus.

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STUDY METHODOLOGY AND FINDINGS

Hengli Tang, PhD, a virologist and professor of biological science at Florida State University (FSU), has been studying similar RNA viruses, including the dengue virus, and already had many of the tools in place to study Zika. Johns Hopkins University neuroscientists Guo-li Ming, MD, PhD, a professor of neurology, neuroscience, and psychiatry and behavioral science at the school's Institute for Cell Engineering, and her husband Hongiun Song, PhD, a professor of neurology and neuroscience in the Institute for Cell Engineering, had been researching neurodevelopmental diseases and had recently become interested in genetic forms of microcephaly.

The researchers at FSU and Johns Hopkins, who had worked together in graduate school, started collaborating in January. The Hopkins labs used a 3-dimensional culture comprising human pluripotent stem cells to grow what they called mini-brains; the cells start to form brain structures that are similar to what is taking place in the developing brain, the investigators explained.

Drs. Ming and Song sent the human induced pluripotent stem cells down to Florida, and a few days later, a post-doctoral fellow from their lab flew down to help prepare the cells for differentiation into neural progenitor cells. Dr. Tang and his team in Florida obtained Zika stock from an infected rhesus Macaca cell line (an older cell line), passaged the virus in a mosquito cell line, and then tittered the virus onto a cell line that is commonly used to grow virus. They added the Zika virus to several human cell lines to see what would happen.

First, they wanted to determine if the virus can infect forebrain-specific human neural progenitor cells and cortical neurons. They put in a small dose of virus and removed the broth containing the virus inoculum after incubating it in the dish for two hours. They measured infection rates during a three-day period with RT-PCR and immunochemistry to test for the presence of Zika.

During that period, the virus had spread by 65 to 90 percent of the human neural progenitor cells. They repeated the study several times to make sure what they were seeing was not an artifact. They also used human embryonic stem cells, human induced pluripotent stem cells, and immature cortical neurons as control cells; when Zika was added to the culture there was very little infectivity.

“This finding told us that human neural progenitor cells are a direct cell target,” said Dr. Tang. “The cells are producing virus, and that is allowing the virus to spread. These cells are critical in the developing embryonic brain.”

The scientists also wanted determine what the infection was doing to the human neural progenitor cells. They found a 30 percent reduction in the total number of viable cells between 66 and 72 hours after the virus was added to the cell culture. By three days, there was a significantly higher caspace-3 activation when compared to cells that were exposed to another mock infection.

Dr. Tang said that this suggests that more cells were dying after exposure to the virus. “We found 15 percent of cells died during the first three days compared with only 2 percent of cell death in non-infected cells.”

There was also significant cell-cycle dysregulation, he said.

The scientists reached out to colleagues at Emory University who agreed to conduct global transcriptome analyses. The Emory researchers, led by geneticist Peng Jin, PhD, conducted genome-wide analyses to see how the virus alters patterns of gene expression in the human neural progenitor cells.

They found a down-regulation of a number of genes involved with cell-cycle pathways. They also observed an up-regulation of genes involved in transcription, protein transport, and catabolic processes. The gene expression of caspace-3 proteins was also enriched.

“This is a first step in a long line of planned studies,” said Dr. Tang. “That the infected cells release viral particles is concerning, because that could impact the development of therapies to arrest or limit the infection.”

Dr. Tang said that it is still not clear how Zika passes from the mother into the fetal brain. “We have a lot of questions that need answers,” he added.

Dr. Tang said he hopes that their model will trigger more work in understanding Zika's effect on neural development. The model also opens up the opportunity to conduct high-throughput screens to prevent Zika infection or reduce some of these pathological effects that may take place during neural development.

The researchers are now repeating these studies and looking for changes in brain development over a longer period of time. They want to understand how the virus perturbs the cell cycle in developing brain cells. They are also in talks with the National Institutes of Health to obtain compounds and test them against Zika-infected human neural progenitor cells. And their collaboration has now expanded to include six laboratories.

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“While this study doesn't definitely prove that Zika virus causes microcephaly, it's very telling that the cells that form the cortex are potentially susceptible to the virus, and their growth could be disrupted by the virus,” said Dr. Ming. “Our study is the first step in trying to understand the relationship between the Zika virus and microcephaly.”

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EXPERTS COMMENT

“This paper shows that Zika virus is able to infect neural stem cells and cause dysfunction or death under laboratory conditions,” said Daniel Pastula, MD, MHS, a neurologist and medical epidemiologist at the University of Colorado School of Medicine. “This is certainly concerning if it is what can happen in vivo, in real life. This finding is building evidence that Zika may be harming the developing human brain.”

He said that it is still possible that there is another factor that is working in tandem with the Zika virus to trigger microcephaly. “We just don't know yet,” he added. “Of course, we need a lot more studies in this area.”

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His colleague, J. David Beckham, MD, an associate professor of medicine and infectious diseases at the University of Colorado School of Medicine, has developed a mouse model of the Zika virus, and has started a series of studies to see if there is a window of time during pregnancy where the cells are more vulnerable. He and his colleagues are also trying to understand how the virus passes through the placenta.

What he finds most intriguing in the Cell Stem Cell paper is that the “neural stem cells are susceptible to infection but the early cells before they go down the neural differentiation pathway are not. As the neural cells become more mature, they become less susceptible again.”

“This gives us a clue into what may be going on in the developing human brain,” he said. He added that there is growing scientific support that the virus is directly killing cells rather than triggering an inflammatory event that damages or destroys them.

One downside to the new paper is that they used an old strain of the Zika virus from Uganda. Dr. Beckham's lab just obtained new isolates from an infected American who had returned from a trip to Puerto Rico. They also have the older Uganda isolates and will be testing both strains to see whether there are differences in the way they target developing brain cells.

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MORE RESEARCH ON THE ASSOCIATION BETWEEN ZIKA AND MICROCEPHALY

In February, the Centers for Disease Control and Prevention (CDC) published a Morbidity Mortality Weekly Report by Roosecelis Brasil Martines, MD, PhD, a pathologist at the federal agency, and her colleagues, outlining the findings from four specimens culled from fetuses and newborns.

The Brazilian government sent tissue samples from two newborns (born at 36 and 38 weeks gestation) who died within the first day of birth and two samples from miscarried fetuses that were lost at 11 and 13 weeks gestation. All of the four mothers had signs of a mild infection during their first trimester. They were not tested for antibodies to Zika.

The CDC samples included brain and other autopsy tissues from the two newborns, a placenta from one of the newborns, and products of conception from the two miscarriages. The tissue from all four samples tested positive for exposure to the Zika virus. They also tested for another mosquito born infection, dengue, and the results were negative.

The pathological changes in the brains of the two newborns included parenchymal calcification, microglial nodules, gliosis, and cell degeneration, and necrosis. They also noted pathological changes in the placenta from one of the mothers who miscarried.

According to the CDC report, more than 4,700 suspected causes of suspected microcephaly were reported from mid-2015 through January 2016. The Brazil Ministry of Health is working with the CDC to determine the connection between microcephaly and exposure to Zika in the womb.

—Jamie Talan

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LINK UP FOR MORE INFORMATION:

•. Tang H, Hammack C, Ogden SC, et al. Zika virus infects human cortical neural progenitors and attenuates their growth http://www.cell.com/cell-stem-cell/abstract/S1934-5909(16)00106-5. Cell Stem Cell 2016; Epub 2016 Mar. 4.
    •. Martines RB, Bhatnagar J, Keating MK, et al. Notes from the field: Evidence of Zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses — Brazil, 2015 http://www.cdc.gov/mmwr/volumes/65/wr/mm6506e1.htm. MMWR Morb Mortal Wkly Rep 2016; 65(6):159–160.
      © 2016 American Academy of Neurology