Resisting Zika

Undeterred, researchers are tackling a global crisis from multiple directions.

By Tamara Bhandari

Robert Boston
Jennifer Govero, PhD, a senior scientist and lab manager, and virologist Michael S. Diamond, MD, PhD, analyze a plaque assay of the Zika virus.

Throughout 2015, increasingly worrisome reports trickled out of Brazil about an obscure virus called Zika. A member of the flavivirus family, which includes dengue and West Nile, Zika was a new arrival in the Americas. Identified almost 70 years earlier in Africa, the virus was thought to cause only mild disease. In Brazil, however, it became associated with birth defects and a progressive form of paralysis known as Guillain-Barré syndrome.

As the Zika epidemic took hold, leaders at the National Institutes of Health (NIH) realized they needed to learn about the virus quickly. They started phoning select scientists, and offered funding for Zika research.

One of those researchers, Michael S. Diamond, MD, PhD, the Herbert S. Gasser Professor of Internal Medicine and Infectious Diseases, is known for his studies on flaviviruses. When the NIH called, the virologist already had been working on Zika for six months, collaborating with experts across the School of Medicine — in neurobiology, reproductive biology, structural biology, immunology and other fields — to determine what damage the virus could do and what could be done to stop it.

Less than a year later, the School of Medicine is one of the hotspots of Zika research. Researchers have developed two highly useful mouse models of Zika infection; identified potential drug targets to block the spread of the virus; showed that Zika can persist in the eye and testis; and identified specific anti-Zika antibodies that could be the basis for vaccines, diagnosis or treatment.

These accomplishments are impressive, but not reassuring. The more researchers learn about the virus, the more dangerous it looks. “Zika hits us where it hurts. It interferes with our ability to have healthy babies. It damages our babies’ brains. It persists in people long after the initial infection, so it affects our reproductive options in the future,” said Indira Mysorekar, PhD, an associate professor of obstetrics and gynecology, who evaluated a pregnant mouse model of Zika infection. “These are all things that affect our humanity at a very fundamental level.”

A chance conversation

It was a chance conversation at an international scientific meeting devoted to another virus that set Diamond on a path to studying Zika. “The meeting was focused on chikungunya, but at the breaks, people get together and talk about all kinds of things. A colleague from Brazil mentioned that they were seeing unusual cases associated with another RNA virus, called Zika,” recalled Diamond. “So when I got back, members of my laboratory started some pilot projects.”

At the time, in June 2015, very little was known about Zika.“There was a non-human primate experiment done when they isolated the virus in 1947, and one or two other mouse experiments done in the ’70s, and that was about it,” Diamond said. “It seemed to be associated with Guillain-Barré syndrome and congenital defects, but causality had not been established.”

“These are all things that affect our humanity at a very fundamental level.”

Over the next few months, as the Zika outbreak escalated thousands of miles away, Diamond acquired samples of the viruses, learned how to grow them and, along with Helen Lazear, PhD, now an assistant professor at University of North Carolina, and Jonathan J. Miner, MD, PhD, an instructor in medicine, started injecting them into mice, in the hopes of creating an animal model of Zika infection.

The project hit a bump right away: Unlike humans, the mice were able to resist Zika infection. Diamond, Lazear and Miner learned that they had to knock out or block a part of the animals’ immune systems before the virus could gain a foothold.

With this realization, they were able to create a mouse model of Zika infection, showing that the virus multiplied in the brain, spinal cord and testes, and developed a model of in utero transmission of Zika virus to fetuses. Meanwhile, Diamond teamed up with Mysorekar to evaluate the sequence of events that enable Zika virus to be transmitted during pregnancy.

“We showed that Zika kills placental progenitor cells that are important for maintaining the health of the fetus while it’s in utero,” Mysorekar said, referring to a kind of cell that is capable of dividing and reproducing into one or more kinds of cells. “It also infects the fetal brain directly. This provides strong evidence that Zika is causing the brain damage we see in human newborns.”

The virus seemed to have an affinity, or tropism, for not just placental progenitor cells but other progenitor cells as well. Kevin K. Noguchi, PhD, an assistant professor of psychiatry, thinks that Zika has a tropism for neural progenitor cells, which may explain the neurological damage that occurs when fetuses are infected.

“If you kill off a neuron, you have a net loss of one cell. But if you kill off a neural progenitor cell, you kill off every cell that it would have produced in the future, which can be thousands or even millions of neurons,” said Noguchi, who studies the effect of toxic exposures on the fetal brain, and recently began collaborating with Diamond and Miner to study Zika. “You can see how a virus that kills off neural progenitor cells could cause microcephaly.”

The kind of damage done by Zika would be difficult — maybe impossible — to treat. “It’s really hard to reverse something that disrupts neurodevelopment because you can’t go back and reinitiate that development again,” Noguchi said. “Other parts of the brain may be able to compensate for some of the defects, but you can’t reverse the brain damage that’s already there.”

Dodging a bullet

The Centers for Disease Control and Prevention estimates that lifetime costs to care for one child with severe microcephaly could total $10 million, not to mention the emotional toll on families. The risks in Central/South America and in the Caribbean are particularly high: infections in women who are pregnant or become pregnant in the near future, or in male sex partners, potentially could result in severe birth defects. Despite this very large global health problem, the overall risk in the U.S. is much lower. Even in Florida’s Miami-Dade County, the heart of the U.S. epidemic, fewer than 1 out of every 20,000 people has contracted the disease locally.

“It hasn’t had much impact in the continental U.S. compared to developing countries, and I don’t expect it to,” said Steven Lawrence, MD, an infectious disease specialist and associate professor of medicine. “The most problematic mosquito vector — Aedes aegypti — isn’t found frequently anywhere in the U.S. other than along the Gulf Coast and in parts of the Southwest. We have relatively few people living without window screens or access to bug spray, so there just aren’t as many opportunities for people to come in contact with infected mosquitoes.”

The same cannot be said of tropical, developing countries such as Haiti. Only a few dozen cases of Zika in pregnant women have been reported in Haiti, but experts agree that the number vastly underestimates the problem. The Dominican Republic, which shares an island with Haiti and has better public health surveillance, has reported more than 1,000 cases in pregnant women.

“The WHO-approved test for Zika is based on amplifying the RNA from a blood sample, and very few clinical labs in Haiti are set up to do that,” said Sarah Brown Riley, PhD, an assistant professor of pediatrics and of pathology and immunology. “It’s not that the virus isn’t there; it’s just that we can’t detect it.”

Brown Riley, who travels to Haiti four times a year, is part of a team of Haitian and American medical professionals, public health experts and scientists designing a strategy to identify possible cases of Zika throughout the country. “Right now, I am trying to figure out whether we can diagnose Zika from dried blood spots,” Brown Riley said. “Then we could just take a drop of blood, dry it onto a card, and then send it by motorcycle, without refrigeration, from anywhere in the country to one of the government labs that is set up to do Zika testing.”

The Haitian government is focusing on low-tech prevention measures, encouraging residents to eliminate potential breeding grounds for mosquitoes by draining standing water near their homes. Local governments in the parts of the U.S. with Aedes aegypti mosquitoes are exhorting their residents to do the same, and in addition, some also are spraying insecticides. But the best hope for a permanent solution is a vaccine.

battlefronts

Daved H. Fremont, PhD, a professor of pathology and immunology, has been working on developing a Zika vaccine. A vaccine made from a live but weakened virus would be relatively simple to create, but it could not be used in pregnant women because the virus, although weakened, could still be strong enough to infect the fetus and cause disease.

Earlier this year, Fremont identified a portion of a Zika protein that elicits a strong, protective antibody response. He plans to collaborate with a primate research facility in Oregon to test whether the engineered vaccines protect non-human primates against Zika infection.

Diamond, in collaboration with Fremont, Mysorekar and researchers at Vanderbilt University, recently has shown that human antibodies can protect developing fetuses from Zika infection and adults from Zika disease, at least in mice. The discovery suggests that treating pregnant women with anti-Zika antibodies may prevent the worst outcomes — microcephaly and other birth defects — and that a vaccine eliciting similar antibodies could do the same.

 

Where next?

Despite the enormous progress made by the ever-growing community of Zika researchers, there is much work left to be done, including:

• Determining whether Zika can cause brain damage in children and adults. Neural progenitor cells, which are abundant in fetuses, are still present, although at much lower numbers, after birth. Robyn S. Klein, MD, PhD, a professor of infectious diseases and neuroscience, studies the impact of the virus on neurodevelopment, with an emphasis on learning and memory.

•  Identifying other routes of transmission. Haina Shin, PhD, an assistant professor of medicine, is studying protective immunity against sexually transmitted Zika, as well as risk factors that may make women more susceptible to this form of transmission. Mysorekar, an associate director of the Center for Reproductive Health Sciences, is researching how the virus crosses the placental barrier, which separates maternal and fetal bodily fluids. Rajendra S. Apte, MD, PhD, the Paul A. Cibis Distinguished Professor of Ophthalmology and Visual Science, co-led a team (with Diamond and Miner) that characterized Zika in the eye and demonstrated presence of virus in tears. He is now studying whether corneal transplants could transmit the virus. This is the most common transplantation surgery in the U.S., with about 40,000 performed yearly.

•  Determining the effect of Zika virus on fertility. See story below.

The list of unanswered questions is daunting, but Diamond is undeterred. The lesson of Zika is not that epidemics can spring up out of nowhere, although that is true, he said. The true lesson is that the scientific community has shown itself to be up to the task of responding to such epidemics.

“While it was true that few were studying Zika before last year, it’s not true that we weren’t prepared for this outbreak. Within a very short period of time we’ve generated vaccine candidates, therapeutic candidates, and animal models both in mice and non-human primates,” Diamond said. “It shows that by studying basic properties of pathogens, whether it is bacteria, viruses, or otherwise, we’ll learn enough so that if something does happen, we’ll be prepared, poised and nimble enough to move into the field very quickly to make significant progress. And that’s what’s been done in Zika.”

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Zika reduces fertility in male mice

Studies needed to determine whether men are similarly affected

Robert Boston
Kelle H. Moley, MD, Andrea Drury, research technician, and Prabagaran Esakky, PhD, the study’s first author and instructor in obstetrics and gynecology, meet to discuss the latest Zika findings.

Most Zika research focuses on how the virus affects pregnant women and causes severe birth defects. A study in mice indicates that the virus also targets the male reproductive system, suggesting  that Zika may have consequences  for men. The study was published  last October in Nature.

“While our study was in mice – and with the caveat that we don’t yet know whether Zika has the same effect in men – it does suggest that men might face low testosterone levels and low sperm counts after Zika infection, affecting their fertility,” said Michael Diamond, MD, PhD, a co-senior author on the study and the Herbert S. Gasser Professor of Medicine.

The testicles of male mice showed cellular damage and shrinkage three weeks after Zika infection. On the left is a healthy mouse testicle; on the right, a testicle following Zika infection.
The testicles of male mice showed cellular damage and shrinkage three weeks after Zika infection. On the left is a healthy mouse testicle; on the right, a testicle following Zika infection.

To find out how the Zika virus affects males, Diamond, co-senior author Kelle H. Moley, MD, the James P. Crane Professor of Obstetrics and Gynecology, and colleagues injected male mice with the Zika virus. After one week, the virus had migrated to the testes, which bore microscopic signs of inflammation. In three weeks, their testicles had shrunk to one-tenth of their normal size and the internal structure was completely destroyed. Sex hormone levels had dropped and fertility was reduced. These mice were less likely to impregnate female mice.

The mice were monitored until six weeks, and in that time their testicles did not heal, even after the mice had cleared the virus from their bloodstreams. “We don’t know for certain if the damage is irreversible, but I expect so, because the cells that hold the internal structure in place have been infected and destroyed,” Diamond said.

“This is the only virus I know of that causes such severe symptoms of infertility,” said Moley, a fertility specialist and director of the university’s Center for Reproductive Health Sciences.

Diamond and Moley said human studies in areas with high rates of Zika infection are needed to determine the impact of the virus on men’s reproductive health.

Published: January 3, 2017