How Zika Spiraled Out of Control

The world's biggest collection of Zika virus is housed in a tan concrete building, rising up from the flat campus of the University of Texas Medical Branch (UTMB) here in Galveston. Inside, armed guards watch the lobby, and access to certain floors requires special clearance. These safeguards are in place because other viruses, including those that cause Ebola and severe acute respiratory syndrome (SARS), are also on the premises.

Zika is not as easily spread as deadly Ebola, so the laboratories that work with the mosquito-carried virus do not require spacesuitlike protection gear. On a recent visit I held a clear plastic bag containing the complete virus collection in my bare hands. Inside the bag the 17 vials of pure, freeze-dried virus resembled old glue. They did not look like the material of a global health crisis that has panicked entire nations. (The scientist who handed me the bag did keep one hand hovering nearby, presumably ready to catch if I dropped it.) Despite its modest appearance, experiments with the collection may be researchers' best hope of understanding how the virus got so out of control—and what to do about it.

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The virus was first detected in Uganda, and for nearly 70 years it stayed small and scientists took little notice, believing all it did was cause symptoms on par with a mild flu. But since 2015 Zika has ricocheted to over 40 countries, transmitted by mosquito bite or human sexual contact. [Editor's note: As of September 2016, the number had increased to more than 50.] Researchers now blame it for terrible birth defects, including microcephaly, when babies are born with abnormally tiny heads, and Guillain-Barré syndrome, an autoimmune disease that can affect patients of any age. “The more we learn, the worse it gets,” says Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases at the National Institutes of Health.

Virologists now need to figure out which of two potential causes is responsible for the Zika explosion. One possibility is that genetic changes within the virus supercharged its ability to infect people and cause disease. The second scenario is that the change happened outside the virus: after decades of relative isolation, it reached dense population areas, providing more fertile ground for its spread. If researchers identify new mutations within the virus that also allow it to breach the placenta and cause microcephaly in fetuses, then they could be on the lookout for ways to combat that transmission, too.

If the outbreaks are just the result of environmental changes, perhaps Zika was always capable of causing serious effects, but they were rare in small populations and only became obvious when the virus hit a lot of people. “It could just be a numbers game,” says Erin Staples, a U.S. Centers for Disease Control and Prevention medical epidemiologist. (There is a third, less accepted theory suggesting that previous exposure to another virus called dengue could somehow make people more susceptible to Zika. But there is little support for this compared with the other two ideas.)

Galveston is an ideal place to tackle this inside versus outside debate because the Medical Branch research center is home to the massive World Reference Center for Emerging Viruses and Arboviruses (the latter are viruses carried by ticks, mosquitoes or other arthropods). The collection includes more than 7,000 pathogens. Investigators also have a variety of specialized resources for testing virus infectivity in various bugs and animals, sequencing viral genes and imaging pathogen structures.

Spitting Image

For choosing among competing theories about what went wrong with Zika, it also helps that arbovirus expert Scott Weaver, the university's director of the Institute for Human Infections and Immunity, and his colleagues have become good at a rather arcane skill: making mosquitoes spit.

For the past few months Weaver's team has fed lab-grown mosquitoes—related to the insects spreading disease in Brazil and the Dominican Republic—blood meals spiked with either a 2015 Zika strain from Mexico, a 2010 strain from Cambodia or a 1984 strain from Senegal. The scientists wait a bit, then coax the mosquitoes to spit, and examine that saliva for the amount of Zika virus particles it contains. Comparing mosquitoes that carry different Zika strains lets the researchers test whether newer strains travel more quickly from insects' guts to their saliva than do older strains. Such increased speed in a virus is often caused by a genetic change. If the virus now found in the Americas and Caribbean moves into the bugs' spit faster than other strains do, it could fuel a larger, faster-moving outbreak in humans—and favor the inside version in the inside/outside hypotheses duel.

Doctoral student Christopher Roundy has become the main drool wrangler. “Basically I spend my days collecting mosquito spit,” Roundy says. “It doesn't sound that glamorous.” He begins the tests in Weaver's lab by mixing a specific strain of Zika with human blood donated by a local hospital. (The blood was getting too old for use in human transfusions.) Next he pours the spiked blood into a metal canister that he covers with skin from a mouse—mosquitoes are more likely to bite through skin. The other side of the canister is hooked to a heater because mosquitoes also prefer warm meals. The whole setup is then placed, skin-side down, over the opening of a small Styrofoam container holding dozens of female mosquitoes (males do not bite). Frenzied by the smell of blood, the females stab their proboscises through the skin, sometimes two or three times. Once they are full, their stomachs become red and engorged—making it clear that they have eaten.

For some, this is their last meal. Every day afterward a small number of those bugs are killed and taken apart. Their bodies, sans legs, are blended into a slurry; the legs are similarly pureed and studied separately. Both batches of bug bits are then scrutinized for signs of the virus—indicators of how far the pathogen traveled in the body.

After eight days Roundy turns his attention to the bugs' spit. He stuns a handful of the remaining live mosquitoes by placing them in the freezer for about a minute. Then Roundy uses tweezers and a magnifying glass to gently guide each of their proboscises into a small tube containing a salt solution. That exposure to the salty mix forces them to drool. He adds that spit to dishes of cells and mixes in chemicals that cause Zika-infected cells to turn a purplish color. The purple cells are visible to the naked eye. Under a microscope, Roundy also examines the spit for tiny purple blotches—viral particles—that he might have missed.

If there are genetic changes in newer Zika strains, Roundy, Weaver and UTMB pathologist Nikos Vasilakis are particularly interested in any that might help the virus infiltrate human cells or aid its journey via mosquito bite. They also want to know if mutations may explain its connections to microcephaly. More virus in any single drop of blood, for example—thanks to boosted replication rates of the organism—could perhaps help the virus to more readily cross the placental barrier and reach a developing fetus. If there are no such changes, that is telling, too. It shifts the balance toward the “outside” environmental idea.

Searching for inside/outside evidence using this method has worked before. The same Galveston team used a similar approach to determine that another mosquito-borne disease, chikungunya, had mutated in the past. By studying spit, they found that specific genetic adaptations allowed that virus, normally spread by the Aedes aegypti mosquito, to expand its range by jumping to a different carrier, the Aedes albopictus mosquito. A single amino acid change in one of the virus's glycoproteins, for example, allowed the virus to replicate about 40 times easier in the bug than it once did. The information prompted health workers to expand warnings about the disease to more areas of the Pacific rather than limiting them to those locations at risk from one of the species. (It is unlikely that Zika made a similar species jump because the virus is already thriving in areas with A. aegypti, Weaver says.)

Moving Target

The Zika test results were finalized in late April. After tracking different strains of the virus via thousands of bugs, the Galveston team came to a quiet conclusion: the latest Zika strain does not appear to be more active or more easily transmissible than are prior strains. In fact, older Zika from Africa in the 1980s is a bit quicker traveling through the mosquito body. The discovery does not completely eliminate the idea of harmful genetic changes, yet this evidence tips the scales of blame toward the environment.

For scientists such as Weaver who are concerned about public health, the environmental tilt is disquieting. “It's bad news in a way because that tells us that the strain that is circulating today in Asia and Africa probably has at least the same capacity to initiate urban epidemics,” he says. (Immunity in some of those populations from past outbreaks, however, may be protecting them from large epidemics.) The serious symptoms became more apparent as more people grew ill, and public health officials saw enough of an uptick in microcephaly, other birth defects and Guillain-Barré to fuel concern and further investigation.

Kathryn Hanley, a biology professor at New Mexico State University who investigates viruses such as dengue and Zika, notes the environment and internal mutations actually could be working together. “These are not mutually exclusive hypotheses,” she says. “It could be that the virus previously had little access to urban populations, but on the way to getting that access it also acquired mutations that increased its transmission.” Researchers at Galveston and elsewhere plan further tests on insects and animals to explore this and other ideas.

There are still many unknowns about the virus. For instance, the way Zika infects human cells and hijacks their machinery to copy itself many times has proved elusive. And because this virus looks so much like dengue, testing for Zika has remained a serious challenge, complicating efforts to accurately track the spread. The first large outbreak—in 2007 on Yap Island in Micronesia—infected 70 percent of the island's population but was only identified because scientists had been deployed there on the assumption the isle was under siege by dengue. Other Zika cases have undoubtedly been missed altogether, scientists say.

That suggests researchers have underestimated this virus all along. Perhaps other benign-seeming threats could be more dangerous than previously thought as well, they worry. “You should not necessarily be scared, but you have to be open-minded and quick to respond,” says the cdc's Staples. If Zika and other emerging viruses have unexpected effects as they reach different populations, it is a serious problem. Without better global surveillance of diseases in remote locations, health agencies will find it hard to foresee or prepare for an organism that could be quickly transported to an urban area and spiral out of control.

Such surveillance is currently not done: many developing nations have little health infrastructure and few available resources to strengthen it. Moreover, most of the World Health Organization's annual budget comes from donor countries that earmark it for specific projects such as HIV prevention or polio eradication, not surveillance of amorphous threats.

Humanity is not without defenses, however. Some insecticides and larvicides work on the A. aegypti mosquito, Zika's preferred host, so disease-control experts know which mosquitoes to study and target. Still, says Ann Powers, a laboratory chief in the cdc's Division of Vector-Borne Diseases, “we need to be more vigilant.” All it takes to change the profile of a disease, she knows, is a few extra mosquito bites.