Until now, most Zika research has chronicled damage to newborns who were inadvertently, and haphazardly, exposed to the virus. Now scientists have taken a different tack: They have deliberately infected developing mice pups and tiny clusters of lab-grown brain cells so they could observe and measure the damage that ensued.
The results of these experiments nail down the causal link between Zika and the rash of birth defects seen over the past year in Brazil. And they begin to show how a virus thought to be little more than a pest crosses the placental barrier and makes a beeline for a fetus' brain.
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In doing so, they give scientists some insight into why a mother's infection with Zika early in pregnancy appears to result in far more extensive damage than when her infection occurs at a later stage.
The new studies, published Wednesday in the journals Nature, Cell and Cell Stem Cell, suggest that when the Zika virus takes hold in the first trimester, it makes its way quickly to the uterus and to cells that line and normally help protect the placenta.
While that firewall between mother and child is still immature, Zika attacks those cells and penetrates the placental barrier. As it does so, the virus also disrupts the growth of placental blood vessels, limiting blood flow to the baby and stunting fetal development.
If the fetus survives the onslaught, the virus will make its way to its developing brain. There it will wreak destruction on the stem cells that are meant to develop into a mature organ. As Zika replicates madly, these stem cells and immature neurons die off in droves, the researchers found. The differentiation of tissues that normally produces a healthy brain goes awry — sometimes subtly, sometimes horribly.
In humans, scientists have observed microcephaly, diffuse calcium deposits and brain structures that are either abnormally large or small. In surviving mouse pups, researchers found that brains infected in utero were abnormal in a variety of ways, not all of them identical to effects seen in humans.
But the relationship between Zika infection and fetal abnormality was direct and consistent, and that says something important about the virus, said Dr. Michael S. Diamond, who studies infectious diseases as Washington University in St. Louis and was lead author of the report in Cell.
"Zika alone is enough" to cause these effects, Diamond said.
That collective finding puts to rest many scientists' suspicions that Zika might need to interact with some other factor — say, a past infection with dengue fever — for brain damage to result in a fetus.
"That's not to say there aren't other factors that may predispose people to get Zika," Diamond said. "But you don't need all those things" for brain damage to occur, he added.
Mice are not naturally vulnerable to Zika and must be treated, bred or genetically altered to become so. Human and mouse fetal development happens on vastly different timescales and levels of complexity.
"A mouse model is evolutionarily far removed from a human, so you have to be cautious when you extrapolate" findings to humans, said Dr. Anthony Fauci, director of the National Institute on Allergies and Infectious Diseases.
But mice capable of contracting Zika provide researchers the chance to see the exact same virus' behavior in thousands of virtually identical animals — and all in a mouse's roughly 20-day gestation period, Fauci said.
Even more important, he added, pregnant mice that can be infected with Zika and reliably give birth to damaged babies offer scientists a vast and valuable experimental population. Unlike humans or other primates, mice can be used in large numbers to screen drugs that might confer protection against the virus' effects.
The researchers who conducted the Nature study infected mice with a cloned sample of a Zika virus harvested in 2015 from a feverish patient in the state of Paraiba, Brazil. The newborn pups "displayed clear evidence of whole-body growth delay or intra-uterine growth restriction," the team wrote.
Upon inspection of the pups' brains, "we noticed cortical malformations in the surviving animals, with reduced cell number and cortical layer thickness, signs associated with microcephaly in humans," they added.
The researchers also took clumps of human cells captured at a very early stage of development and used them to grow brain "organoids," allowing them to study a facsimile of a fetal brain in its first trimester. Within four days, organoids infected with Zika had significantly fewer neurons in the nascent cortex than their uninfected counterparts.
In the Cell study, researchers infected pregnant mice around day 7 of gestation with a Zika strain that circulated in French Polynesia in 2013. The resulting offspring were abnormally small and had brains that were full of Zika virus. But they did not have microcephaly.
Diamond said the absence of microcephaly did not undermine the value of the work.
"We infected early and harvested early," he said. In mice, whose brain development starts late and continues after birth, waiting a few extra days to infect a pregnant mother would probably have resulted in more obvious abnormalities, he said.
The third study, in Cell Stem Cell, did just that. Researchers from the Chinese Academy of Sciences injected a strain of Zika isolated from a patient in Samoa into a mouse fetus on the 13th day of gestation. The virus was delivered directly to the developing brain's fluid-filled ventricles.
Three to five days later, the virus had replicated copiously and the scientists noticed structural abnormalities similar to those seen in human babies with microcephaly, including thinner cortical structures and larger fluid reservoirs.
Their efforts to inspect the mouse pups' brains after birth, however, ran afoul of nature. Within two days of birth, lactating mothers ate their infected newborn pups.
Fauci cautioned that the new findings likely do not give an exact picture of how Zika infection affects pregnant humans, since all are based on observations of Zika-infected mice and of Zika's behavior in laboratory tissues.
The urgency among scientists to make and report their discoveries has resulted in a torrent of research that has drawn together virologists, neuroscientists, stem cell biologists, obstetricians and child health specialists to collaborate.
Their work is motivated by the tragic outcomes in Brazil, the mysterious odyssey of this virus first identified in Uganda in 1947, and by the fact that Zika is easier and less deadly to work with than a virus like Ebola.
"I know the public is anxious to have vaccines and therapeutics," Diamond said. "But seeing research progress at this rate is very remarkable. This does not always happen."
"This is what happens when you get good people pursuing a new problem," he said. But he warned that without an infusion of $1.9 billion in emergency Zika funds proposed by the Obama administration, this pace of discovery can't be sustained.