StemCell Therapy

Monkeys and mosquitoes hold clues to when and where the virus could resurge in Brazil.

Researchers Bruna Sena Nascimento (foreground) and Hlio Saraiva climb a tree to capture mosquitoes in the canopy in Santa Barbara, Brazil.

The howler monkeys slink like cats through the canopy, swiveling their heads to look for danger. They have reason to be wary. In the jungle below, biologists armed with a black tranquilizer rifle are hunting them, flashes of khaki through the foliage.

The scientists signal each other with snaps and whistles and pivot directions frequently; they often cross trails perpendicularly instead of following them. As they maneuver for clean shots, thick leaves come rattling down through the branches. In the distance, marmosets whistle.

The scientists are on Ilha Grande, an island off southern Brazil that once housed a notorious maximum security prison. Resorts dot the perimeter today, but untouched jungle dominates the interior. Brazil recently experienced its worst outbreak of yellow fever in decades with hundreds of cases, some just east of Ilha Grande. So scientists plan to test local howler monkeys (called bugios) for the virus to learn whether it has spread here, too. If they can catch one.

Suddenly, a shota sharp hiss. A 20-centimeter dart with a red tuft for a tail rockets upward. The shooter is Filipe Abreu, a Ph.D. student at the Oswaldo Cruz Foundation (Fiocruz), a research institute in Rio de Janeiro, Brazil. Abreu looks as if he's been in the wild for weeks: muddy pants, scruffy beard, machete dangling from his belt. He's an entomologist, but the intricate transmission cycle of yellow fever, which involves both monkeys and mosquitoes, compels him to study primates, too.

The shot misses. Instead of leaves, the dart comes clattering down. A second shot also goes awry, striking a branch and bending the needle tip 45.

The party regroups, and Abreu hands the rifle to Marcelo Quintela Gomes, a technician at Fiocruz. The hunters thread through vines and shinny down trees into a gully. Every so often Gomes raises the rifle, then drops it. He eventually takes three shots, all misses. Everyone agrees that the bugios are unusually skittish.

They're not the only ones. Yellow fever has left the people of Brazil feeling edgy as well. Over the past 8 months, they've endured vaccine shortages, lurid media stories, and the occasional slaughter of innocent monkeys. To stem such problems, scientists have scoured this massive country to figure out where the virus lurks and how it's spreadingknowledge vital to fighting future outbreaks as well. But fear remains, especially the fear that yellow fever will ravage the megacities along Brazil's east coastor push even beyond that.

We've seen Zika march up to the United States, says Seth Berkley, CEO of Gavi, the Vaccine Alliance, in Geneva, Switzerland. So have dengue fever, chikungunya, and other mosquito-borne diseases. There's no reason to think yellow fever won't do the same, he says.

YELLOW FEVER WAS ONCE the world's most feared disease. Most victims suffer only fevers and aching joints, but 15% get walloped: Their eyes and skin turn yellow, and they bleed from the mouth and eyes. (The Spanish name for the disease, vomito negro, means black vomit.) No drugs exist to treat it, and half of those who develop the serious form die as the virus destroys their livers.

Native to Africa, yellow fever spread to the Americas with the slave trade. In 1793, then-President George Washington fled Philadelphia, Pennsylvania (then the U.S. capital), during an epidemic that killed 5000. Later outbreaks nearly derailed the construction of the Panama Canal.

A vaccine developed in the 1930s largely eliminated the disease in North America. Africa and South America, meanwhile, continue to suffer. Sub-Saharan Africa accounts for 90% of cases, including a 2016 outbreak centered in Angola with thousands of victims. But in some ways yellow fever in South America is scarier: Because of the low natural immunity there, it kills one-third of those who contract it.

Brazil seems particularly vulnerable. Yellow fever long ago disappeared into the interior, and given the mere handful of cases most years (2014 saw none at all), few people outside endemic areas get vaccines. But in December 2016, victims began appearing in the east for the first time since 1942. The government confirmed 792 cases in 130 cities nationwide, with several hundred more under investigation. More than half occurred in Minas Gerais (population: 21 million), a southeastern state bordering the states of Rio de Janeiro (17 million) and So Paulo (44 million). One victim reportedly died within 40 kilometers of the city of Rio.

State health ministries, which administer vaccines, have ordered 26.3 million emergency doses since December, but some places still ran short. During Carnival in February, a squirrel monkey died of yellow fever in Utinga State Park in Belm in northern Brazil. After panicked stories in the press, people stampeded vaccine clinics, which had to open extra rooms to contain the crowds. After 12,000 inoculations in 5 days, Belm's clinics exhausted their supply.

Although the onset of winter slowed the outbreak, scientists fear a resurgence next year. When and where it might reappear depends on the erratic and often maddening behavior of three animals: mosquitoes, monkeys, and humans.

TWO TRANSMISSION CYCLES of yellow fever exist in South America. In the sylvatic (jungle) cycle, mosquitoes from two generaSabethes and Haemagogusspread the virus mostly between monkeys, with people (usually loggers or miners in remote areas) bitten and infected only incidentally. But the possibility of an urban cycle looms. Here, a different mosquito, the infamous Aedes aegypti, spreads the disease directly from person to person, and cases quickly multiply. All the recent cases in Brazil have been sylvatica slight misnomer because some have occurred inside citiesbut no human-to-human transmission has occurred.

Bruna Sena Nascimento brings the mosquitoes she catches in the forest (top) back to a lab at the Evandro Chagas Institute in Belm, Brazil (bottom), where she identifies the species and checks for signs of the yellow fever virus.

Pinning down which mosquito species carry the virus can yield clues about who is at risk and where. So on a recent June morning, entomologists from the Evandro Chagas Institute in Belm don green jumpsuits and drive north to the jungle. On the way, they swap stories about all the mosquito-borne diseases they've endured. Leathery old Hlio Saraiva has had three strains of dengue.

They park at a dilapidated banana farm and start gathering equipment. Just in case, bring the big knife, someone says. Saraiva grabs a 45-centimeter machete with ENTOMOLOGIA on the scabbard. As they head off, the farm's resident Dalmatian trots up to join the adventure.

After several turns, they stop. Twelve meters above is a platformor rather, four ragged planks. Yellow fever mosquitoes dine on monkeys in the canopy, so researchers have to scale trees, too. There's no ladder, just a few dozen nails pounded into the trunk to hoist yourself up. It's a drenching climb in the heat.

An ignorant reporter asks what they use for mosquito bait. Bruna Sena Nascimento, an animated entomologist, laughs. We are the bait. Oh. Mosquitoes are attracted to the carbon dioxide that primates exhale. So unlike with monkeys, hunting mosquitoes is easy: Just sit and waitand hold off on the bug spray.

In the old days, researchers sometimes sat on platforms in their underwear, counting bites and noting what parts of the body mosquitoes preferred. Now, fully clothed scientists use short white nets to nab mosquitoes in the air. They then slip tubes into the nets, suck the bugs up with their lungs, and puff them out into collection vesselsanything from sawed-off sections of PVC pipe to plastic Teenage Mutant Ninja Turtles cups with lids. (Entomology is a cheap science, Abreu says. You improvise.) A half-day in the canopy can net a few hundred skeeters.

Back at the lab in Belm, Nascimento freezes and sorts her mosquitoes, focusing on the abdominal stripes or sweeping back legs that distinguish various species. After the sorting, the mosquitoes go into vials filled with saline and a BB. An automatic agitator then liquefies the bugs, producing a foamy gray ink with floating legs. The fluid is added to cell cultures to test for yellow fever.

Although scientists know the disease's main vectors in Brazil, they're still investigating whether others play a role. One mystery is how outbreaks shift from sylvatic to urban and what species drive that shift. Perhaps miners or loggers simply carry the virus back to cities. But certain mosquitoes might play a role, too. Abreu suspects that A. albopictus, the Asian tiger mosquito, which often straddles jungle and urban areas, might harbor the virus and could bridge the cycles by introducing cases into new areas.

Even the known vectors wreak plenty of havoc, though. Says Thomas Monath, a yellow fever expert at NewLink Genetics, a biotech company in Ames, Iowa: In Brazil, they've drawn a line that supposedly demarcates risk. We vaccinate people on one side and don't vaccinate people living on the other side. But mosquitoes aren't observing that demarcation.

IF MOSQUITOES ARE THE VILLAINS of yellow fever, monkeys are the main victims. A lack of natural immunity leads to devastating outbreaks for many South American species, especially howler monkeys. In the throes of the disease, Abreu says, they often descend from trees to riverbanks to slake their thirst, but lack the strength to climb up again. They end up shivering in the dirt, wracked with pain.

During the Brazilian outbreak, health officials nationwide asked people to report sightings of sick or dead monkeys so they could test for yellow fever. They received reports of 5300 dead monkeys since the outbreak started, with an unknown number related to yellow fever. Biologists also carried out active vigilance campaigns, such as the hunts on Ilha Grande.

Shooting monkeys on those hunts is only half the challenge. After the tranquilizer hits, scientists have about 5 minutes before the monkey passes out and plummetsduring which time they're scrambling about with a net, hoping like crazy it doesn't wander into some inaccessible area. Once it lands safely, they check its gums, eyes, and genitals for yellow discoloration, then take blood from the groin to test for virus. Although the monkey usually wakes within an hour, it might remain loopy for several more, rendering it vulnerable to falling or a jaguar attack. So scientists usually detain itone group uses a red dog carrieruntil it recovers its wits.

Most people in Brazil know that monkeys can catch yellow fever. Alas, many also believe that monkeys infect people, which isn't true. (Mosquitoes do.) As a result, people in rural areas sometimes shoot monkeys or leave poisoned fruit around, adding dozens of casualties to an already devastating outbreak. Killing monkeys ends up harming humans as well, says Jlio Bicca-Marques, a primatologist at the Pontifical Catholic University of Rio Grande do Sul in Porto Alegre, Brazil: The death of monkeys from yellow fever is the first warning that the virus is circulating. In other words, monkeys are sentinels, he says, early detection systems. Without the monkeys in our forests, we'll be blind.

HUMAN BEINGS, the third actor in this drama, are the least predictable. Miners and loggers, some with fake immunization cards, jet between sylvatic regions and cities in just hours, potentially transferring the virus to new areas. Cutting down forests also exposes people to mosquitoes that would otherwise remain hidden.

Building homes in forest clearings, a common practice in Brazil, is especially risky. On one side of the road might sit shanties with chickens wandering about and satellite dishes anchored onto mossy boulders. On the other, a wall of thick jungle with vibrant red tropical birds and playful monkeys. Jozelene Beckmann, who lives in Belm, home of the state park where the squirrel monkey died, says that monkeys used to cavort along her rooftop and slip inside her home. Unfortunately, where monkeys go, mosquitoes followas does yellow fever. After years of ignoring recommendations, Beckmann finally got vaccinated in mid-June.

Briefly anesthetized, a monkey has its blood drawn to check for yellow fever virus.

Vaccinating infants for yellow fever is already mandatory in northern and Amazonian states, where the disease is endemic, and some scientists propose extending the law nationwide. But because vaccination carries a small riskone in 300,000 people develops full-blown yellow fever after vaccination and another one in 100,000 develops meningitisthe government has stopped short of mandating it for everyone, instead merely recommending it. Many people in Rio and other cities ignore the recommendation. One problem is that most vaccine clinics keep short hours, and with the Brazilian economy struggling, people can't take time off work to visit.

The low compliance frustrates scientists because inoculation could basically eliminate yellow fever nationwide, and do so at a pittance. A single dose given as an infant that costs a dollar is effective for life. That's extraordinary, Monath says. During the Angolan emergency last year, health officials stretched vaccine supplies with fractional doses of one-fifth the normal amount, and even that seemed to offer short-term protection.

Still, making the vaccine has its challenges. It's hard to scale up production, for one thing, partly because many steps are still performed by hand, the same basic way they were in the 1930s. Manufacturers grow the virus in chicken eggs, which workers must candle one by one with hand lamps to check for signs of live embryosveins, eyes, movement. (Dead eggs are discarded.) A single technician might examine 8000 eggs per week. Technicians also must burn holes in the eggshells with torches to introduce the virus, and later burn more holes around the waist of the egg to extract the infected embryos for grinding in blenders. It's not quite artisanal vaccine production, but it's close.

Another problem is more perverse: The vaccine is almost too good at preventing yellow fever. Because it costs so little and protects people for life, making it isn't profitable. Worldwide, just four institutes bother, including Fiocruz. Manufacturers also dislike the uneven market for it: low demand most years, followed by outbreaks that leave them scrambling. Production at Fiocruz, for instance, jumped from 25 million doses in 2016 to a planned 70 million this yearwhich forced the institute to cut back severely on its vaccine for measles, mumps, and rubella.

The market could soon change, however. The World Health Organization (WHO) recently brokered deals in several African and South American countries to vaccinate 584 million people for yellow fever over the next decade, with a goal of eliminating all outbreaks worldwide by 2026. That campaign should steady the market by ensuring stable demand. To boost supply, Fiocruz is also building a new $1 billion plant west of Rio that, in a few years, could produce up to 100 million doses of yellow fever vaccine annually.

Still, WHO notes that production of vaccine almost always lags behind projections. Lack of urgency among political leaders could also weaken the campaign. Public health authorities, says Monath, can basically forget about [the disease] in between these horrible episodes. So although the new Fiocruz facility will help, eliminating outbreaks worldwide remains an ambitious goal. The quick spread of Zika and other mosquito-borne diseases from South America up to Texas and Florida also suggests that yellow fever will be hard to tame.

During the yellow fever outbreak this year, public health clinics in Belm, Brazil, vaccinated hundreds of people daily.

But the danger of yellow fever in North America pales in comparison with the disaster that could arise if it ever spread to Asia. Even though southern Asia has all the prerequisites for yellow fevermonkeys, mosquitoes, a warm climatethe disease has never gained a foothold on the world's most populous continent. Perhaps, some scientists speculate, other diseases there provide cross-immunity. Or perhaps humans have simply been lucky. But last year, 11 Chinese men working in Angola developed yellow fever upon returning home, the first confirmed cases in Asian history. Thankfully, the disease spread no further. But if the urban cycle ever got established there, 1.8 billion peoplevirtually all unvaccinated and presumably with high genetic susceptibilitycould be vulnerable.

Moreover, the struggle to contain yellow fever, a familiar disease with an effective vaccine, does not augur well for our ability to combat the new diseases that will almost certainly emerge as people push deeper into jungles. As Jon Abramson, a pediatrician at Wake Forest University in Winston-Salem, North Carolina, and a yellow fever adviser for WHO, puts it: If we can't stop outbreaks with yellow fever, we're in a lot of trouble with other outbreaks.

IN BRAZIL, the quest to stop the current outbreak continues. On Ilha Grande, after the howler monkeys scatter, the hunting party takes a break to catch insects. Sometimes we get mosquitoes and can't get monkeys; other times, we get monkeys but can't get mosquitoes, Abreu says philosophically. Both are important.

But after tracking those two creatures all morning, Abreu and Gomes now switch their attention to the final animal in the yellow fever triad: humans. To that end, they visit the abandoned prison nearby, now a museum.

Locals there are gossiping about a tourist who recently found a dead monkey nearby and posted a picture on Facebook. Unfortunately, something dragged the carcass off before authorities could test it for yellow fever.

Abreu befriends the museum guard, whose backyard is a popular crossing point for monkeys. She's quite taken with the tranquilizer rifle, and he shows her how it works. Afterward they exchange contact information, so she can alert him if she sees sick or dead monkeys. She then asks Abreu whether it's true that monkeys give yellow fever to humans. He assures her that they don't.

Late the next morning, Gomes finally bags a bugio, a female. Instead of tumbling from the canopy, she slides down a thick shaft of bamboo like that pole the firemen come down, Abreu says. But she is unharmed, and they get a good blood sample.

A few weeks later, the results come back: negative for yellow fever. That's a reliefno sign yet that this ancient scourge has infiltrated the island paradise. But with nearby mainland cities confirming cases and tourists pouring in, it might not be long. As the rest of Brazil has shown, exemption from yellow fever today is no guarantee of a safe tomorrow.

Link:
On the trail of yellow fever - Science Magazine

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