Peace for the World

Peace for the World
First democratic leader of Justice the Godfather of the Sri Lankan Tamil Struggle: Honourable Samuel James Veluppillai Chelvanayakam

Wednesday, July 12, 2017

How a Tiny Bacterium Called Wolbachia Could Defeat Dengue

Scientists are immunizing mosquitoes against disease with the help of a common microbe
Credit: Illustration by Bill Mayer
The best time of day to release mosquitoes in northern Australia is midmorning. Later in the day, winds might sweep the insects away and dash any hope that they will find a mate. Earlier than that, the workers who drive around and release containers full of mosquitoes would have to get overtime pay. And so, on a sweltering January morning at the height of the Australian summer, I climbed into my white van with thousands of mosquitoes stowed in Tupperware cups on the backseat.

Once a week, for about three months in 2011, we made trips like this to release mosquitoes. We concentrated on two communities in the city of Cairns, a popular tourism spot near the Great Barrier Reef. At every fourth house, where residents had agreed to participate in our study, we would grab a cup of mosquitoes from the van, peel off the lid, and set 50 or so insects free.

These were not your garden-variety mosquitoes. Each one was infected with a microbe called Wolbachia, a common bacterium that lives in insect cells. For our purposes, the most interesting characteristic of Wolbachia is that it appears to block the dengue virus from replicating in the tissues of mosquitoes. Because the virus cannot replicate, the insects do not transmit it to their victims, and the disease does not spread.

Infecting mosquitoes with a bacterium is a roundabout way to fight dengue, but we do it because otherwise the options are few. Dengue, nicknamed “breakbone fever” for the crippling pain it causes, infects 390 million people every year. Because there is no cure or treatment, the chief strategy has been to attack Aedes aegypti, the mosquito that transmits the virus. Yet common insecticides such as temephos have lost much of their effectiveness as mosquitoes have developed resistance. Bed nets are almost useless, too, because A. aegypti typically feed during the day. At present, one of the most promising tools for halting the spread of dengue—and perhaps malaria and other mosquito-borne illnesses—appears to be spreading Wolbachia among wild mosquitoes.

Wolbachia is not an obvious choice as a dengue fighter. It does not naturally occur in the mosquitoes that most often transmit dengue. We actually have had to infect those mosquitoes artificially, in the laboratory. In other words, we use Wolbachia to immunize the mosquitoes against dengue and then set them loose in the wild, where (we hope) those mosquitoes will pass the bacterium to their offspring. Wolbachia is largely benign for mosquitoes and the environment, although it may reduce the insects' egg production. But the potential benefits for humans are clear: if mosquitoes infected with Wolbachia become predominant in the wild, we expect dengue infection rates among people to drop.

Pest control

Mosquitoes are among the deadliest creatures on earth. Yellow fever, also transmitted by A. aegypti, took out more U.S. troops than enemy fire during the Spanish-American War in 1898. Malaria, transmitted by a parasite harbored in mosquitoes, killed approximately 627,000 people in 2012 alone. Now A. aegypti is rapidly spreading dengue around the globe. About half of the world's population is at risk of contracting the disease, according to the World Health Organization. A. aegypti, which is recognizable by the white stripes on its legs and the lyre pattern on its thorax, can breed in any pool of standing water, which makes it particularly hard to control. The mosquito is found in tropical and subtropical climates around the world—in Africa, the Americas, the Eastern Mediterranean, Southeast Asia and the Western Pacific. Dengue, however, does not naturally occur in these creatures: the mosquitoes get dengue from us.

The mechanism of dengue infection is simple. Female mosquitoes bite humans because they need the protein found in our blood to produce eggs. (Male mosquitoes do not bite.) If the mosquito bites someone with dengue—and then, after the virus's roughly eight- to 12-day replication period, bites someone else—it passes dengue into its next victim's bloodstream. Wolbachia, however, disrupts this process by preventing replication from ever taking place.

Wolbachia was first identified in 1924 during dissections of household mosquitoes. Interest in the bacterium waned until the 1970s, when researchers noticed that under certain circumstances, it could prevent mosquito eggs from hatching, which suggested the bacterium could be used for insect control. In the 1990s scientists learned that some strains of Wolbachia could also shorten insect life span, which presented another way to limit disease transmission by insects.

I was introduced to Wolbachia as a Ph.D. student in the mid-1980s. Back then I wondered if we could use it to stop mosquitoes from transmitting human diseases. If we could reduce the life spans of mosquitoes by even a modest amount, it could seriously reduce the ability of the insects to spread disease among humans.

The catch, of course, was Wolbachia's lack of affinity for A. aegypti. The bacterium is common in up to 60 percent of insect species—including some mosquitoes that bite humans—but the infection does not easily pass between species. The challenge was finding a way to transfer different strains of Wolbachia from another insect—the fruit fly—into this dengue-carrying mosquito. It was a tedious process that took us more than a decade.

How to infect mosquitoes

Imagine taking a knitting needle and poking it into a balloon. Next, you have to remove the needle without popping the balloon. That pretty well sums up the process of infecting mosquito eggs with Wolbachia. In the lab, my team uses microscopic needles to take the microbe from the fruit fly and inject it directly into young mosquito eggs. At first, like balloons pierced with knitting needles, the eggs would burst. We tried with many thousands of eggs before we were successful.
Once we managed to infect mosquito eggs without destroying them, we had other problems to solve. Wolbachia would often disappear after a generation or two of mosquito breeding, which meant there was no way the bacterium would spread in the wild the way we wanted it to. We eventually found that we had to condition the microbes before injecting them into mosquitoes—to get these bacteria, which were used to living in fruit flies, accustomed to their new hosts. To do so, we extracted Wolbachia from fruit flies and then grew it in mosquito cell lines. In 2005 we finally prevailed: we infected mosquitoes with Wolbachia and watched them pass the bacterium from generation to generation—13 in all. Since then, Wolbachia has flourished in all subsequent generations. As we expected, at least one strain of Wolbachia shortens the life of A. aegypti.
Yet it turns out that Wolbachia is even better at fighting dengue than we thought. For reasons we do not fully understand, the dengue virus has trouble growing in Wolbachia-infected mosquitoes. We figured this out a few years after successfully transplanting Wolbachia into A. aegypti, when separate work I had been part of revealed that in fruit flies the bacterium also blocks replication of the Drosophila C virus, which is deadly to flies. My team injected dengue directly into our Wolbachiamosquitoes, and to our delight, dengue no longer replicated in their bodies. We repeated the experiment a number of times—each time with dozens of mosquitoes—and discovered that our results were consistent.

These days we use a strain of Wolbachia that blocks dengue transmission but does nothing to shorten the mosquitoes' lives. After all, we want our mosquitoes to live as long as possible and lay as many Wolbachia-infected eggs as they can. We have known since my time as a graduate student that female mosquitoes infected with Wolbachia pass the bacterium on to nearly all of their offspring. It takes only a few generations after the introduction of Wolbachia before almost every mosquito in a population carries the bacterium.

One of our experiments in northern Australia showed that after releasing approximately 10 mosquitoes per house per week for 10 weeks, more than 80 percent of the wild mosquitoes in the area had Wolbachia—and they still had it when we tested them two months after we had stopped releasing mosquitoes. Because Wolbachia passes so well through successive generations, we should not have to do any repeat releases. Wolbachia should spread on its own.

Into the wild

Before we could release Wolbachia mosquitoes into the wild, we had to address a lot of concerns in the community. We spent months going door-to-door to ask permission to release mosquitoes near people's homes. We conducted formal informational meetings as well as impromptu chats outside shopping centers. Australian federal officials also checked our method for safety before approving the release of the infected mosquitoes.

To humans, Wolbachia poses no apparent threat. Our own lab experiments have found that the bacterium cannot be passed on to humans, because it is too big to travel down the mosquitoes' salivary duct and into the human bloodstream. We have also conducted safety tests looking for antibodies in human volunteers, but after three years of letting mosquitoes bite volunteers, the humans still have no sign of the microbe. Our lab staff and volunteers have frequently rolled up their sleeves and spent 15 minutes in the mosquito cages, allowing the insects to drink their fill.
There has been no sign that Wolbachia harms the environment, either. Since we started releasing mosquitoes with Wolbachia in 2011, we have been studying the animals and insects that encounter them, and our work has reaffirmed that the bacterium resides solely within the cells of insects and other arthropods. Moreover, we do not think that Wolbachia would survive even if it were to find a way into the bloodstream of humans or other mammals. Indeed, Wolbachia is already found in many other mosquito species, including a number that regularly bite people. Tests conducted on spiders and geckos that have eaten Wolbachia mosquitoes showed no ill effects from the exposure and no sign of the bacterium in the tissues of those animals.

Before the first Wolbachia mosquito releases in 2011, we commissioned an independent risk assessment by the Commonwealth Scientific and Industrial Research Organization (CSIRO),
Australia's national science agency. Teams of experts identified and evaluated potential hazards associated with the release of Wolbachia mosquitoes, ranging from possible ecological impacts to effects on communities. That agency scrutinized existing studies and interviewed experts in evolutionary biology. Tough issues were involved: changes in mosquito density, the possibility of evolution of the dengue virus, the nuisance of increasing numbers of biting mosquitoes and changes in the community's perceptions of the risks associated with dengue. But CSIRO's final report concluded that the release of Wolbachia mosquitoes would have negligible risk to people and the environment—the lowest possible rating.

Wolbachia goes global

In addition to the field trials we have been doing in Australia for the past four years, trials are under way in Vietnam and Indonesia. Last September we also started releasing the mosquitoes in Brazil. We have found that Wolbachia can establish itself in wild mosquito populations within small communities. Now we are going to attempt to do the same over larger areas. Scaling up our operations may require some tweaks in our methods. Rearing enough adult Wolbachia mosquitoes, for example, will be too labor-intensive. In Cairns, we are instead testing the effectiveness of putting Wolbachia mosquito eggs into the environment.

Meanwhile other researchers are developing alternative approaches to mosquito control. One entails releasing male mosquitoes that have been genetically modified so that the sperm cells of males carry a lethal gene. When those mosquitoes mate with females in the wild, their offspring die. This approach is innovative and potentially powerful, but it could also be costly. To be effective on a large scale, it could be necessary to constantly release modified mosquitoes; otherwise, unmodified mosquitoes from surrounding areas would move into the area and replenish the population. The use of transgenic mosquitoes also faces strong opposition from critics of genetic modification.

In contrast, the costs of Wolbachia-based dengue control are front-loaded: after the initial investment in bacterium-infected mosquitoes, the process takes care of itself. It could be a relatively inexpensive way to tackle dengue, which is especially important in the poor tropical countries where the disease is most prevalent. Another benefit of our approach is that it involves no gene modification—although it still took years to get off the ground because of the work necessary to assure communities of its safety.

We still have a significant hurdle ahead of us: measuring the reduction in dengue that occurs when we introduce Wolbachia into communities. This step will be difficult for several reasons. In the areas where we work, reliable data on dengue cases are largely nonexistent, and infection rates can vary widely from year to year. To firmly establish the effectiveness of our method, we will need to compare dengue rates in areas where we have released Wolbachia mosquitoes against those where we have not. Doing so will require taking lots of blood samples, which will be laborious.

Yet we believe the work will be worthwhile and not only for fighting dengue. These mosquitoes—or rather the microbes inside them—show promise against other diseases as well. We have seen evidence that Wolbachia may also reduce the ability of mosquitoes to transmit chikungunya, which first appeared in the mainland U.S. last July, and yellow fever. Researchers are also attempting to use Wolbachia-infected mosquitoes to slow the transmission of malaria and lymphatic filariasis, a profoundly disfiguring disease caused by worms.

The new observations are exciting. For the time being, however, our group will remain focused on evaluating the method against dengue. It is where we first started our research and where we are closest to seeing a real-world impact. One day, we hope, a mosquito bite will leave nothing more consequential than an itchy bump.