A Tiny but Mighty Helper Stops Mosquito Viruses in Their Tracks

ABOVE: Researchers join forces with a bacterial ally, Wolbachia, to combat mosquito-borne viruses in the field. © istock.com, 3DFOX

Luciano Moreira, a vector biologist and the project lead of the World Mosquito Program in Brazil, has always been driven by solving people’s problems. As a kid, he concocted blends of house-cleaning products to eliminate common home pests; in adulthood, Moreira channeled his problem-solving tendencies into finding innovative ways to tackle mosquito-borne pathogens for more than two decades. Now he tests these strategies to ease the burden of diseases that affect millions of people in his home country and around the world.   

From Plant Bugs to Human Bugs

Moreira worked on approaches to fight agricultural pests during his graduate studies at the Federal University of Viçosa. He focused on tomato leafminers, insects whose larvae feed on leaves and create white, winding trails called mines on their surfaces. Injured leaves tend to drop prematurely, and infested plants may lose most of their leaves, affecting fruit size and yield. By investigating plants that were resilient against leafminers, Moreira and his colleagues identified markers of resistance located on chromosome two of tomato plants.¹

After concluding his graduate studies, he pursued a postdoctoral position at the laboratory of molecular entomologist Marcelo Jacobs-Lorena, now an emeritus researcher at John Hopkins University. Jacobs-Lorena and his team were interested in using transgenic mosquitoes to control the transmission of the single-celled parasite Plasmodium, the causative agent of malaria. “We did lots of molecular biology, trying to find genes and promoters of the genes that would block Plasmodium inside the mosquitoes,” Moreira said.

They got the communities involved years before they were ready to actually do a release. That’s how these things need to be done, as opposed to developing the technology and then going out and saying, ‘hey, we want to do this. So, we’re going to talk to you before we do it.’ That’s not the way to do it.

 —Jason Rasgon, Pennsylvania State University

To alter the insect’s capacity to harbor the malaria parasite, the team first needed to identify promoters that could drive gene expression in a tissue- and stage-specific manner. Since mosquito-borne pathogens are transmitted through the insect’s bites, the team zoomed in on the arthropod’s gut to find those promoters. In their previous work, Jacobs-Lorena and his team found that blood meal stimulated the expression of the carboxypeptidase gene specifically in the midgut of mosquitoes.^2,3 To test if the promoter of this enzyme could be used to express antimalarial genes, Moreira and his colleagues microinjected Aedes aegypti mosquito embryos with constructs containing a luciferase reporter gene linked to a carboxypeptidase promoter region of either A. aegypti or Anopheles gambiae mosquitoes. They inserted the construct in the mosquito genome using transposons and assessed their expressions in different transgenic mosquitoes. The enzyme’s promoter drove the expression of the luciferase gene only in the mosquito gut, suggesting their potential use to control the expression of genes that would interfere with the development of the parasite in that tissue.⁴

The team next tested whether A. gambiae carboxypeptidase promoter could drive the expression of a synthetic gene designed to disrupt Plasmodium development in Anopheles mosquitoes.⁵ When they let wild type and transgenic mosquitoes feed on Plasmodium-infected mice, the researchers observed inhibition of the parasite development inside the transgenic insects.⁶ Additionally, many of the antimalarial mosquitoes were unable to transmit the parasite to uninfected mice. 

For this strategy to work against the malaria parasite, the transgenes should not impose a higher fitness cost on the mosquitoes, allowing them to survive, reproduce, and pass the transgenes on to subsequent generations.⁷ When they assessed the effects of the transgene on mosquito’s fitness, Moreira and his colleagues found that their survival, fertility, and fecundity remained unaffected.⁸ 

In the early 2000s, Moreira returned to Brazil and established a research group at the Oswaldo Cruz Foundation to continue studying transgenic mosquitoes and transgenes that could block malaria parasite transmission. Across the Pacific Ocean, at the University of Queensland, biologist Scott O’Neill, founder and chief executive officer of the World Mosquito Program, was investigating another potential strategy of vector control centered on the endosymbiotic bacteria Wolbachia. 

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A Mighty Endosymbiont Blocks Viruses

Wolbachia nests in the cells of around half of all arthropod species, and studies in the late 1990s suggested that some strains of the bacteria reduced the lifespan of their natural arthropod hosts.^9,10 O’Neill saw in this life-shortening effect an opportunity to use the endosymbiont as a tool for vector control. 

Since mosquitoes are important vectors of disease-causing pathogens, they were a clear target to test Wolbachia’s effects. O’Neill and his team first showed that the life-shortening Wolbachia strain originally identified in Drosophila melanogaster could inhabit the cells of a mosquito host.¹¹ The researchers then microinjected the mosquito-adapted endosymbiont in A. aegypti embryos to create mosquitoes that naturally harbored the bacterial strain. They found that the life-shortening Wolbachia reduced approximately by half the lifespan of female mosquitoes while inducing phenotypic changes that ensured the bacteria dissemination into the insect’s offspring.¹²

Moreira joined O’Neill’s group just as the researchers were concluding the arduous process of transfecting A. aegypti mosquitoes with the life-reducing Wolbachia strain. Around the same time, O’Neill was also exploring Wolbachia’s effects on fruit fly viruses in collaboration with University of Queensland molecular virologist Karyn Johnson. The researchers compared flies that harbored the endosymbiont with those that did not and found that the ones that did survived longer after exposure to deadly fruit fly viruses.¹³ These results, supported by others a few months after, suggested that Wolbachia could induce resistance to natural viral pathogens.¹⁴

O’Neill and Moreira wondered whether the Wolbachia-induced viral resistance would be generalizable to other insects, particularly those that transmit disease-causing pathogens to humans. To test this idea, the team exposed two groups of A. aegypti mosquitoes, those infected with the life-reducing bacterial strain and those without infection, to dengue and chikungunya viruses, the causative agents of two epidemiologically relevant mosquito-borne diseases. By assessing the presence of the viruses in different parts of the mosquitoes, they found that Wolbachia-infected A. aegypti had lower levels of the viruses’ genetic materials.¹⁵ “[When] we had the results, they were astonishing,” Moreira said.

Concurrently, the team also found that the endosymbiont induced behavioral changes in A. aegypti. In older mosquitoes, which are the primary transmitters of viruses, Wolbachia infection impaired the insect’s capacity to successfully obtain a blood meal, which could also diminish their ability to transmit disease-causing viruses.^16,17

For O’Neill, having Moreira in his lab at that time was a great experience. “He’s a very gentle guy, very curious, very hardworking,” O’Neill said. “We worked well together and closely for all the time he was in the group.”

Excited about their findings on the effects of Wolbachia in arboviruses transmitted by mosquitoes, O’Neill and Moreira saw the endosymbiont as a promising biological control strategy. Yet, the Wolbachia strain they were studying in the lab, while effective at blocking human pathogens from infecting their mosquito hosts, imposed high fitness costs on the arthropod vectors, potentially hindering the strain’s ability to invade a wild mosquito population. 

To circumvent that limitation, the researchers looked for another Wolbachia strain and found that one called wMel had limited impact on A. aegypti fitness as it did not significantly alter the mosquito’s fecundity, egg viability, and lifespan.¹⁸ Additionally, the bacterial strain could invade a new, uninfected mosquito population in cage experiments, and when wMel-carrying mosquitoes were fed dengue virus-infected blood, there was also strong inhibition of virus replication inside the vectors. 

With the new strain in hand, the scientists felt ready to test Wolbachia’s biocontrol capabilities outside the laboratory.

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World Mosquito Program: Controlling Disease Vectors in the Field

Although researchers first identified dengue virus more than 70 years ago, the impact of dengue fever has not dwindled. It is estimated that half of the world’s population is at risk of contracting this disease, with dengue infections being a major health problem in many Latin American and Asian countries.¹⁹ “We haven’t really had effective tools for many years, and what we currently do isn’t working,” O’Neill said. “We need new approaches.”

In the early 2010s, O’Neill and his team were ready to put Wolbachia’s biocontrol potential to the test. After two years of community engagement work and preparation, the researchers released Wolbachia-infected mosquitoes in two locations in Australia and found that the wMel Wolbachia strain was able to invade natural mosquito populations and that mosquitoes collected from these sites resisted dengue virus infections and carried Wolbachia even after a year of the initial releases.^20,21 

There are other approaches that are being developed at the moment. We have vaccines coming on board, we have new vector control tools. Hopefully with all of these tools, we can make a positive impact on the viruses this mosquito transmits.

 —Scott O’Neill, World Mosquito Program

Back in Brazil, Moreira was excited about those promising field results and wanted to bring the Wolbachia-based control approach to his home country. In 2012, he became the project leader of the Eliminate Dengue Program, now World Mosquito Program, in Brazil, and started working to gain support from national research institutions and health authorities to begin small-scale trials in the country. Before releasing the Wolbachia-carrying mosquitoes, which Brazilians later nicknamed “wolbitos”, Moreira’s team backcrossed the Australian wMel mosquitoes with wild type Brazilian A. aegypti to generate a Wolbachia-infected line with the genetic background of the local vectors. The new mosquitoes showed the main Wolbachia-related features needed to disseminate in a population: high vertical transmission from females to their offspring and strong cytoplasmic incompatibility, a reproductive manipulation associated with Wolbachia that gives Wolbachia-infected females an advantage in a given population.²² 

Generating wolbitos with a local genetic background also proved necessary as the researchers found that the native A. aegypti populations had developed insecticide resistance due to the overuse of these products by local residents.²³ 

Following these initial trials, the researchers began large-scale wolbito deployments in other locations in the state of Rio de Janeiro. In one of these releases conducted in Niterói, a city with a population of about 400,000 residents, the team found that the release of Wolbachia-carrying mosquitoes was associated with a decrease in the incidence of Aedes-borne diseases, including dengue, even though the endosymbiont did not penetrate the mosquito populations in the different release zones to the same extent.²⁴ 

“When you do field releases, it is not homogeneous,” Moreira explained. “In the field there are lots of factors that could influence Wolbachia establishment.” A better understanding of these factors is central to Jason Rasgon, an entomologist and disease epidemiologist at Pennsylvania State University, who has followed the work of the World Mosquito Program over the years. “What are the parameters and things that actually govern why it’s blocking here, but it’s not blocking here or why it’s invading here, why it’s not invading here,” said Rasgon, who cited the discovery of the insecticide effect on the mosquitoes as an example of the importance of identifying these factors. 

Rasgon also noted that the involvement of the community has played a key role in the World Mosquito Program’s widespread implementation in many different countries. “[Their community engagement] wasn’t an afterthought; it was really baked into the foundation of their strategy,” he said. “They got the communities involved years before they were ready to actually do a release. That’s how these things need to be done, as opposed to developing the technology and then going out and saying, ‘hey, we want to do this. So, we’re going to talk to you before we do it.’ That’s not the way to do it.”

Even though Wolbachia’s virus-blocking effects were described more than 10 years ago, the mechanisms behind it are still poorly understood, noted William Sullivan, a cell biologist at the University of California, Santa Cruz, who has delved into the biology of Wolbachia for more than two decades. “The million-dollar question is the mechanism of virus protection, and there are lots of models out there,” said Sullivan, who emphasized that more research efforts should be devoted to uncovering the basic molecular and cellular biology of Wolbachia interactions with their hosts, as well as to examining the effects of long-term Wolbachia exposure on the potential selection of resistant dengue virus strains.   

For O’Neill, Moreira has been a central character in the development of the World Mosquito Program strategy. “Luciano has been heavily involved in scaling up and taking those laboratory results and putting them into the field and showing that it can be deployed in complex settings,” said O’Neill, who is also hopeful that this strategy will contribute to easing the burden of many mosquito-borne diseases in Brazil and other places in the world. “There are other approaches that are being developed at the moment. We have vaccines coming on board, we have new vector control tools. Hopefully with all these tools, we can make a positive impact on the viruses this mosquito transmits.”