Could Wolbachia be an alternative to dengue-fighting GMOsquitos?

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By all accounts, dengue fever has returned to the US. The symptoms of the disease are reminiscent of the flu: fever, chills, vomiting, and headache, while the characteristically severe muscle and joint paint lends it its other name–“breakback fever.” Under rare circumstances dengue fever can turn deadly, morphing into either dengue hemorrhagic fever or dengue shock syndrome. There are four types (serotypes) of dengue viruses that cause dengue fever, all of which are spread by a mosquito’s bite.

Worldwide, dengue ranks second behind malaria in terms of mosquito-borne diseases with an estimated 50-100 million cases a year. The World Health Organiziation reports that dengue is endemic in at least 100 countries, representing about 40% of the world’s population. And dengue is spreading as mosquitoes that can transmit the disease invade new territory. In addition, recent research suggests that dengue virus has the sinister ability to cause changes in gene expression in the mosquito to suit the virus’ needs (1). This serves not only to make conditions for viral replication more favorable but also to alter the mosquito’s feeding behavior–making viral transmission to a human host more likely.

Although not as deadly as malaria, dengue is still a worldwide public health burden because of the hospitalization required to treat patients. Furthermore, there are no antiviral drugs or vaccines specific for treating or preventing dengue fever. Last year, a clinical trial for a dengue vaccine was conducted in Thailand with high hopes, but yielded mixed results:

“The vaccine proved fairly effective in preventing illness from three serotypes, cutting infection rates by 55.6% for serotype 1, 75.3% for serotype 3, and 100% for serotype 4 after three injections. But it provided near-zero protection against dengue serotype 2, which is the most prevalent one in the region and is most responsible for severe illness throughout the world.”

Aedes aegypti biting human

Aedes aegypti biting human

In the US, the primary dengue-spreading culprit is Aedes aegypti, a non-native, invasive species of mosquitos brought here most likely by slave ships from Africa. Aedes albopictus, also known as the Asian Tiger mosquito, is a more recent mosquito migrant from Asia and can also transmit dengue, albeit to a lesser degree. Southern US states have been particularly accommodating in terms of climate and habitat to both species, but they have also been found as far north as Chicago and New York. Because no vaccine exists, prevention is primarily aimed at eliminating mosquitoes through pesticide spraying. But this carries with it the particularly thorny problem of affecting other insects and animals. And there’s always the specter of insecticide resistance, which as has been reported for  A. aegypti populations on the island of Martinique (2).


Aedes albopictus (Asian Tiger mosquito)

Florida, however, is exploring a different approach to rid the state of their mosquito problem. Last December, mosquito control officials requested FDA approval to use genetically-modified (GM) mosquitoes to eradicate A. aegypti populations in the Florida Keys. These altered A. aegypti  mosquitoes, engineered by British biotech company Oxitec and given the bleak name OX513A, carry a gene that when passed on will kill the next generation of mosquitoes. The protein that is produced from this gene is lethal to mosquito larvae. The gene has also been engineered, however, so that the drug tetracycline can shut down the production of the deadly protein. This allows scientists to raise the mosquitoes in the lab by adding tetracycline to the diet of the larvae. Without a source of tetracycline in the wild, however, any mosquito offspring that inherits the gene will live not beyond the larval stage.

Aedes aegypti larva

Aedes aegypti larva

So, the idea here is to flood the market with genetically-modified male mosquitoes that will mate with females in the wild. The resulting offspring that inherit the lethal gene from their GM fathers will die leading to a population crash. While Oxitec calls this strategy Release of Insects carrying a Dominant Lethal (RIDL), you could think of the males as being effectively “sterile.” In 2010, field tests of RIDL conducted in the Cayman Islands demonstrated that the strategy was effective in driving down the island population of A. aegypti by 80% (3).

The idea for RIDL has its roots in an earlier technique involving radiation to sterilize mosquitoes. But radiation is inconvenient because every generation has to be sterilized and the process often left adult mosquitoes too weak to compete against fertile mosquitoes in the wild. With RIDL, however, every adult born is inherently “sterile.” RIDL also provides an advantage over insecticides because its effects are species-specific since OX513A can only mate with A. egypti. Furthermore, only males are released–it’s the females that are the biters and spreaders of disease.

The proposal, however, is being met with stiff resistance. Many residents of Key West and environmentalists are uncomfortable with releasing millions of genetically-modified mosquitoes into their backyards. In many ways, concerns over the GM mosquitoes mirror those over GM foods. Environmentalists are uneasy about the ecological consequences of both introducing GM mosquitoes and eradicating entire mosquito populations (keep in mind, both A. aegypti and A. albopictus are species not native to the US). And as one Key West resident quoted in the New Yorker bluntly put it, “You are not going to cram something down my throat that I don’t want. I am no guinea pig.”


Transmission electron micrograph of Wolbachia within an insect cell

If public opinion can’t be turned, however, there is perhaps an alternative. Enter Wolbachia, a genus of symbiotic bacteria that can rapidly and stably infect insects–an infection that is passed down from mother to offspring. One particular Wolbachia strain, wMel, which naturally infects Drosophila (fruit fly) but not A. aegypti, has been of interest because it protects Drosophila against infection by certain RNA viruses. Since the dengue viruses are of the RNA variety, this raised the possibility of using Wolbachia as a means to prevent dengue virus infection in mosquitoes.

To test this hypothesis, a team of scientists recently established an A. aegypti mosquito population infected with the wMel strain of Wolbachia (4). The scientists then fed dengue virus-contaminated blood to the wMel-infected mosquitoes and their Wolbachiauninfected counterparts. The dengue virus was allowed to replicate in the mosquitos for fourteens days after feeding and then the amount of dengue infection was determined by measuring the amount of dengue viral nucleic acid in both groups of mosquitoes. As it turns out, the wMel-infected mosquitoes were protected against subsequent infection by dengue virus. The scientists found that the amount of dengue viral nucleic acid in the bodies of wMel-infected mosquitoes was 1500 fold fewer than in the Wolbachia-uninfected mosquitoes.

The scientists also measured the amount of dengue in mosquito saliva since dengue virus is spread through the saliva of mosquitoes when they bite humans. Dengue was found in only 4.2% of saliva samples taken from the wMel-infected mosquitoes compared to 80.2% of saliva samples taken from “unprotected” mosquitoes. The wMel strain has also been found to similarly block dengue infection in the Asian Tiger mosquito (A. albopictus) (5). Interestingly, infection by the closely related Wolbachia strain, wMelPop, has been shown to also have the dual effect of reducing both the lifespan of A. aegypti and its blood-feeding behavior–effects that could dramatically reduce the transmission of dengue (6).

The use of a Wolbachia infection-based strategy in controlling the spread of dengue in the US, however, does come with its own set of questions and concerns. It is not clear exactly how Wolbachia guards mosquitoes from dengue infection, but studies indicate that Wolbachia infection seemingly boosts or manipulates the mosquito’s immune system to interfere with replication of the dengue virus (7 & 8). Furthermore, protection against dengue virus appears to be dependent on the extent of Wolbachia infection in the mosquito (9). For instance, the wAlbB strain of Wolbachia, which naturally infects A. albopictus, does not provide A. albopictus any protection against dengue. However, scientists have found that when wAlbB infects A. aegypti it can prevent dengue infection in its newly-infected host species. The key appears to be that wAlbB is capable of surviving in higher numbers in its non-native host, A. aegypti, than it can in its normal host, A. albopictus. This suggests that Wobachia infection must reach a critical, minimum level in order to guard against dengue infection.

One potential explanation for the low level of wAlbB infection in A. albopictus is that A. albopictus, as wAlbB’s natural host, has had sufficient time to adapt to wAlbB and keep the level of infection low. Therefore, it’s possible that over time, as a mosquito species becomes adapted to Wolbachia infection, the corresponding protection against dengue  may wear off. There is also the question of how to replace wAlbB infections in A. albopictus with an infection by a dengue-protective Wolbachia strain. In the lab, Wolbachia infection can be cleared by antibiotics, but that may not be practical in the field. Lastly, since the research cited here have tested Wolbachia protection against only the serotype 2 dengue virus, the ability of Wolbachia infection to protect against the other 3 dengue serotypes still needs to be studied. This is where the OX513A strategy has the upper hand since it eradicates the mosquito while paying no mind to the serotype of the virus being carried.

In addition to balancing the pros and cons of either strategy, it’s also important to evaluate the risk of a future dengue outbreak. Are we overestimating the threat of dengue fever? The relatively low number of recent dengue fever cases in the US compared to figures in countries where dengue is endemic certainly gives that impression. It wouldn’t come as surprise to me, either, if some of the objections to using OX513A in Key West stems from the perception that dengue isn’t a real danger to local residents. The pieces for a larger outbreak, however, are in place. As Maryn Mckenna writes:

“Could more dengue outbreaks happen? To spark one, you need three things. First, imported virus: check. Second, a population with no immunity. The United States has that, since dengue was last widespread in the 1940s. And third, mosquitoes that can transmit it. Those are already widespread.”

And one of the strategies discussed here may end up being instrumental in preventing such an outbreak of dengue from happening in the US.

1) Sim S, Ramirez JL, Dimopoulos G (2012) Dengue Virus Infection of the Aedes aegypti Salivary Gland and Chemosensory Apparatus Induces Genes that Modulate Infection and Blood-Feeding Behavior. PLoS Pathog 8(3): e1002631. doi:10.1371/journal.ppat.1002631

2) Marcombe S, Mathieu RB, Pocquet N, Riaz M-A, Poupardin R, et al. (2012) Insecticide Resistance in the Dengue Vector Aedes aegypti from Martinique: Distribution, Mechanisms and Relations with Environmental Factors. PLoS ONE 7(2): e30989. doi:10.1371/journal.pone.0030989

3) Harris AF, McKemey AR, Nimmo D, Curtis Z, Black I, Morgan SA, Oviedo MN, Lacroix R, Naish N, Morrison NI, Collado A, Stevenson J, Scaife S, Dafa’alla T, Fu G, Phillips C, Miles A, Raduan N, Kelly N, Beech C, Donnelly CA, Petrie WD, Alphey L. Successful suppression of a field mosquito population by sustained release of engineered male mosquitoes. Nat Biotechnol. 2012 Sep;30(9):828-30. doi: 10.1038/nbt.2350. PubMed PMID: 22965050.

4) Walker T, Johnson PH, Moreira LA, Iturbe-Ormaetxe I, Frentiu FD, McMeniman CJ, Leong YS, Dong Y, Axford J, Kriesner P, Lloyd AL, Ritchie SA, O’Neill SL, Hoffmann AA. The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature. 2011 Aug 24;476(7361):450-3. doi: 10.1038/nature10355. PubMed PMID: 21866159.

5) Blagrove MS, Arias-Goeta C, Failloux AB, Sinkins SP. Wolbachia strain wMel induces cytoplasmic incompatibility and blocks dengue transmission in Aedes albopictus. Proc Natl Acad Sci U S A. 2012 Jan 3;109(1):255-60. doi: 10.1073/pnas.1112021108. Epub 2011 Nov 28. PubMed PMID: 22123944; PubMed Central PMCID: PMC3252941.

6) Turley AP, Moreira LA, O’Neill SL, McGraw EA (2009) Wolbachia Infection Reduces Blood-Feeding Success in the Dengue Fever Mosquito, Aedes aegypti.PLoS Negl Trop Dis 3(9): e516. doi:10.1371/journal.pntd.0000516

7) Rancès E, Ye YH, Woolfit M, McGraw EA, O’Neill SL (2012) The Relative Importance of Innate Immune Priming in Wolbachia-Mediated Dengue Interference.PLoS Pathog 8(2): e1002548. doi:10.1371/journal.ppat.1002548

8)Pan X, Zhou G, Wu J, Bian G, Lu P, Raikhel AS, Xi Z. Wolbachia induces reactive oxygen species (ROS)-dependent activation of the Toll pathway to control dengue virus in the mosquito Aedes aegypti. Proc Natl Acad Sci U S A. 2012 Jan 3;109(1):E23-31. doi: 10.1073/pnas.1116932108. Epub 2011 Nov 28. PubMed PMID: 22123956; PubMed Central PMCID: PMC3252928.

9) Lu P, Bian G, Pan X, Xi Z (2012) Wolbachia Induces Density-Dependent Inhibition to Dengue Virus in Mosquito Cells. PLoS Negl Trop Dis 6(7): e1754.doi:10.1371/journal.pntd.0001754

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