Background: Aedes aegypti mosquitoes infected with Wolbachia pipientis ( w Mel strain) have reduced potential to transmit dengue viruses. Methods: We conducted a cluster randomised trial of deployments of w Mel-infected Ae. aegypti for control of dengue in Yogyakarta City, Indonesia. Twenty-four geographic clusters were randomly allocated to receive w Mel deployments as an adjunct to local mosquito control measures; or to continue with local mosquito control measures only. A test-negative design was used to measure efficacy. Study participants were persons 3–45 years old attending primary care clinics with acute undifferentiated fever. Laboratory testing identified virologically-confirmed dengue cases and test-negative controls. The primary endpoint was efficacy of w Mel in reducing the incidence of symptomatic, virologically-confirmed dengue, caused by any dengue virus serotype. Results: Following successful introgression of w Mel in intervention clusters, 8144 participants were enrolled; 3721 from w Mel-treated clusters and 4423 from untreated clusters. In the ITT analysis virologically-confirmed dengue occurred in 67 of 2905 (2.3%) participants in the w Mel-treated and 318 of 3401 (9.4%) in the untreated arm (OR 0.23, 95% CI, 0.15 to 0.35; P=0.004): protective efficacy of 77.1% (95% CI, 65.3 to 84.9). Protective efficacy was similar for the four serotypes. Hospitalisation for virologically-confirmed dengue was less frequent for participants resident in the w Mel-treated (13/2905, 2.8%) compared to the untreated arm (102/3401, 6.3%): protective efficacy 86.2% (95% CI, 66.2 to 94.3) Conclusions: w Mel introgression into Ae. aegypti populations was efficacious in reducing the incidence of symptomatic dengue, and also led to fewer dengue hospitalisations. Trial registration number: ClinicalTrials.gov Identifier: NCT03055585 and INA-A7OB6TW

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachiarevealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool forWolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction ofWolbachia with its host.

Dengue-suppressing Wolbachia strains are promising tools for arbovirus control, particularly as they have the potential to self-spread following local introductions. To test this, we followed the frequency of the transinfected Wolbachia strain wMel through Ae. aegypti in Cairns, Australia, following releases at 3 nonisolated locations within the city in early 2013. Spatial spread was analysed graphically using interpolation and by fitting a statistical model describing the position and width of the wave. For the larger 2 of the 3 releases (covering 0.97 km2 and 0.52 km2), we observed slow but steady spatial spread, at about 100–200 m per year, roughly consistent with theoretical predictions. In contrast, the smallest release (0.11 km2) produced erratic temporal and spatial dynamics, with little evidence of spread after 2 years. This is consistent with the prediction concerning fitness-decreasing Wolbachia transinfections that a minimum release area is needed to achieve stable local establishment and spread in continuous habitats. Our graphical and likelihood analyses produced broadly consistent estimates of wave speed and wave width. Spread at all sites was spatially heterogeneous, suggesting that environmental heterogeneity will affect large-scale Wolbachia transformations of urban mosquito populations. The persistence and spread of Wolbachia in release areas meeting minimum area requirements indicates the promise of successful large-scale population transformation.

Background: A number of new technologies are under development for the control of mosquito transmitted viruses, such as dengue, chikungunya and Zika that all require the release of modified mosquitoes into the environment. None of these technologies has been able to demonstrate evidence that they can be implemented at a scale beyond small pilots. Here we report the first successful citywide scaled deployment of Wolbachia in the northern Australian city of Townsville. Methods: The wMel strain of Wolbachia was backcrossed into a local Aedes aegypti genotype and mass reared mosquitoes were deployed as eggs using mosquito release containers (MRCs). In initial stages these releases were undertaken by program staff but in later stages this was replaced by direct community release including the development of a school program that saw children undertake releases. Mosquito monitoring was undertaken with Biogents Sentinel (BGS) traps and individual mosquitoes were screened for the presence of Wolbachia with a Taqman qPCR or LAMP diagnostic assay. Dengue case notifications from Queensland Health Communicable Disease Branch were used to track dengue cases in the city before and after release. Results: Wolbachia was successfully established into local Ae. aegypti mosquitoes across 66 km 2 in four stages over 28 months with full community support.  A feature of the program was the development of a scaled approach to community engagement. Wolbachia frequencies have remained stable since deployment and to date no local dengue transmission has been confirmed in any area of Townsville after Wolbachia has established, despite local transmission events every year for the prior 13 years and an epidemiological context of increasing imported cases. Conclusion: Deployment of Wolbachia into Ae. aegypti populations can be readily scaled to areas of ~60km 2 quickly and cost effectively and appears in this context to be effective at stopping local dengue transmission

Wolbachia pipientis is an endosymbiotic bacterium estimated to chronically infect between 40–75% of all arthropod species. Aedes aegypti, the principle mosquito vector of dengue virus (DENV), is not a natural host of Wolbachia. The transinfection of Wolbachia strains such as wAlbB, wMel and wMelPop-CLA into Ae. aegypti has been shown to significantly reduce the vector competence of this mosquito for a range of human pathogens in the laboratory. This has led to wMel-transinfected Ae. aegypti currently being released in five countries to evaluate its effectiveness to control dengue disease in human populations. Here we describe the generation of a superinfected Ae. aegypti mosquito line simultaneously infected with two avirulent Wolbachia strains, wMel and wAlbB. The line carries a high overall Wolbachia density and tissue localisation of the individual strains is very similar to each respective single infected parental line. The superinfected line induces unidirectional cytoplasmic incompatibility (CI) when crossed to each single infected parental line, suggesting that the superinfection would have the capacity to replace either of the single constituent infections already present in a mosquito population. No significant differences in fitness parameters were observed between the superinfected line and the parental lines under the experimental conditions tested. Finally, the superinfected line blocks DENV replication more efficiently than the single wMel strain when challenged with blood meals from viremic dengue patients. These results suggest that the deployment of superinfections could be used to replace single infections and may represent an effective strategy to help manage potential resistance by DENV to field deployments of single infected strains.

Aedes mosquito-transmitted diseases, such as dengue, Zika and chikungunya, are becoming major global health emergencies while old threats, such as yellow fever, are re-emerging. Traditional control methods, which have focused on reducing mosquito populations through the application of insecticides or preventing breeding through removal of larval habitat, are largely ineffective, as evidenced by the increasing global disease burden. Here, we review novel mosquito population reduction and population modification approaches with a focus on control methods based on the release of mosquitoes, including the release of Wolbachia-infected mosquitoes and strategies to genetically modify the vector, that are currently under development and have the potential to contribute to a reversal of the current alarming disease trends.

BackgroundIntroduced Wolbachia bacteria can influence the susceptibility of Aedes aegypti mosquitoes to arboviral infections as well as having detrimental effects on host fitness. Previous field trials demonstrated that the wMel strain of Wolbachia effectively and durably invades Ae. aegypti populations. Here we report on trials of a second strain, wMelPop-PGYP Wolbachia, in field sites in northern Australia (Machans Beach and Babinda) and central Vietnam (Tri Nguyen, Hon Mieu Island), each with contrasting natural Ae. aegypti densities.MethodsMosquitoes were released at the adult or pupal stages for different lengths of time at the sites depending on changes in Wolbachia frequency as assessed through PCR assays of material collected through Biogents-Sentinel (BG-S) traps and ovitraps. Adult numbers were also monitored through BG-S traps. Changes in Wolbachia frequency were compared across hamlets or house blocks.ResultsReleases of adult wMelPop-Ae. aegypti resulted in the transient invasion of wMelPop in all three field sites. Invasion at the Australian sites was heterogeneous, reflecting a slower rate of invasion in locations where background mosquito numbers were high. In contrast, invasion across Tri Nguyen was relatively uniform. After cessation of releases, the frequency of wMelPop declined in all sites, most rapidly in Babinda and Tri Nguyen. Within Machans Beach the rate of decrease varied among areas, and wMelPop was detected for several months in an area with a relatively low mosquito density.ConclusionsThese findings highlight challenges associated with releasing Wolbachia-Ae. aegypti combinations with low fitness, albeit strong virus interference properties, as a means of sustainable control of dengue virus transmission.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1174-x) contains supplementary material, which is available to authorized users.

BackgroundTraditional vector control approaches such as source reduction and insecticide spraying have limited effect on reducing Aedes aegypti population. The endosymbiont Wolbachia is pointed as a promising tool to mitigate arbovirus transmission and has been deployed worldwide. Models predict a rapid increase on the frequency of Wolbachia-positive Ae. aegypti mosquitoes in local settings, supported by cytoplasmic incompatibility (CI) and high maternal transmission rate associated with the wMelBr strain.Methodology/principle findingsWolbachia wMelBr strain was released for 20 consecutive weeks after receiving >87% approval of householders of the isolated community of Tubiacanga, Rio de Janeiro. wMelBr frequency plateued~40% during weeks 7–19, peaked 65% but dropped as releases stopped. A high (97.56%) maternal transmission was observed. Doubling releases and deploying mosquitoes with large wing length and low laboratory mortality produced no detectable effects on invasion trend. By investigating the lab colony maintenance procedures backwardly, pyrethroid resistant genotypes in wMelBr decreased from 68% to 3.5% after 17 generations. Therefore, we initially released susceptible mosquitoes in a local population highly resistant to pyrethroids which, associated with the over use of insecticides by householders, ended jeopardizing Wolbachia invasion. A new strain (wMelRio) was produced after backcrossing wMelBr females with males from field to introduce mostly pyrethroid resistance alleles. The new strain increased mosquito survival but produced relevant negative effects on Ae. aegypti fecundity traits, reducing egg clutche size and egg hatch. Despite the cost on fitness, wMelRio successful established where wMelBr failed, revealing that matching the local population genetics, especially insecticide resistance background, is critical to achieve invasion.Conclusions/significanceLocal householders support was constantly high, reaching 90% backing on the second release (wMelRio strain). Notwithstanding the drought summer, the harsh temperature recorded (daily average above 30°C) did not seem to affect the expression of maternal transmission of wMel on a Brazilian background. Wolbachia deployment should match the insecticide resistance profile of the wild population to achieve invasion. Considering pyrethroid-resistance is a widely distributed phenotype in natural Ae. aegypti populations, future Wolbachia deployments must pay special attention in maintaining insecticide resistance in lab colonies for releases.