The AWED trial (Applying Wolbachia to Eliminate Dengue) to assess the efficacy of Wolbachia-infected mosquito deployments to reduce dengue incidence in Yogyakarta, Indonesia: study protocol for a cluster randomised controlled trial.

BACKGROUND: Dengue and other arboviruses transmitted by Aedes aegypti mosquitoes, including Zika and chikungunya, present an increasing public health challenge in tropical regions. Current vector control strategies have failed to curb disease transmission, but continue to be employed despite the absence of robust evidence for their effectiveness or optimal implementation. The World Mosquito Program has developed a novel approach to arbovirus control using Ae. aegypti stably transfected with Wolbachia bacterium, with a significantly reduced ability to transmit dengue, Zika and chikungunya in laboratory experiments. Modelling predicts this will translate to local elimination of dengue in most epidemiological settings. This study protocol describes the first trial to measure the efficacy of Wolbachia in reducing dengue virus transmission in the field. METHODS/DESIGN: The study is a parallel, two-arm, non-blinded cluster randomised controlled trial conducted in a single site in Yogyakarta, Indonesia. The aim is to determine whether large-scale deployment of Wolbachia-infected Ae. aegypti mosquitoes leads to a measurable reduction in dengue incidence in treated versus untreated areas. The primary endpoint is symptomatic, virologically confirmed dengue virus infection of any severity. The 26 km2 study area was subdivided into 24 contiguous clusters, allocated randomly 1:1 to receive Wolbachia deployments or no intervention. We use a novel epidemiological study design, the cluster-randomised test-negative design trial, in which dengue cases and arbovirus-negative controls are sampled concurrently from among febrile patients presenting to a network of primary care clinics, with case or control status classified retrospectively based on the results of laboratory diagnostic testing. Efficacy is estimated from the odds ratio of Wolbachia exposure distribution (probability of living in a Wolbachia-treated area) among virologically confirmed dengue cases compared to test-negative controls. A secondary per-protocol analysis allows for individual Wolbachia exposure levels to be assessed to account for movements outside the cluster and the heterogeneity in local Wolbachia prevalence among treated clusters. DISCUSSION: The findings from this study will provide the first experimental evidence for the efficacy of Wolbachia in reducing dengue incidence. Together with observational evidence that is accumulating from pragmatic deployments of Wolbachia in other field sites, this will provide valuable data to estimate the effectiveness of this novel approach to arbovirus control, inform future cost-effectiveness estimates, and guide plans for large-scale deployments in other endemic settings. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT03055585 . Registered on 14 February 2017

Étude des déterminants génétiques bactériens impliqués dans les interactions Wolbachia-hôtes (analyse fonctionnelle du système de sécrétion de type IV)

Wolbachia est une bactérie intra-cellulaire stricte qui infecte certains nématodes filaires et un grand nombre d'arthropodes. Chez la plupart des insectes, Wolbachia est considérée comme parasite de la reproduction. Néanmoins, chez l'hyménoptère Asobara tabida, la bactérie est devenue obligatoire à l'accomplissement de l'ovogenèse. Malgré l'intétêt scientifique porté à Wolbachia, les mécanismes moléculaires impliqués dans les interactions avec ses hôtes peu connus. Le séquençage des génomes de souches Wolbachia a mis en évidence la présence des gènes codant le système de sécrétion de type IV ( SST4) souvent engagé dans le dialogue bactéries-hôtes. Dans ce contexte, le travail de cette thèse consistait à développer des outils permettant la manipulation de la bactérie puis à étudier la fonctionnalité du SST4 chez Wolbachia. Un anticorps ciblant la protéine VirB6 du SST4 a été produit et a permis de confirmer la fontionnalité du translocon in cellulo et in insectaWolbachia is a strict intracellular bacterium which infects certain filarial nematodes and a great number of arthropods. For the marjority of insects, Wolbachia is considered a reproductive parasite. Nevertheless, in hymenoptera Asobara tabida, the bacterium became obligatory for insect oogenesis. Despite the scientific interest carried with Wolbachia, the molecular mechanisms implied in the interactions with its hosts are poorly knwn. The sequencing Wolbachia genomes highlighted the presence of genes encoding the type IV secretion system ( T4SS) often involved in the dialogue between bacteria and hosts. In the context, this study consisted in developing tools to handle the bacterium, then to decipher the functionality of the T4SS in Wolbachia. Polyclonal antibodies raised against the VirB6 protein was produced and confirmed the functionality of the T4SS translocon in cellulo and in insectaLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Competition for amino acids between Wolbachia and the mosquito host, Aedes aegypti

The endosymbiont Wolbachia represents a promising method of dengue control, as it reduces the ability of the primary vector, the mosquito Aedes aegypti, to transmit viruses. When mosquitoes infected with the virulent Wolbachia strain wMelPop are fed non-human blood, there is a drastic reduction in mosquito fecundity and egg viability. Wolbachia has a reduced genome and is clearly dependent on its host for a wide range of nutritional needs. The fitness defects seen in wMelPop-infected A. aegypti could be explained by competition between the mosquito and the symbiont for essential blood meal nutrients, the profiles of which are suboptimal in non-human blood. Here, we examine cholesterol and amino acids as candidate molecules for competition, as they have critical roles in egg structural development and are known to vary between blood sources. We found that Wolbachia infection reduces total cholesterol levels in mosquitoes by 15-25 %. We then showed that cholesterol supplementation of a rat blood meal did not improve fecundity or egg viability deficits. Conversely, amino acid supplementation of sucrose before and after a sheep blood meal led to statistically significant increases in fecundity of approximately 15-20 eggs per female and egg viability of 30-40 %. This mosquito system provides the first empirical evidence of competition between Wolbachia and a host over amino acids and may suggest a general feature of Wolbachia-insect associations. These competitive processes could affect many aspects of host physiology and potentially mosquito fitness, a key concern for Wolbachia-based mosquito biocontrol

The Toll and Imd pathways are not required for Wolbachia-mediated dengue virus interference

Wolbachia blocks dengue virus replication in Drosophila melanogaster as well as in Aedes aegypti. Using the Drosophila model and mutations in the Toll and Imd pathways, we showed that neither pathway is required for expression of the dengue virusblocking phenotype in the Drosophila host. This provides additional evidence that the mechanistic basis of Wolbachia-mediated dengue virus blocking in insects is more complex than simple priming of the host insect innate immune system

A native Wolbachia endosymbiont does not limit dengue virus infection in the mosquito Aedes notoscriptus (Diptera: Culicidae)

The endosymbiotic bacterium Wolbachia pipientis infects many species of insects and has been transinfected into the mosquito Aedes aegypti (L.), the primary vector of dengue virus (DENV). Recently, it has been shown that Wolbachia blocks the replication and transmission of RNA viruses, such as DENV, in a number of mosquito species including Ae. aegypti and Aedes albopictus (Skuse), which is naturally infected with Wolbachia and considered a secondary vector for DENV. The mosquito species Aedes notoscriptus (Skuse) is highly prevalent in Australia, including in areas where DENV outbreaks have been recorded. The mosquito has been implicated in the transmission of Ross River and Barmah Forest viruses, but not DENV. We investigated whether Wolbachia naturally infects this mosquito species and whether it has an impact on the ability of Ae. notoscriptus to transmit DENV. We show, for the first time, that Ae. notoscriptus is naturally infected with a strain of Wolbachia that belongs to supergroup B and is localized only in the ovaries. However, Wolbachia infection in Ae. notoscriptus did not induce resistance to DENV and had no effect on overall DENV infection rate or titer. The presence of a native Wolbachia in Ae. notoscriptus cannot explain why this mosquito is an ineffective vector of DENV

Genomic evolution of the pathogenic Wolbachia strain, wMelPop

Most strains of the widespread endosymbiotic bacterium Wolbachia pipientis are benign or behave as reproductive parasites. The pathogenic strain wMelPop is a striking exception, however: it overreplicates in its insect hosts and causes severe life shortening. The mechanism of this pathogenesis is currently unknown. We have sequenced the genomes of three variants of wMelPop and of the closely related nonpathogenic strain wMelCS. We show that the genomes of wMelCS and wMelPop appear to be identical in the nonrepeat regions of the genome and differ detectably only by the triplication of a 19-kb region that is unlikely to be associated with life shortening, demonstrating that dramatic differences in the host phenotype caused by this endosymbiont may be the result of only minor genetic changes. We also compare the genomes of the original wMelPop strain from Drosophila melanogaster and two sequential derivatives, wMelPop-CLA and wMelPop-PGYP. To develop wMelPop as a novel biocontrol agent, it was first transinfected into and passaged in mosquito cell lines for approximately 3.5 years, generating wMelPop-CLA. This cell line-passaged strain was then transinfected into Aedes aegypti mosquitoes, creating wMelPop-PGYP, which was sequenced after 4 years in the insect host. We observe a rapid burst of genomic changes during cell line passaging, but no further mutations were detected after transinfection into mosquitoes, indicating either that host preadaptation had occurred in cell lines, that cell lines are a more selectively permissive environment than animal hosts, or both. Our results provide valuable data on the rates of genomic and phenotypic change in Wolbachia associated with host shifts over short time scales

Large-scale releases and establishment of wMel Wolbachia in Aedes aegypti mosquitoes throughout the Cities of Bello, Medellín and Itagüí, Colombia.

BackgroundThe wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and has been shown to reduce the transmission of dengue and other Aedes-borne viruses. Here we report the entomological results from phased, large-scale releases of Wolbachia infected Ae. aegypti mosquitoes throughout three contiguous cities located in the Aburrá Valley, Colombia.Methodology/principal findingsLocal wMel Wolbachia-infected Ae. aegypti mosquitoes were generated and then released in an initial release pilot area in 2015-2016, which resulted in the establishment of Wolbachia in the local mosquito populations. Subsequent large-scale releases, mainly involving vehicle-based releases of adult mosquitoes along publicly accessible roads and streets, were undertaken across 29 comunas throughout Bello, Medellín and Itagüí Colombia between 2017-2022. In 9 comunas these were supplemented by egg releases that were undertaken by staff or community members. By the most recent monitoring, Wolbachia was found to be stable and established at consistent levels in local mosquito populations (>60% prevalence) in the majority (67%) of areas.ConclusionThese results, from the largest contiguous releases of wMel Wolbachia mosquitoes to date, highlight the operational feasibility of implementing the method in large urban settings. Based on results from previous studies, we expect that Wolbachia establishment will be sustained long term. Ongoing monitoring will confirm Wolbachia persistence in local mosquito populations and track its establishment in the remaining areas