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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.

Dengue viruses cause more human morbidity and mortality than any other arthropod-borne virus. Dengue prevention relies mainly on vector control; however, the failure of traditional methods has promoted the development of novel entomological approaches. Although use of the intracellular bacterium wolbachia to control mosquito populations was proposed 50 years ago, only in the past decade has its use as a potential agent of dengue control gained substantial interest. Here, we review evidence that supports a practical approach for dengue reduction through field release of wolbachia-infected mosquitoes and discuss the additional studies that have to be done before the strategy can be validated and implemented. A crucial next step is to assess the efficacy of wolbachia in reducing dengue virus transmission. We argue that a cluster randomised trial is at this time premature because choice of wolbachia strain for release and deployment strategies are still being optimised. We therefore present a pragmatic approach to acquiring preliminary evidence of efficacy through various complementary methods including a prospective cohort study, a geographical cluster investigation, virus phylogenetic analysis, virus surveillance in mosquitoes, and vector competence assays. This multipronged approach could provide valuable intermediate evidence of efficacy to justify a future cluster randomised trial.

Background: The introduction of Wolbachia (wMel strain) into Aedes aegypti mosquitoes reduces their capacity to transmit dengue and other arboviruses. Randomised and non-randomised studies in multiple countries have shown significant reductions in dengue incidence following field releases of wMel-infected Ae. aegypti. We report the public health outcomes from phased, large-scale releases of wMel-Ae. aegypti mosquitoes throughout three contiguous cities in the Aburrá Valley, Colombia. Methodology/Principal findings: Following pilot releases in 2015–2016, staged city-wide wMel-Ae. aegypti deployments were undertaken in the cities of Bello, Medellín and Itagüí (3.3 million people) between October 2016 and April 2022. The impact of the Wolbachia intervention on dengue incidence was evaluated in two parallel studies. A quasi-experimental study using interrupted time series analysis showed notified dengue case incidence was reduced by 95% in Bello and Medellín and 97% in Itagüí, following establishment of wMel at ≥60% prevalence, compared to the pre-intervention period and after adjusting for seasonal trends. A concurrent clinic-based case-control study with a test-negative design was unable to attain the target sample size of 63 enrolled virologically-confirmed dengue (VCD) cases between May 2019 and December 2021, consistent with low dengue incidence throughout the Aburrá Valley following wMel deployments. Nevertheless, VCD incidence was 45% lower (OR 0.55 [95% CI 0.25, 1.17]) and combined VCD/presumptive dengue incidence was 47% lower (OR 0.53 [95% CI 0.30, 0.93]) among participants resident in wMel-treated versus untreated neighbourhoods. Conclusions/Significance: Stable introduction of wMel into local Ae. aegypti populations was associated with a significant and sustained reduction in dengue incidence across three Colombian cities. These results from the largest contiguous Wolbachia releases to-date demonstrate the real-world effectiveness of the method across large urban populations and, alongside previously published results, support the reproducibility of this effectiveness across different ecological settings. Trial registration: NCT03631719. Author summary: Dengue fever is a viral disease transmitted by Aedes aegypti mosquitoes and is an increasing public health concern globally. A novel evidence-based tool for dengue control involves the release of Ae. aegypti mosquitoes infected with a naturally-occurring insect bacteria called Wolbachia (wMel strain). wMel significantly reduces the mosquito's ability to transmit dengue and other viruses and can establish long-term in local Ae. aegypti populations following short-term releases. Previous studies have shown that establishment of wMel in local mosquito populations significantly reduces local dengue incidence. In Colombia, wMel-infected Ae. aegypti were released throughout the cities of Bello, Medellín, and Itagüí in the Aburrá Valley between 2015 and 2022, covering a population of 3.3 million people. The rate of dengue case notifications in the three cities declined by 95–97% after Wolbachia releases, compared to the prior decade, and dengue case numbers since 2020 have been the lowest in twenty years. A case-control study in Medellin in 2019–2021 showed that laboratory-confirmed dengue incidence was significantly lower in neighbourhoods with Wolbachia releases compared to untreated neighbourhoods. These results confirm the real-world effectiveness of the Wolbachia method, and show that it can be implemented at a city-wide scale to protect communities against dengue. [ABSTRACT FROM AUTHOR]

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the alpha-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel-D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.

Wolbachia is an endosymbiotic bacterium that can restrict the transmission of human pathogenic viruses by Aedes aegypti mosquitoes. Recent field trials have shown that dengue incidence is significantly reduced when Wolbachia is introgressed into the local Ae. aegypti population. Female Ae. aegypti are anautogenous and feed on human blood to produce viable eggs. Herein, we tested whether people who reside on Tri Nguyen Island (TNI), Vietnam developed antibodies to Wolbachia Surface Protein (WSP) following release of Wolbachia-infected Ae. aegypti, as a measure of exposure to Wolbachia. Paired blood samples were collected from 105 participants before and after mosquito releases and anti-WSP titres were measured by ELISA. We determined no change in anti-WSP titres after ~30 weeks of high levels of Wolbachia-Ae. aegypti on TNI. These data suggest that humans are not exposed to the major Wolbachia surface antigen, WSP, following introgression of Wolbachia-infected Ae. aegypti mosquitoes. [ABSTRACT FROM AUTHOR]

Abstract Background Strains of the endosymbiotic bacterium Wolbachia pipientis are extremely diverse both genotypically and in terms of their induced phenotypes in invertebrate hosts. Despite extensive molecular characterisation of Wolbachia diversity, little is known about the actual genomic diversity within or between closely related strains that group tightly on the basis of existing gene marker systems, including Multiple Locus Sequence Typing (MLST). There is an urgent need for higher resolution fingerprinting markers of Wolbachia for studies of population genetics, horizontal transmission and experimental evolution. Results The genome of the wMel Wolbachia strain that infects Drosophila melanogaster contains inter- and intragenic tandem repeats that may evolve through expansion or contraction. We identified hypervariable regions in wMel, including intergenic Variable Number Tandem Repeats (VNTRs), and genes encoding ankyrin (ANK) repeat domains. We amplified these markers from 14 related Wolbachia strains belonging to supergroup A and were successful in differentiating size polymorphic alleles. Because of their tandemly repeated structure and length polymorphism, the markers can be used in a PCR-diagnostic multilocus typing approach, analogous to the Multiple Locus VNTR Analysis (MLVA) established for many other bacteria and organisms. The isolated markers are highly specific for supergroup A and not informative for other supergroups. However, in silico analysis of completed genomes from other supergroups revealed the presence of tandem repeats that are variable and could therefore be useful for typing target strains. Conclusions Wolbachia genomes contain inter- and intragenic tandem repeats that evolve through expansion or contraction. A selection of polymorphic tandem repeats is a novel and useful PCR diagnostic extension to the existing MLST typing system of Wolbachia, as it allows rapid and inexpensive high-throughput fingerprinting of closely related strains for which polymorphic markers were previously lacking.

Cytoplasmic incompatibility (CI) is the most widespread reproductive modification induced in insects by the maternally inherited intracellular bacteria, Wolbachia. Expression of CI in Drosophila melanogasteris quite variable. Published papers typically show that CI expression is weak and often varies between different Drosophila lines and different labs reporting the results. The basis for this variability is not well understood but is often considered to be due to unspecified host genotype interactions with Wolbachia. Here, we show that male development time can greatly influence CI expression in D. melanogaster. In a given family, males that develop fastest express very strong CI. The 'younger brothers' of these males (males that take longer to undergo larval development) quickly lose their ability to express the CI phenotype as a function of development time. This effect is independent of male age effects and is enhanced when flies are reared under crowded conditions. No correlation is seen between this effect and Wolbachia densities in testes, suggesting that a more subtle interaction between host and symbiont is responsible. The observed younger brother effect may explain much of the reported variability in CI expression in this species. When male development time is controlled, it is possible to obtain consistently high levels of CI expression, which will benefit future studies that wish to use D. melanogaster as a model host to unravel CI mechanisms.

The endosymbiotic bacterium Wolbachia pipientis infects a wide range of arthropods, in which it induces a variety of reproductive phenotypes, including cytoplasmic incompatibility (CI), parthenogenesis, male killing, and reversal of genetic sex determination. The recent sequencing and annotation of the first Wolbachia genome revealed an unusually high number of genes encoding ankyrin domain (ANK) repeats. These ANK genes are likely to be important in mediating the Wolbachia-host interaction. In this work we determined the distribution and expression of the different ANK genes found in the sequenced Wolbachia wMel genome in nine Wolbachia strains that induce different phenotypic effects in their hosts. A comparison of the ANK genes of wMel and the non-CI-inducing wAu Wolbachia strain revealed significant differences between the strains. This was reflected in sequence variability in shared genes that could result in alterations in the encoded proteins, such as motif deletions, amino acid insertions, and in some cases disruptions due to insertion of transposable elements and premature stops. In addition, one wMel ANK gene, which is part of an operon, was absent in the wAu genome. These variations are likely to affect the affinity, function, and cellular location of the predicted proteins encoded by these genes.

We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.

Research has been conducted on the maternally transmitted bacteria of the Wolbachia genus. The use of the shell vial technique in establishing Wolbachia infections and examining the host cell range is described.

The genome size of Wolbachia strains varies considerably particularly between the parasitic group A that infect insects and mutualistic C and D strains that infect nematodes. Data reveal that the genome is circular and rules out the occurrence of an extrachromosomal plasmid DNA in any strain.