Elimination of dengue by community programs using Mesocyclops(Copepoda) against Aedes aegypti in central Vietnam

From September 2000 to June 2003, a community-based program for dengue control using local predacious copepods of the genus Mesocyclops was conducted in three rural communes in the central Vietnam provinces of Quang Nam, Quang Ngai, and Khanh Hoa. Post-project, three subsequent entomologic surveys were conducted until March 2004. The number of households and residents in the communes were 5,913 and 27,167, respectively, and dengue notification rates for these communes from 1996 were as high as 2,418.5 per 100,000 persons. Following knowledge, attitude, and practice evaluations, surveys of water storage containers indicated that Mesocyclops spp. already occurred in 3-17% and that large tanks up to 2,000 liters, 130-300-liter jars, wells, and some 220-liter metal drums were the most productive habitats for Aedes aegypti. With technical support, the programs were driven by communal management committees, health collaborators, schoolteachers, and pupils. From quantitative estimates of the standing crop of third and fourth instars from 100 households, Ae. aegypti were reduced by approximately 90% by year 1, 92.3-98.6% by year 2, and Ae. aegypti immature forms had been eliminated from two of three communes by June 2003. Similarly, from resting adult collections from 100 households, densities were reduced to 0-1 per commune. By March 2004, two communes with no larvae had small numbers but the third was negative; one adult was collected in each of two communes while one became negative. Absolute estimates of third and fourth instars at the three intervention communes and one left untreated had significant correlations (P = 0.009-< 0.001) with numbers of adults aspirated from inside houses on each of 15 survey periods. By year 1, the incidence of dengue disease in the treated communes was reduced by 76.7% compared with non-intervention communes within the same districts, and no dengue was evident in 2002 and 2003, compared with 112.8 and 14.4 cases per 100,000 at district level. Since we had similar success in northern Vietnam from 1998 to 2000, this study demonstrates that this control model is broadly acceptable and achievable at community level but vigilance is required post-project to prevent reinfestation.

The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations

Dengue fever is the most important mosquito-borne viral disease of humans with more than 50 million cases estimated annually in more than 100 countries. Disturbingly, the geographic range of dengue is currently expanding and the severity of outbreaks is increasing. Control options for dengue are very limited and currently focus on reducing population abundance of the major mosquito vector, Aedes aegypti. These strategies are failing to reduce dengue incidence in tropical communities and there is an urgent need for effective alternatives. It has been proposed that endosymbiotic bacterial Wolbachia infections of insects might be used in novel strategies for dengue control. For example, the wMelPop-CLA Wolbachia strain reduces the lifespan of adult A. aegypti mosquitoes in stably transinfected lines. This life-shortening phenotype was predicted to reduce the potential for dengue transmission. The recent discovery that several Wolbachia infections, including wMelPop-CLA, can also directly influence the susceptibility of insects to infection with a range of insect and human pathogens has markedly changed the potential for Wolbachia infections to control human diseases. Here we describe the successful transinfection of A. aegypti with the avirulent wMel strain of Wolbachia, which induces the reproductive phenotype cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, providing optimal phenotypic effects for invasion. Under semi-field conditions, the wMel strain increased from an initial starting frequency of 0.65 to near fixation within a few generations, invading A. aegypti populations at an accelerated rate relative to trials with the wMelPop-CLA strain. We also show that wMel and wMelPop-CLA strains block transmission of dengue serotype 2 (DENV-2) in A. aegypti, forming the basis of a practical approach to dengue suppression.

Wolbachia uses host microRNAs to manipulate host gene expression and facilitate colonization of the dengue vector Aedes aegypti

The obligate endosymbiont Wolbachia pipientis is found in a wide range of invertebrates where they are best known for manipulating host reproduction. Recent studies have shown that Wolbachia also can modulate the lifespan of host insects and interfere with the development of human pathogens in mosquito vectors. Despite considerable study, very little is known about the molecular interactions between Wolbachia and its hosts that might mediate these effects. Using microarrays, we show that the microRNA (miRNA) profile of the mosquito, Aedes aegypti, is significantly altered by the wMelPop-CLA strain of W. pipientis. We found that a host miRNA (aae-miR-2940) is induced after Wolbachia infection in both mosquitoes and cell lines. One target of aae-miR-2940 is the Ae. aegypti metalloprotease gene. Interestingly, expression of the target gene was induced after Wolbachia infection, ectopic expression of the miRNA independent of Wolbachia, or transfection of an artificial mimic of the miRNA into mosquito cells. We also confirmed the interaction of aae-miR-2940 with the target sequences using GFP as a reporter gene. Silencing of the metalloprotease gene in both Wolbachia-infected cells and adult mosquitoes led to a significant reduction in Wolbachia density, as did inhibition of the miRNA in cells. These results indicate that manipulation of the mosquito metalloprotease gene via aae-miR-2940 is crucial for efficient maintenance of the endosymbiont. This report shows how Wolbachia alters the host miRNA profile and provides insight into the mechanisms of host manipulation used by this widespread endosymbiont.

Community-based control of Aedes aegypti by using Mesocyclops in southern Vietnam

We previously reported a new community-based mosquito control strategy that resulted in elimination of Aedes aegypti (Linn.) in 40 of 46 communes in northern and central Vietnam, and with annual recurrent total costs (direct and indirect) of only $0.28-$0.89 international dollars per person. This control strategy was extended to four provinces in southern Vietnam in Long An and Hau Giang (2004-2007) and to Long An, Ben Tre, and Vinh Long (2005-2010). In a total of 14 communes with 124,743 residents, the mean ± SD of adult female Ae. aegypti was reduced from 0.93 ± 0.62 to 0.06 ± 0.09, and the reduction of immature Ae. aegypti averaged 98.8%. By the final survey, no adults could be collected in 6 of 14 communes, and one commune, Binh Thanh, also had no immature forms. Although the community-based programs also involved community education and clean-up campaigns, the prevalence of Mesocyclops in large water storage containers > 50 liters increased from 12.77 ± 8.39 to 75.69 ± 9.17% over periods of 15-45 months. At the conclusion of the study, no confirmed dengue cases were detected in four of the five communes for which diagnostic serologic analysis was performed. The rate of progress was faster in communes that were added in stages to the program but the reason for this finding was unclear. At the completion of the formal project, sustainability funds were set up to provide each commune with the financial means to ensure that community-based dengue control activities continued.

Impact of Wolbachia on infection with chikungunya and yellow fever viruses in the mosquito vector Aedes aegypti

Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 10(4) times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression.

Limited dengue virus replication in field-collected Aedes aegypti mosquitoes infected with Wolbachia

Introduction Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus. Methodology/Principal Findings Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes. Conclusions/Significance Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.

Characterising the spatial dynamics of sympatric Aedes aegypti and Aedes albopictus populations in the Philippines

Entomological surveillance and control are essential to the management of dengue fever (DF). Hence, understanding the spatial and temporal patterns of DF vectors, Aedes (Stegomyia) aegypti (L.) and Ae. (Stegomyia) albopictus (Skuse), is paramount. In the Philippines, resources are limited and entomological surveillance and control are generally commenced during epidemics, when transmission is difficult to control. Recent improvements in spatial epidemiological tools and methods offer opportunities to explore more efficient DF surveillance and control solutions: however, there are few examples in the literature from resource-poor settings. The objectives of this study were to: (i) explore spatial patterns of Aedes populations and (ii) predict areas of high and low vector density to inform DF control in San Jose village, Muntinlupa city, Philippines. Fortnightly, adult female Aedes mosquitoes were collected from 50 double-sticky ovitraps (SOs) located in San Jose village for the period June-November 2011. Spatial clustering analysis was performed to identify high and low density clusters of Ae. aegypti and Ae. albopictus mosquitoes. Spatial autocorrelation was assessed by examination of semivariograms, and ordinary kriging was undertaken to create a smoothed surface of predicted vector density in the study area. Our results show that both Ae. aegypti and Ae. albopictus were present in San Jose village during the study period. However, one Aedes species was dominant in a given geographic area at a time, suggesting differing habitat preferences and interspecies competition between vectors. Density maps provide information to direct entomological control activities and advocate the development of geographically enhanced surveillance and control systems to improve DF management in the Philippines.

Abundance and prevalence of Aedes aegypti immatures and relationships with household water storage in rural areas in southern Viet Nam

Since 2000, the Government of Viet Nam has committed to provide rural communities with increased access to safe water through a variety of household water supply schemes (wells, ferrocement tanks and jars) and piped water schemes. One possible, unintended consequence of these schemes is the concomitant increase in water containers that may serve as habitats for dengue mosquito immatures, principally Aedes aegypti. To assess these possible impacts we undertook detailed household surveys of Ae. aegypti immatures, water storage containers and various socioeconomic factors in three rural communes in southern Viet Nam. Positive relationships between the numbers of household water storage containers and the prevalence and abundance of Ae. aegypti immatures were found. Overall, water storage containers accounted for 92–97% and 93–96% of the standing crops of III/IV instars and pupae, respectively. Interestingly, households with higher socioeconomic levels had significantly higher numbers of water storage containers and therefore greater risk of Ae. aegypti infestation. Even after provision of piped water to houses, householders continued to store water in containers and there was no observed decrease in water storage container abundance in these houses, compared to those that relied entirely on stored water. These findings highlight the householders’ concerns about the limited availability of water and their strong behavoural patterns associated with storage of water. We conclude that household water storage container availability is a major risk factor for infestation with Ae. aegypti immatures, and that recent investment in rural water supply infrastructure are unlikely to mitigate this risk, at least in the short term.

Wolbachia reduces the transmission potential of Dengue-infected Aedes aegypti

BACKGROUND: Dengue viruses (DENV) are the causative agents of dengue, the world's most prevalent arthropod-borne disease with around 40% of the world's population at risk of infection annually. Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits replication of the virus in the mosquito. The Wolbachia strain wMel, which has been introduced into the mosquito vector, Aedes aegypti, has been shown to invade and spread to near fixation in field releases. Standard measures of Wolbachia's efficacy for blocking virus replication focus on the detection and quantification of virus in mosquito tissues. Examining the saliva provides a more accurate measure of transmission potential and can reveal the extrinsic incubation period (EIP), that is, the time it takes virus to arrive in the saliva following the consumption of DENV viremic blood. EIP is a key determinant of a mosquito's ability to transmit DENVs, as the earlier the virus appears in the saliva the more opportunities the mosquito will have to infect humans on subsequent bites. METHODOLOGY/PRINCIPAL FINDINGS: We used a non-destructive assay to repeatedly quantify DENV in saliva from wMel-infected and Wolbachia-free wild-type control mosquitoes following the consumption of a DENV-infected blood meal. We show that wMel lengthens the EIP, reduces the frequency at which the virus is expectorated and decreases the dengue copy number in mosquito saliva as compared to wild-type mosquitoes. These observations can at least be partially explained by an overall reduction in saliva produced by wMel mosquitoes. More generally, we found that the concentration of DENV in a blood meal is a determinant of the length of EIP, saliva virus titer and mosquito survival. CONCLUSIONS/SIGNIFICANCE: The saliva-based traits reported here offer more disease-relevant measures of Wolbachia's effects on the vector and the virus. The lengthening of EIP highlights another means, in addition to the reduction of infection frequencies and DENV titers in mosquitoes, by which Wolbachia should operate to reduce DENV transmission in the field.

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.

Wolbachia-mediated resistance to dengue virus infection and death at the cellular level

Dengue is currently the most important arthropod-borne viral disease of humans. Recent work has shown dengue virus displays limited replication in its primary vector, the mosquito Aedes aegypti, when the insect harbors the endosymbiotic bacterium Wolbachia pipientis. Wolbachia-mediated inhibition of virus replication may lead to novel methods of arboviral control, yet the functional and cellular mechanisms that underpin it are unknown.

Comparative susceptibility of mosquito populations in North Queensland, Australia to oral infection with dengue virus

Dengue is the most prevalent arthropod-borne virus, with at least 40% of the world’s population at risk of infection each year. In Australia, dengue is not endemic, but viremic travelers trigger outbreaks involving hundreds of cases. We compared the susceptibility of Aedes aegypti mosquitoes from two geographically isolated populations with two strains of dengue virus serotype 2. We found, interestingly, that mosquitoes from a city with no history of dengue were more susceptible to virus than mosquitoes from an outbreak-prone region, particularly with respect to one dengue strain. These findings suggest recent evolution of population-based differences in vector competence or different historical origins. Future genomic comparisons of these populations could reveal the genetic basis of vector competence and the relative role of selection and stochastic processes in shaping their differences. Lastly, we show the novel finding of a correlation between midgut dengue titer and titer in tissues colonized after dissemination.

Spatiotemporal patterns of Aedes aegyptipopulations in Cairns, Australia : assessing drivers of dengue transmission

OBJECTIVES To identify the meteorological drivers of dengue vector density and determine high- and low-risk transmission zones for dengue prevention and control in Cairns, Australia. METHODS Weekly adult female Ae. aegypti data were obtained from 79 double sticky ovitraps (SOs) located in Cairns for the period September 2007-May 2012. Maximum temperature, total rainfall and average relative humidity data were obtained from the Australian Bureau of Meteorology for the study period. Time series-distributed lag nonlinear models were used to assess the relationship between meteorological variables and vector density. Spatial autocorrelation was assessed via semivariography, and ordinary kriging was undertaken to predict vector density in Cairns. RESULTS Ae. aegypti density was associated with temperature and rainfall. However, these relationships differed between short (0-6 weeks) and long (0-30 weeks) lag periods. Semivariograms showed that vector distributions were spatially autocorrelated in September 2007-May 2008 and January 2009-May 2009, and vector density maps identified high transmission zones in the most populated parts of Cairns city, as well as Machans Beach. CONCLUSION Spatiotemporal patterns of Ae. aegypti in Cairns are complex, showing spatial autocorrelation and associations with temperature and rainfall. Sticky ovitraps should be placed no more than 1.2 km apart to ensure entomological coverage and efficient use of resources. Vector density maps provide evidence for the targeting of prevention and control activities. Further research is needed to explore the possibility of developing an early warning system of dengue based on meteorological and environmental factors.

Experimental Infection of Culex Annulirostris, Culex Gelidus, and Aedes Vigilax With a Yellow Fever/Japanese Encephalitis Virus Vaccine Chimera (Chimerivax TM-JE)

Australian mosquitoes from which Japanese encephalitis virus (JEV) has been recovered (Culex annulirostris, Culex gelidus, and Aedes vigilax) were assessed for their ability to be infected with the ChimeriVax-JE vaccine, with yellow fever vaccine virus 17D (YF 17D) from which the backbone of ChimeriVax-JE vaccine is derived and with JEV-Nakayama. None of the mosquitoes became infected after being fed orally with 6.1 log(10) plaque-forming units (PFU)/mL of ChimeriVax-JE vaccine, which is greater than the peak viremia in vaccinees (mean peak viremia = 4.8 PFU/mL, range = 0-30 PFU/mL of 0.9 days mean duration, range = 0-11 days). Some members of all three species of mosquito became infected when fed on JEV-Nakayama, but only Ae. vigilax was infected when fed on YF 17D. The results suggest that none of these three species of mosquito are likely to set up secondary cycles of transmission of ChimeriVax-JE in Australia after feeding on a viremic vaccinee.