A portable approach for the surveillance of dengue virus-infected mosquitoes

Dengue virus is the most significant human viral pathogen spread by the bite of an infected mosquito. With no vaccine or antiviral therapy currently available, disease prevention relies largely on surveillance and mosquito control. Preventing the onset of dengue outbreaks and effective vector management would be considerably enhanced through surveillance of dengue virus prevalence in natural mosquito populations. However, current approaches to the identification of virus in field-caught mosquitoes require relatively slow and labor intensive techniques such as virus isolation or RT-PCR involving specialized facilities and personnel. A rapid and portable method for detecting dengue virus-infected mosquitoes is described. Using a hand held battery operated homogenizer and a dengue diagnostic rapid strip the viral protein NS1 was detected as a marker of dengue virus infection. This method could be performed in less than 30 min in the field, requiring no downstream processing, and is able to detect a single infected mosquito in a pool of at least 50 uninfected mosquitoes. The method described in this study allows rapid, real-time monitoring of dengue virus presence in mosquito populations and could be a useful addition to effective monitoring and vector control responses.

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.

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.

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.

One-year delayed effect of fog on malaria transmission : a time-series analysis in the rain forest area of Mengla County, south-west China

Background: Malaria is a major public health burden in the tropics with the potential to significantly increase in response to climate change. Analyses of data from the recent past can elucidate how short-term variations in weather factors affect malaria transmission. This study explored the impact of climate variability on the transmission of malaria in the tropical rain forest area of Mengla County, south-west China. Methods: Ecological time-series analysis was performed on data collected between 1971 and 1999. Auto-regressive integrated moving average (ARIMA) models were used to evaluate the relationship between weather factors and malaria incidence. Results: At the time scale of months, the predictors for malaria incidence included: minimum temperature, maximum temperature, and fog day frequency. The effect of minimum temperature on malaria incidence was greater in the cool months than in the hot months. The fog day frequency in October had a positive effect on malaria incidence in May of the following year. At the time scale of years, the annual fog day frequency was the only weather predictor of the annual incidence of malaria. Conclusion: Fog day frequency was for the first time found to be a predictor of malaria incidence in a rain forest area. The one-year delayed effect of fog on malaria transmission may involve providing water input and maintaining aquatic breeding sites for mosquitoes in vulnerable times when there is little rainfall in the 6-month dry seasons. These findings should be considered in the prediction of future patterns of malaria for similar tropical rain forest areas worldwide.

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.

A case of Kunjin virus encephalitis in a traveller returning from the Northern Territory

On 6 May 2001, a 67-year-old Australian born, Caucasian male presented to the Emergency Department of the Austin and Repatriation Medical Centre (A&RMC) with a 3 day history of fever, lethargy and confusion. This occurred one week after returning from a trip to the Northern Territory. His previous medical problems included ischaemic heart disease, a repaired abdominal aortic aneurysm, hypertension, hyperlipidaemia and congestive cardiac failure. He smoked 20 cigarettes per day and had a history of heavy alcohol consumption. He had no history of diabetes. His medications were aspirin, frusemide, lisinopril, simvastatin, and a nitroglycerol patch. Fifty years ago, he had an adverse reaction to penicillin with angioedema and an urticarial rash. Four weeks before admission he went on a fishing trip in the Northern Territory. He travelled by road, through outback regions of Victoria, New South Wales, Queensland, the Northern Territory and South Australia, spending time in Daly River, Coolum, Darwin, Dunmarra, Avon Downs, Innaminka and Mataranka. He was away for 3 weeks and camped in tents or outside in a swag throughout the trip. He recalls numerous times where he was exposed to mosquitoes with large numbers of bites at Dunmarra. During the time away, he remained well as did his 5 travelling companions. There was...

Recasting the theory of mosquito-borne pathogen transmission dynamics and control

Mosquito-borne diseases pose some of the greatest challenges in public health, especially in tropical and sub-tropical regions of theworld. Efforts to control these diseases have been underpinned by a theoretical framework developed for malaria by Ross and Macdonald, including models, metrics for measuring transmission, and theory of control that identifies key vulnerabilities in the transmission cycle. That framework, especially Macdonald’s formula for R0 and its entomological derivative, vectorial capacity, are nowused to study dynamics and design interventions for many mosquito-borne diseases. A systematic review of 388 models published between 1970 and 2010 found that the vast majority adopted the Ross–Macdonald assumption of homogeneous transmission in a well-mixed population. Studies comparing models and data question these assumptions and point to the capacity to model heterogeneous, focal transmission as the most important but relatively unexplored component in current theory. Fine-scale heterogeneity causes transmission dynamics to be nonlinear, and poses problems for modeling, epidemiology and measurement. Novel mathematical approaches show how heterogeneity arises from the biology and the landscape on which the processes of mosquito biting and pathogen transmission unfold. Emerging theory focuses attention on the ecological and social context formosquito blood feeding, themovement of both hosts and mosquitoes, and the relevant spatial scales for measuring transmission and for modeling dynamics and control.

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.

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.

Role of enhanced vector transmission of a new West Nile virus strain in an outbreak of equine disease in Australia in 2011

Background In 2011, a variant of West Nile virus Kunjin strain (WNVKUN) caused an unprecedented epidemic of neurological disease in horses in southeast Australia, resulting in almost 1,000 cases and a 9% fatality rate. We investigated whether increased fitness of the virus in the primary vector, Culex annulirostris, and another potential vector, Culex australicus, contributed to the widespread nature of the outbreak. Methods Mosquitoes were exposed to infectious blood meals containing either the virus strain responsible for the outbreak, designated WNVKUN2011, or WNVKUN2009, a strain of low virulence that is typical of historical strains of this virus. WNVKUN infection in mosquito samples was detected using a fixed cell culture enzyme immunoassay and a WNVKUN- specific monoclonal antibody. Probit analysis was used to determine mosquito susceptibility to infection. Infection, dissemination and transmission rates for selected days post-exposure were compared using Fisher’s exact test. Virus titers in bodies and saliva expectorates were compared using t-tests. Results There were few significant differences between the two virus strains in the susceptibility of Cx. annulirostris to infection, the kinetics of virus replication and the ability of this mosquito species to transmit either strain. Both strains were transmitted by Cx. annulirostris for the first time on day 5 post-exposure. The highest transmission rates (proportion of mosquitoes with virus detected in saliva) observed were 68% for WNVKUN2011 on day 12 and 72% for WNVKUN2009 on day 14. On days 12 and 14 post-exposure, significantly more WNVKUN2011 than WNVKUN2009 was expectorated by infected mosquitoes. Infection, dissemination and transmission rates of the two strains were not significantly different in Culex australicus. However, transmission rates and the amount of virus expectorated were significantly lower in Cx. australicus than Cx. annulirostris. Conclusions The higher amount of WNVKUN2011 expectorated by infected mosquitoes may be an indication that this virus strain is transmitted more efficiently by Cx. annulirostris compared to other WNVKUN strains. Combined with other factors, such as a convergence of abundant mosquito and wading bird populations, and mammalian and avian feeding behaviour by Cx. annulirostris, this may have contributed to the scale of the 2011 equine epidemic.

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.