From the Cover: Alphavirus-derived small RNAs modulate pathogenesis in disease vector mosquitoes

Mosquito-borne viruses cause significant levels of morbidity and mortality in humans and domesticated animals. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. Although the vertebrate infection is acute and often associated with disease, continual transmission of these viruses in nature depends on the establishment of a persistent, nonpathogenic infection in the mosquito vector. An antiviral RNAi response has been shown to limit the replication of RNA viruses in flies. However, the importance of the RNAi pathway as an antiviral defense in mammals is unclear. Differences in the immune responses of mammals and mosquitoes may explain why these viruses are not generally associated with pathology in the invertebrate host. We identified virus-derived small interfering RNAs (viRNAs), 21 nt in length, in Aedes aegypti infected with the mosquito-borne virus, Sindbis (SINV). viRNAs had an asymmetric distribution that spanned the length of the SINV genome. To determine the role of viRNAs in controlling pathogenic potential, mosquitoes were infected with recombinant alphaviruses expressing suppressors of RNA silencing. Decreased survival was observed in mosquitoes in which the accumulation of viRNAs was suppressed. These results suggest that an exogenous siRNA pathway is essential to the survival of mosquitoes infected with alphaviruses and, thus, the maintenance of these viruses in nature.     

Asymptomatic humans transmit dengue virus to mosquitoes

Three-quarters of the estimated 390 million dengue virus (DENV) infections each year are clinically inapparent. People with inapparent dengue virus infections are generally considered dead-end hosts for transmission because they do not reach sufficiently high viremia levels to infect mosquitoes. Here, we show that, despite their lower average level of viremia, asymptomatic people can be infectious to mosquitoes. Moreover, at a given level of viremia, DENV-infected people with no detectable symptoms or before the onset of symptoms are significantly more infectious to mosquitoes than people with symptomatic infections. Because DENV viremic people without clinical symptoms may be exposed to more mosquitoes through their undisrupted daily routines than sick people and represent the bulk of DENV infections, our data indicate that they have the potential to contribute significantly more to virus transmission to mosquitoes than previously recognized.With 3.97 billion people living in 128 countries currently at risk for infection, dengue viruses (DENV-1 to -4) cause more human morbidity and mortality worldwide than any other arthropod-borne virus (1, 2). Aedes aegypti mosquitoes are the primary vectors of DENV throughout the tropics (3). Dengue prevention relies on the control of Ae. aegypti populations, which is failing in most parts of the world due to lack of resources, lack of political will, and/or ineffective implementation (4).Virus transmission from infected humans to mosquitoes is a critical step in dengue epidemiology, but due to logistical constraints it has been directly examined only in a handful of studies to date (5). In initial experimental infections of human volunteers during the 1920s (6, 7), the onset of clinical symptoms occurred 4–9 d after virus inoculation by mosquito bite (8). DENV-infected humans were infectious to mosquitoes from 2 d before to 2 d after the onset of symptoms, and Ae. aegypti fed on viremic people were able to transmit virus to another person after at least 11 d of extrinsic incubation (8). Results from later studies indicated that, for naturally infected people with clinically apparent dengue, the duration of detectable viremia was on average 4–5 d after the onset of symptoms, but could range from 2 to 12 d (9, 10). Investigators in Vietnam fed Ae. aegypti directly on 208 symptomatic, hospitalized dengue patients and reported that the probability of successful human-to-mosquito DENV transmission was coincident with the kinetics of viremia (11). Dengue patients were infectious up to 5 d after the onset of symptoms, which generally corresponded with “defervescence” (11).All previous studies on human-to-mosquito DENV transmission were limited to people with overt illness and did not consider subclinical infections. An estimated 300 million of the total 390 million DENV infections per year are clinically inapparent or mildly symptomatic, i.e., no illness that disrupted a person’s daily routine (1). Following Grange et al. (12), we use “inapparent” or “subclinical” interchangeably to denote infections confirmed by virus detection or seroconversion, but with insufficient symptoms to be detected by existing surveillance systems and health care providers. “Asymptomatic” refers to a confirmed DENV infection in the complete absence of reported or detected symptoms. Inapparent human DENV infections are a potentially important component of the overall burden of dengue because they can serve as a previously unrecognized source of mosquito infection (12). Epidemic transmission of DENV associated with low viremia levels and mild illness has been reported (13). It has long been assumed, but not empirically verified, that people with inapparent infections fail to infect mosquitoes because they do not reach sufficiently high viremia levels (5). This assumption is based on the observation that disease severity is positively correlated with the magnitude of DENV viremia (10, 11, 14). To our knowledge, the only study that quantified viral RNA levels in a limited number of asymptomatic DENV infections in humans did not detect a significantly lower viremia (15), but infectiousness to mosquitoes was not evaluated. The aim of the present study was to document variation in DENV infectiousness of naturally infected humans across the spectrum of disease manifestations, including fully asymptomatic infections, and to verify the assumption that people with inapparent infections are not infectious to mosquitoes.     

Distribution of Mosquito Larvae on Kosrae Island,Kosrae State,the Federated States of Micronesia

Surveys of mosquito larvae were carried out in six areas of Kosrae Island, Kosrae State, the Federated States of Micronesia in December 2009 and June 2012. A total of 962 larvae of six species were collected from 106 natural and artificial habitats. They were identified as Aedes aegypti, Ae. albopictus, Ae. marshallensis, Culex quinquefasciatus, Cx. annulirostris, and Cx. kusaiensis. This is the first report from Kosrae Island for three of these species—Ae. marshallensis, Cx. quinquefasciatus, and Cx. annulirostris. The most abundant species was Ae. albopictus, followed by Ae. marshallensis, and these two species were found in all areas. Relatively large numbers of Cx. quinquefasciatus and Cx. kusaiensis were found in five areas. Fewer Cx. annulirostris were found, and only in three areas. Aedes aegypti larvae were collected from a single habitat at Tafunsak in 2009. To prevent the outbreak of dengue fever, environmental management should focus on the destruction, alteration, disposal and recycling of containers that produce larger numbers of adult Aedes mosquitoes.     

VERTICAL TRANSMISSION OF DENGUE VIRUS IN Aedes aegypti COLLECTED IN PUERTO IGUAZú, MISIONES,ARGENTINA

A finding of vertical transmission of the DEN 3 virus in male specimens ofAedes aegypti, collected in the 2009 fall-winter period, inPuerto Iguazú city, Misiones, Argentina, using the RT-PCR technique in a 15-specimenpool is reported. This result is analyzed within the context of the epidemiologicalsituation of Argentina''s northeast border.     

CPB1 of Aedes aegypti Interacts with DENV2 E Protein and Regulates Intracellular Viral Accumulation and Release from Midgut Cells

Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV). To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H) screenings against DENV2 envelope (E) protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1) was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER) of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network.     

LABORATORY EVALUATION OF THE DEVELOPMENT OF Aedes aegypti IN TWO SEASONS: INFLUENCE OF DIFFERENT PLACES AND DIFFERENT DENSITIES

Aedes aegypti is an important vector in Brazil being the main vector of the dengue-fever. This paper employs survival curves to describe the time in days from larvae to adult forms of Aedes aegypti raised, individually and collectively, and compares it during winter and spring when positioned inside and outside a laboratory. The study was conducted in São Vicente, a coastal city in Southeastern Brazil. The lowest water temperature in winter and in spring was 20 °C and the highest was 26 °C in spring. Higher and more stable temperatures were measured in the intra compared to the peri in both seasons. Consequently, larvae positioned in the intra resulted in the lowest median time to develop in the individual and collective experiment (nine and ten days, respectively). At least 25% of the larvae positioned in the intra in the individual experiment in the spring took only seven days to reach adulthood. Sex ratios and the median time development by sex did not show significant differences. These results indicate that efforts to control Aedes aegypti must be continuous and directed mainly to prevent the intra-domiciliary sites that can be infested in a week in order to reduce the human-vector contact.     

Spatial analysis of Aedes aegypti immatures in Northern Argentina: Clusters and temporal instability

The objective of this study was to analyze the spatio-temporal patterns of Aedes aegypti immatures based on four entomological surveys that inspected over 6000 households in a large neighborhood of the city of Clorinda between 2007 and 2008. Global and local spatial point pattern analyses of immature presence or absence, habitat quality (estimated using a previously obtained statistical model) and pupal production were performed. Global analyses showed aggregation of both infestation and habitat quality up to 10 times bigger than previously described, ranging from 150 to 400 m between surveys. Pupal production was also clustered but at smaller scales than infestation presence/absence. The location of the clusters was temporally unstable between surveys. There was no spatial structure related to the control strategy; lots treated with temephos and lots uninspected (i.e., closed or refusing) were randomly distributed. These results suggest a combination of exogenous (the aggregation of better quality habitats) and endogenous (dispersal) processes explaining the observed patterns of larger-scale infestation. A spatial targeting strategy at the neighborhood scale would not be as cost-effective in Clorinda as in other sites where stable smaller-scale clusters permit the identification of key premises.     

An evolutionary conserved function of the JAK-STAT pathway in anti-dengue defense

Here, we show that the major mosquito vector for dengue virus uses the JAK-STAT pathway to control virus infection. Dengue virus infection in Aedes aegypti mosquitoes activates the JAK-STAT immune signaling pathway. The mosquito''s susceptibility to dengue virus infection increases when the JAK-STAT pathway is suppressed through RNAi depletion of its receptor Domeless (Dome) and the Janus kinase (Hop), whereas mosquitoes become more resistant to the virus when the negative regulator of the JAK-STAT pathway, PIAS, is silenced. The JAK-STAT pathway exerts its anti-dengue activity presumably through one or several STAT-regulated effectors. We have identified, and partially characterized, two JAK-STAT pathway-regulated and infection-responsive dengue virus restriction factors (DVRFs) that contain putative STAT-binding sites in their promoter regions. Our data suggest that the JAK-STAT pathway is part of the A. aegypti mosquito''s anti-dengue defense and may act independently of the Toll pathway and the RNAi-mediated antiviral defenses.     

Seasonal and nonseasonal dynamics of Aedes aegypti in Rio de Janeiro,Brazil: Fitting mathematical models to trap data

Mathematical models suggest that seasonal transmission and temporary cross-immunity between serotypes can determine the characteristic multi-year dynamics of dengue fever. Seasonal transmission is attributed to the effect of climate on mosquito abundance and within host virus dynamics. In this study, we validate a set of temperature and density dependent entomological models that are built-in components of most dengue models by fitting them to time series of ovitrap data from three distinct neighborhoods in Rio de Janeiro, Brazil. The results indicate that neighborhoods differ in the strength of the seasonal component and that commonly used models tend to assume more seasonal structure than found in data. Future dengue models should investigate the impact of heterogeneous levels of seasonality on dengue dynamics as it may affect virus maintenance from year to year, as well as the risk of disease outbreaks.     

Toxicity effect of Delonix elata (Yellow Gulmohr) and predatory efficiency of Copepod,Mesocyclops aspericornis for the control of dengue vector,Aedes aegypti

Objective

To evaluate the toxicity, predatory efficiency of Delonix elata (D. elata) and Mesocyclops aspericornis (M. aspericornis) against dengue vector, Aedes aegypti (Ae. aegypti).

Methods

A mosquitocidal bioassay was conducted at different concentration of plant extract followed by WHO standard method. The probit analysis of each tested concentration and control were observed by using software SPSS 11 version package. The each tested concentration variable was assessed by DMRT method. The predatory efficiency of copepod was followed by Deo et al., 1988. The predator, M. aspericornis was observed for mortality, abnormalities, survival and swimming activity after 24 h treatment of plant and also predation on the mosquito larvae were observed.

Results

D. elata were tested for biological activity against the larvae, and pupae of Ae. aegypti. Significant mortality effects were observed in each life stage. The percentage of mortality was 100% in first and second instars whereas 96%, 92% in third and fourth instars. Fitted probit-mortality curves for larvae indicated the median and 90% lethal concentrations of D. elata for instars 1-4 to be 4.91 (8.13), 5.16 (8.44), 5.95 (7.76) and 6.87 (11.23), respectively. The results indicate that leaf extract exhibits significant biological activity against life stages. The present study revealed that D. elata is potentially important in the control of Ae. aegypti. Similar studies were conducted for predatory efficiency of Copepod, M. aspericornis against mosquito vector Ae. Aegypti. This study reported that the predatory copepod fed on 39% and 25% in I and III instar larvae of mosquito and in combined treatment of D. elata and copepod maximum control of mosquito larval states and at 83%, 80%, 75% and 53% in I, II, III and IV instars, respectively.

Conclusions

The combined action of plant extract and predatory copepod to effectively control mosquito population and reduce the dengue transmitting diseases.     

Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut

In this work we show that the lumen of Aedes aegypti midgut is highly colonized by bacteria that were identified by culture-dependent and culture-independent methods. rDNA sequences obtained were compared with those from GenBank and the main bacterial genera identified were: Serratia, Klebsiella, Asaia, Bacillus, Enterococcus, Enterobacter,Kluyvera and Pantoea. All genera were identified in midgut except Enterobacter that was observed only in eggs. Asaia and Pantoea were also identified in eggs and ovary, respectively. In addition two yeast genera were observed in A. aegypti: Pichia isolated from midgut and Candida identified in midgut and ovary. The genus Serratia was dominant in all isolation assays representing 54.5% of the total of microorganisms. Thirty-nine and 24 bacterial clones were successfully obtained from midguts 24 and 48 h after blood feeding (ABF), respectively. The majority of clones obtained were from Serratia sp. (48.7% and 50% for 24 and 48 h ABF, respectively). Light microscopy showed that bacteria were located preferentially in the posterior midgut, around the blood meal and associated with peritrophic matrix. Scanning electron microscopy images showed a high number of bacteria in midgut during blood digestion and the peak of bacterial enumeration was reached 48 h ABF, stage in which lumen was almost totally occupied by bacteria that were also interacting with epithelial microvilli. Our results show the dynamics of microbial colonization and their distribution in midgut during blood digestion.     

High potential risk of dengue transmission during the hot-dry season in Nha Trang City, Vietnam

We visited houses and inspected water-holding containers to determine the potential risks of dengue transmission during different seasons. This survey was conducted in two neighbourhoods of Nha Trang City in July and December 2006, which correspond to the middle of the hot-dry season and the beginning of the cool-wet season, respectively. We inspected a total of 1438 wet containers in 196 premises during both survey periods; 20% of the containers were positive for Aedes aegypti larvae and 8% for A. aegypti pupae. Indoor water-holding containers, which were sparsely distributed, exhibited high pupal productivity and efficiency (pupal productivity of a type of container/prevalence of that type of container) in either the first survey conducted in July, or the second, conducted in December. Although rainfall may not influence the number and distribution of water-holding containers in the city, the high average temperature in the first survey period resulted in a higher potential risk of dengue transmission. Our analysis suggests that if intensive source reduction is conducted in summer and containers with high pupal productivity and efficiency are targeted, the risk of dengue transmission in the city could be effectively reduced.     

Development and application of a simple colorimetric assay reveals widespread distribution of sodium channel mutations in Thai populations of Aedes aegypti

Dengue fever and its more serious complications dengue haemorrhagic fever and dengue shock syndrome are growing public health problems in tropical and subtropical countries. In the absence of a vaccine, most dengue control programmes rely heavily on the use of insecticides to target the Aedes mosquito vectors. As a limited number of insecticides are routinely used in control, monitoring for the presence of resistance is an essential component of dengue prevention programmes. The pyrethroid insecticides target the voltage-gated sodium channel on the insects’ neurons. Substitutions at residue 1016 of this protein have been associated with pyrethroid and DDT resistance in Aedes aegypti populations from Latin America and Asia. Here we report on the development of a simple colorimetric assay to detect these mutations in individual mosquitoes. Evaluation of this diagnostic assay on 180 Ae. aegypti individuals from Thailand revealed the presence of high frequencies of the Val1016Gly mutation throughout the country. The assay requires no specialised equipment and will enable monitoring for insecticide resistance associated alleles to be routinely incorporated into dengue surveillance operations.     

Female-specific flightless phenotype for mosquito control

Dengue and dengue hemorrhagic fever are increasing public health problems with an estimated 50–100 million new infections each year. Aedes aegypti is the major vector of dengue viruses in its range and control of this mosquito would reduce significantly human morbidity and mortality. Present mosquito control methods are not sufficiently effective and new approaches are needed urgently. A “sterile-male-release” strategy based on the release of mosquitoes carrying a conditional dominant lethal gene is an attractive new control methodology. Transgenic strains of Aedes aegypti were engineered to have a repressible female-specific flightless phenotype using either two separate transgenes or a single transgene, based on the use of a female-specific indirect flight muscle promoter from the Aedes aegypti Actin-4 gene. These strains eliminate the need for sterilization by irradiation, permit male-only release (“genetic sexing”), and enable the release of eggs instead of adults. Furthermore, these strains are expected to facilitate area-wide control or elimination of dengue if adopted as part of an integrated pest management strategy.