A dengue receptor as possible genetic marker of vector competence in <Emphasis Type="Italic">Aedes aegypti</Emphasis>

Background  

Vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. Notwithstanding studies of Quantitative Trait Loci (QTL) that influence the ability of Aedes aegypti midgut (MG) to become infected with dengue virus (DENV), no study to date has been undertaken to identify genetic markers of vector competence. Furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. Differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. The first interaction of DENV with the insect is through proteins in the apical membrane of the midgut epithelium resulting in binding and receptor-mediated endocytosis of the virus, and this determines cell permissiveness to infection. The susceptibility of mosquitoes to infection may therefore depend on their specific virus receptors. To study this interaction in Ae. aegypti strains that differ in their vector competence for DENV, we investigated the DS3 strain (susceptible to DENV), the IBO-11 strain (refractory to infection) and the membrane escape barrier strain, DMEB, which is infected exclusively in the midgut epithelial cells.     

Bicluster pattern of codon context usages between flavivirus and vector mosquito Aedes aegypti: relevance to infection and transcriptional response of mosquito genes

The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in most of the subtropical and tropical countries. Besides DENV, yellow fever virus (YFV) is also transmitted by A. aegypti. Susceptibility of A. aegypti to West Nile virus (WNV) has also been confirmed. Although studies have indicated correlation of codon bias between flaviviridae and their animal/insect hosts, it is not clear if codon sequences have any relation to susceptibility of A. aegypti to DENV, YFV and WNV. In the current study, usages of codon context sequences (codon pairs for neighboring amino acids) of the vector (A. aegypti) genome as well as the flaviviral genomes are investigated. We used bioinformatics methods to quantify codon context bias in a genome-wide manner of A. aegypti as well as DENV, WNV and YFV sequences. Mutual information statistics was applied to perform bicluster analysis of codon context bias between vector and flaviviral sequences. Functional relevance of the bicluster pattern was inferred from published microarray data. Our study shows that codon context bias of DENV, WNV and YFV sequences varies in a bicluster manner with that of specific sets of genes of A. aegypti. Many of these mosquito genes are known to be differentially expressed in response to flaviviral infection suggesting that codon context sequences of A. aegypti and the flaviviruses may play a role in the susceptible interaction between flaviviruses and this mosquito. The bias in usages of codon context sequences likely has a functional association with susceptibility of A. aegypti to flaviviral infection. The results from this study will allow us to conduct hypothesis-driven tests to examine the role of codon context bias in evolution of vector–virus interactions at the molecular level.     

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.     

Establishment of a Wolbachia Superinfection in Aedes aegypti Mosquitoes as a Potential Approach for Future Resistance Management

Wolbachia pipientis is an endosymbiotic bacterium estimated to chronically infect between 40–75% of all arthropod species. Aedes aegypti, the principle mosquito vector of dengue virus (DENV), is not a natural host of Wolbachia. The transinfection of Wolbachia strains such as wAlbB, wMel and wMelPop-CLA into Ae. aegypti has been shown to significantly reduce the vector competence of this mosquito for a range of human pathogens in the laboratory. This has led to wMel-transinfected Ae. aegypti currently being released in five countries to evaluate its effectiveness to control dengue disease in human populations. Here we describe the generation of a superinfected Ae. aegypti mosquito line simultaneously infected with two avirulent Wolbachia strains, wMel and wAlbB. The line carries a high overall Wolbachia density and tissue localisation of the individual strains is very similar to each respective single infected parental line. The superinfected line induces unidirectional cytoplasmic incompatibility (CI) when crossed to each single infected parental line, suggesting that the superinfection would have the capacity to replace either of the single constituent infections already present in a mosquito population. No significant differences in fitness parameters were observed between the superinfected line and the parental lines under the experimental conditions tested. Finally, the superinfected line blocks DENV replication more efficiently than the single wMel strain when challenged with blood meals from viremic dengue patients. These results suggest that the deployment of superinfections could be used to replace single infections and may represent an effective strategy to help manage potential resistance by DENV to field deployments of single infected strains.     

The Neovolcanic Axis Is a Barrier to Gene Flow among Aedes aegypti Populations in Mexico That Differ in Vector Competence for Dengue 2 Virus

Background

Aedes aegypti is the main mosquito vector of the four serotypes of dengue virus (DENV). Previous population genetic and vector competence studies have demonstrated substantial genetic structure and major differences in the ability to transmit dengue viruses in Ae. aegypti populations in Mexico.

Methodology/Principal Findings

Population genetic studies revealed that the intersection of the Neovolcanic axis (NVA) with the Gulf of Mexico coast in the state of Veracruz acts as a discrete barrier to gene flow among Ae. aegypti populations north and south of the NVA. The mosquito populations north and south of the NVA also differed in their vector competence (VC) for dengue serotype 2 virus (DENV2). The average VC rate for Ae. aegypti mosquitoes from populations from north of the NVA was 0.55; in contrast the average VC rate for mosquitoes from populations from south of the NVA was 0.20. Most of this variation was attributable to a midgut infection and escape barriers. In Ae. aegypti north of the NVA 21.5% failed to develop midgut infections and 30.3% of those with an infected midgut failed to develop a disseminated infection. In contrast, south of the NVA 45.2% failed to develop midgut infections and 62.8% of those with an infected midgut failed to develop a disseminated infection.

Conclusions

Barriers to gene flow in vector populations may also impact the frequency of genes that condition continuous and epidemiologically relevant traits such as vector competence. Further studies are warranted to determine why the NVA is a barrier to gene flow and to determine whether the differences in vector competence seen north and south of the NVA are stable and epidemiologically significant.     

Rise and fall of vector infectivity during sequential strain displacements by mosquito‐borne dengue virus

Each of the four serotypes of mosquito‐borne dengue virus (DENV‐1‐4) comprises multiple, genetically distinct strains. Competitive displacement between strains within a serotype is a common feature of DENV epidemiology and can trigger outbreaks of dengue disease. We investigated the mechanisms underlying two sequential displacements by DENV‐3 strains in Sri Lanka that each coincided with abrupt increases in dengue haemorrhagic fever (DHF) incidence. First, the post‐DHF strain displaced the pre‐DHF strain in the 1980s. We have previously shown that post‐DHF is more infectious than pre‐DHF for the major DENV vector, Aedes aegypti. Then, the ultra‐DHF strain evolved in situ from post‐DHF and displaced its ancestor in the 2000s. We predicted that ultra‐DHF would be more infectious for Ae. aegypti than post‐DHF but found that ultra‐DHF infected a significantly lower percentage of mosquitoes than post‐DHF. We therefore hypothesized that ultra‐DHF had effected displacement by disseminating in Ae. aegypti more rapidly than post‐DHF, but this was not borne out by a time course of mosquito infection. To elucidate the mechanisms that shape these virus–vector interactions, we tested the impact of RNA interference (RNAi), the principal mosquito defence against DENV, on replication of each of the three DENV strains. Replication of all strains was similar in mosquito cells with dysfunctional RNAi, but in cells with functional RNAi, replication of pre‐DHF was significantly suppressed relative to the other two strains. Thus, differences in susceptibility to RNAi may account for the differences in mosquito infectivity between pre‐DHF and post‐DHF, but other mechanisms underlie the difference between post‐DHF and ultra‐DHF.     

Dengue and Zika virus infection patterns vary among Aedes aegypti field populations from Belo Horizonte,a Brazilian endemic city

Dengue virus (DENV) and Zika virus (ZIKV) belong to the same viral family, the Flaviviridae. They cause recurring threats to the public health systems of tropical countries such as Brazil. The primary Brazilian vector of both viruses is the mosquito Aedes aegypti. After the mosquito ingests a blood meal from an infected person, the viruses infect and replicate in the midgut, disseminate to secondary tissues and reach the salivary gland (SG), where they are ready to be transmitted to a vertebrate host. It is thought that the intrinsic discrepancies among mosquitoes could affect their ability to deal with viral infections. This study confirms that the DENV and ZIKV infection patterns of nine Ae. aegypti field populations found in geographically separate health districts of an endemic Brazilian city vary. We analyzed the infection rate, disseminated infection, vector competence, and viral load through quantitative PCR. Mosquitoes were challenged using the membrane-feeding assay technique and were tested seven and fourteen days post-infection (early and late infection phases, respectively). The infection responses varied among the Ae. aegypti populations for both flaviviruses in the two infection phases. There was no similarity between DENV and ZIKV vector competencies or viral loads. According to the results of our study, the risk of viral transmission overtime after infection either increases or remains unaltered in ZIKV infected vectors. However, the risk may increase, decrease, or remain unaltered in DENV-infected vectors depending on the mosquito population. For both flaviviruses, the viral load persisted in the body even until the late infection phase. In contrast to DENV, the ZIKV accumulated in the SG over time in all the mosquito populations. These findings are novel and may help direct the development of control strategies to fight dengue and Zika outbreaks in endemic regions, and provide a warning about the importance of understanding mosquito responses to arboviral infections.     

Aedes aegypti SNAP and a calcium transporter ATPase influence dengue virus dissemination

Dengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, and is transmitted to humans by Aedes aegypti mosquitoes. Habitat expansion of Aedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world’s population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in the A. aegypti vector. We performed a mass spectrometry assay where we identified a set of A. aegypti salivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication both in vitro and in vivo, observing a ubiquitous expression of these proteins in the mosquito. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector.     

Vector Competence in West African Aedes aegypti Is Flavivirus Species and Genotype Dependent

Background

Vector competence of Aedes aegypti mosquitoes is a quantitative genetic trait that varies among geographic locations and among different flavivirus species and genotypes within species. The subspecies Ae. aegypti formosus, found mostly in sub-Saharan Africa, is considered to be refractory to both dengue (DENV) and yellow fever viruses (YFV) compared to the more globally distributed Ae. aegypti aegypti. Within Senegal, vector competence varies with collection site and DENV-2 viral isolate, but knowledge about the interaction of West African Ae. aegypti with different flaviviruses is lacking. The current study utilizes low passage isolates of dengue-2 (DENV-2-75505 sylvatic genotype) and yellow fever (YFV BA-55 -West African Genotype I, or YFV DAK 1279-West African Genotype II) from West Africa and field derived Ae. aegypti collected throughout Senegal to determine whether vector competence is flavivirus or virus genotype dependent.

Methodology/Principal Findings

Eight collections of 20–30 mosquitoes from different sites were fed a bloodmeal containing either DENV-2 or either isolate of YFV. Midgut and disseminated infection phenotypes were determined 14 days post infection. Collections varied significantly in the rate and intensity of midgut and disseminated infection among the three viruses.

Conclusions/Significance

Overall, vector competence was dependent upon both viral and vector strains. Importantly, contrary to previous studies, sylvatic collections of Ae. aegypti showed high levels of disseminated infection for local isolates of both DENV-2 and YFV.     

Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints

Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients’ sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.     

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.     

Wolbachia Reduces the Transmission Potential of Dengue-Infected Aedes aegypti

BackgroundDengue 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.Conclusions/SignificanceThe 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.     

Assessment of fitness and vector competence of a New Caledonia wMel Aedes aegypti strain before field-release

BackgroundBiological control programs involving Wolbachia-infected Aedes aegypti are currently deployed in different epidemiological settings. New Caledonia (NC) is an ideal location for the implementation and evaluation of such a strategy as the only proven vector for dengue virus (DENV) is Ae. aegypti and dengue outbreaks frequency and severity are increasing. We report the generation of a NC Wolbachia-infected Ae. aegypti strain and the results of experiments to assess the vector competence and fitness of this strain for future implementation as a disease control strategy in Noumea, NC.Methods/principal findingsThe NC Wolbachia strain (NC-wMel) was obtained by backcrossing Australian AUS-wMel females with New Caledonian Wild-Type (NC-WT) males. Blocking of DENV, chikungunya (CHIKV), and Zika (ZIKV) viruses were evaluated via mosquito oral feeding experiments and intrathoracic DENV challenge. Significant reduction in infection rates were observed for NC-wMel Ae. aegypti compared to WT Ae. aegypti. No transmission was observed for NC-wMel Ae. aegypti. Maternal transmission, cytoplasmic incompatibility, fertility, fecundity, wing length, and insecticide resistance were also assessed in laboratory experiments. Ae. aegypti NC-wMel showed complete cytoplasmic incompatibility and a strong maternal transmission. Ae. aegypti NC-wMel fitness seemed to be reduced compared to NC-WT Ae. aegypti and AUS-wMel Ae. aegypti regarding fertility and fecundity. However further experiments are required to assess it accurately.Conclusions/significanceOur results demonstrated that the NC-wMel Ae. aegypti strain is a strong inhibitor of DENV, CHIKV, and ZIKV infection and prevents transmission of infectious viral particles in mosquito saliva. Furthermore, our NC-wMel Ae. aegypti strain induces reproductive cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, supporting field-releases in Noumea, NC.     

Genetic specificity and potential for local adaptation between dengue viruses and mosquito vectors

Background  

Several observations support the hypothesis that vector-driven selection plays an important role in shaping dengue virus (DENV) genetic diversity. Clustering of DENV genetic diversity at a particular location may reflect underlying genetic structure of vector populations, which combined with specific vector genotype × virus genotype (G × G) interactions may promote adaptation of viral lineages to local mosquito vector genotypes. Although spatial structure of vector polymorphism at neutral genetic loci is well-documented, existence of G × G interactions between mosquito and virus genotypes has not been formally demonstrated in natural populations. Here we measure G × G interactions in a system representative of a natural situation in Thailand by challenging three isofemale families from field-derived Aedes aegypti with three contemporaneous low-passage isolates of DENV-1.