Aedes aegypti, the mosquito vector of dengue viruses, utilizes its innate immune system to ward off a variety of pathogens, some of which can cause disease in humans. To date, the features of insects' innate immune defenses against viruses have mainly been studied in the fruit fly Drosophila melanogaster, which appears to utilize different immune pathways against different types of viruses, in addition to an RNA interference–based defense system. We have used the recently released whole-genome sequence of the Ae. aegypti mosquito, in combination with high-throughput gene expression and RNA interference (RNAi)-based reverse genetic analyses, to characterize its response to dengue virus infection in different body compartments. We have further addressed the impact of the mosquito's endogenous microbial flora on virus infection. Our findings indicate a significant role for the Toll pathway in regulating resistance to dengue virus, as indicated by an infection-responsive regulation and functional assessment of several Toll pathway–associated genes. We have also shown that the mosquito's natural microbiota play a role in modulating the dengue virus infection, possibly through basal-level stimulation of the Toll immune pathway. The Aedes aegypti mosquito is largely responsible for the transmission of dengue viruses that cause disease in humans. The virus is taken up with an infected blood meal from which it will first infect the mosquito gut tissue. From the gut it will migrate to other parts of the mosquito, including the salivary glands, from where it can be transmitted to another human upon a second blood meal. In this study we show that the mosquito utilizes its innate immune system to control dengue virus infection. Infection with the virus will result in the activation of mosquito immune responses that are mainly controlled by the Toll pathway. These responses entail antiviral activities that limit infection with the virus. We also show that the mosquito's natural microbial flora play a role in modulating the dengue virus infection, possibly through the stimulation of the mosquito's immune system.

Dengue virus is one of the most important arboviral pathogens and the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. It is transmitted between humans by the mosquitoes Aedes aegypti and Aedes albopictus, and at least 2.5 billion people are at daily risk of infection. During their lifecycle, mosquitoes are exposed to a variety of microbes, some of which are needed for their successful development into adulthood. However, recent studies have suggested that the adult mosquito's midgut microflora is critical in influencing the transmission of human pathogens. In this study we assessed the reciprocal interactions between the mosquito's midgut microbiota and dengue virus infection that are, to a large extent, mediated by the mosquito's innate immune system. We observed a marked decrease in susceptibility to dengue virus infection when mosquitoes harbored certain field-derived bacterial isolates in their midgut. Transcript abundance analysis of selected antimicrobial peptide genes suggested that the mosquito's microbiota elicits a basal immune activity that appears to act against dengue virus infection. Conversely, the elicitation of the mosquito immune response by dengue virus infection itself influences the microbial load of the mosquito midgut. In sum, we show that the mosquito's microbiota influences dengue virus infection of the mosquito, which in turn activates its antibacterial responses. Dengue virus is transmitted by Aedes mosquitoes. During their lifecycle, mosquitoes are exposed to a variety of microbes, and many of them inhabit the mosquito midgut, thereby sharing the same environment with ingested pathogens. The mosquito midgut is the site of multiple reciprocal interactions between the mosquito, its commensal bacteria, and ingested pathogens that will ultimately influence the level of pathogen infection and transmission. In this study the authors addressed the reciprocal interactions between the Aedes immune system, dengue virus and mosquito midgut microbiota using molecular and microbiological assays. The study showed that certain field-derived bacterial isolates of the mosquito midgut exert a detrimental effect on dengue virus infection. This effect is at least partly manifested through the action of the mosquito immune system which is activated by microbes. Conversely, dengue virus infection induces immune responses in the mosquito midgut tissue that act against the natural mosquito midgut microbiota. This study contributes to our understanding of dengue virus infection in Aedes mosquitoes, which may aid towards the development of novel biocontrol strategies to halt dengue transmission.

Dengue fever is endemic in many countries in South and Southeast Asia, Africa and South America, and elsewhere it is an emerging threat. It is caused by one of four dengue viruses — DENV-1, 2, 3 and 4 — transmitted by Aedes sp. mosquitoes; there is no approved vaccine and no effective specific therapy. The dengue viruses are compact flaviviruses likely to require a large number of host factors, and knowledge of those factors could lead to the discovery of potential targets for drugs and new vector control strategies. Now by using a high-throughput RNAi screening approach in DENV-2-infected Drosophila cells — Drosophila is related to the vector species and more amenable to the tools of genomics — more than a hundred candidate dengue virus host factors have been identified, many of them also acting as host factors in human cells. This report identifies host factors required for Dengue virus propagation by using a high throughput genome-wide RNA interference screening approach in Drosophila cells. Dengue fever is the most frequent arthropod-borne viral disease of humans, with almost half of the world’s population at risk of infection1. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make dengue fever a global public health threat1,2. Given their compact genomes, dengue viruses (DENV-1–4) and other flaviviruses probably require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors, have been identified3,4,5,6,7,8,9,10. Here we identify insect host factors required for DENV-2 propagation, by carrying out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 double-stranded RNA library. This screen identified 116 candidate dengue virus host factors (DVHFs). Although some were previously associated with flaviviruses (for example, V-ATPases and α-glucosidases)3,4,5,7,9,10, most of the DVHFs were newly implicated in dengue virus propagation. The dipteran DVHFs had 82 readily recognizable human homologues and, using a targeted short-interfering-RNA screen, we showed that 42 of these are human DVHFs. This indicates notable conservation of required factors between dipteran and human hosts. This work suggests new approaches to control infection in the insect vector and the mammalian host.

Plasmodium and dengue virus, the causative agents of the two most devastating vector-borne diseases, malaria and dengue, are transmitted by the two most important mosquito vectors, Anopheles gambiae and Aedes aegypti, respectively. Insect-bacteria associations have been shown to influence vector competence for human pathogens through multi-faceted actions that include the elicitation of the insect immune system, pathogen sequestration by microbes, and bacteria-produced anti-pathogenic factors. These influences make the mosquito microbiota highly interesting from a disease control perspective. Here we present a bacterium of the genus Chromobacterium (Csp_P), which was isolated from the midgut of field-caught Aedes aegypti. Csp_P can effectively colonize the mosquito midgut when introduced through an artificial nectar meal, and it also inhibits the growth of other members of the midgut microbiota. Csp_P colonization of the midgut tissue activates mosquito immune responses, and Csp_P exposure dramatically reduces the survival of both the larval and adult stages. Ingestion of Csp_P by the mosquito significantly reduces its susceptibility to Plasmodium falciparum and dengue virus infection, thereby compromising the mosquito's vector competence. This bacterium also exerts in vitro anti-Plasmodium and anti-dengue activities, which appear to be mediated through Csp_P -produced stable bioactive factors with transmission-blocking and therapeutic potential. The anti-pathogen and entomopathogenic properties of Csp_P render it a potential candidate for the development of malaria and dengue control strategies. The infectious agents that cause malaria and dengue are transmitted by Anopheles and Aedes mosquitoes, respectively. Bacteria found in the mosquito midgut have the potential to dramatically affect the susceptibility of the mosquito vector to the malaria parasite and dengue virus. In this work, we investigate one such microbe, Chromobacterium sp. (Csp_P), a bacterium isolated from a field-caught Aedes aegypti mosquito. We show that Csp_P can effectively colonize the midguts of Anopheles gambiae and Aedes aegypti mosquitoes and can, when ingested by the mosquito, significantly reduce the mosquito's susceptibility to infection with the malaria parasite and dengue virus. We also show that exposure to, and ingestion of, Csp_P can reduce the lifespan of larval and adult mosquitoes, respectively. We show that Csp_P has anti-Plasmodium and anti-dengue activity independent of the mosquito, suggesting that the bacterium secretes metabolites that could potentially be exploited to prevent disease transmission or to treat infection.

The female Aedes aegypti salivary gland plays a pivotal role in bloodmeal acquisition and reproduction, and thereby dengue virus (DENV) transmission. It produces numerous immune factors, as well as immune-modulatory, vasodilatory, and anti-coagulant molecules that facilitate blood-feeding. To assess the impact of DENV infection on salivary gland physiology and function, we performed a comparative genome-wide microarray analysis of the naïve and DENV infection-responsive A. aegypti salivary gland transcriptomes. DENV infection resulted in the regulation of 147 transcripts that represented a variety of functional classes, including several that are essential for virus transmission, such as immunity, blood-feeding, and host-seeking. RNAi-mediated gene silencing of three DENV infection-responsive genes - a cathepsin B, a putative cystatin, and a hypothetical ankyrin repeat-containing protein - significantly modulated DENV replication in the salivary gland. Furthermore, silencing of two DENV infection-responsive odorant-binding protein genes (OBPs) resulted in an overall compromise in blood acquisition from a single host by increasing the time for initiation of probing and the probing time before a successful bloodmeal. We also show that DENV established an extensive infection in the mosquito's main olfactory organs, the antennae, which resulted in changes of the transcript abundance of key host-seeking genes. DENV infection, however, did not significantly impact probing initiation or probing times in our laboratory infection system. Here we show for the first time that the mosquito salivary gland mounts responses to suppress DENV which, in turn, modulates the expression of chemosensory-related genes that regulate feeding behavior. These reciprocal interactions may have the potential to affect DENV transmission between humans. Dengue virus (DENV) is transmitted between humans through the bite of infected Aedes aegypti mosquitoes. Since the virus is inoculated in saliva, infection of the mosquito salivary gland is an essential requirement for transmission. In addition, the gland also produces numerous biologically active compounds that facilitate blood-feeding. Despite the salivary gland's crucial role in DENV transmission, very little is known about the host-pathogen interactions, at the molecular level, in this organ. In this study, we characterized the A. aegypti salivary gland response to DENV infection at both the gene expression and functional levels. We found that DENV induced the expression of several gene transcripts whose products modulate virus replication in the salivary gland. Unexpectedly, the virus also induced transcripts of two odorant-binding proteins, which we demonstrate to be important for mosquito host-seeking and probing behavior. This is the first study to demonstrate that besides affecting cellular processes that modulate virus replication, DENV also has the potential to alter chemosensory processes in ways that may result in increased virus transmission.