Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

<p>Abstract</p> <p>Background</p> <p>Arthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family <it>Togaviridae</it>; genus <it>Alphavirus</it>) that expresses B2 protein of Flock House virus (FHV; family <it>Nodaviridae</it>; genus <it>Alphanodavirus</it>), a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition.</p> <p>Results</p> <p>B2 protein expression from SINV (TE/3'2J) inhibited the accumulation of non-specific small RNAs in <it>Aedes aegypti </it>mosquito cell culture and virus-specific small RNAs both in infected cell culture and <it>Ae. aegypti </it>mosquitoes. More viral genomic and subgenomic RNA accumulated in cells and mosquitoes infected with TE/3'2J virus expressing B2 (TE/3'2J/B2) compared to TE/3'2J and TE/3'2J virus expressing GFP. TE/3'2J/B2 exhibited increased infection rates, dissemination rates, and infectious virus titers in mosquitoes following oral bloodmeal. Following infectious oral bloodmeal, significantly more mosquitoes died when TE/3'2J/B2 was ingested. The virus was 100% lethal following intrathoracic inoculation of multiple mosquito species and lethality was dose-dependent in <it>Ae. aegypti</it>.</p> <p>Conclusion</p> <p>We show that RNAi is active in <it>Ae. aegypti </it>cell culture and that B2 protein inhibits RNAi in mosquito cells when expressed by a recombinant SINV. Also, SINV more efficiently replicates in mosquito cells when RNAi is inhibited. Finally, TE/3'2J/B2 kills mosquitoes in a dose-dependent manner independent of infection route and mosquito species.</p

Engineered Anopheles Immunity to Plasmodium Infection

A causative agent of human malaria, Plasmodium falciparum, is transmitted by Anopheles mosquitoes. The malaria parasite is under intensive attack from the mosquito's innate immune system during its sporogonic development. We have used genetic engineering to create immune-enhanced Anopheles stephensi mosquitoes through blood meal-inducible expression of a transgene encoding the IMD pathway-controlled NF-kB Rel2 transcription factor in the midgut and fat-body tissue. Transgenic mosquitoes showed greater resistance to Plasmodium and microbial infection as a result of timely concerted tissue-specific immune attacks involving multiple effectors. The relatively weak impact of this genetic modification on mosquito fitness under laboratory conditions encourages further investigation of this approach for malaria control

Midgut microbiota of the malaria mosquito vector Anopheles gambiae and Interactions with plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.Institut de Recherche pour le Developpement (IRD); French Agence Nationale pour la Recherche [ANR-11-BSV7-009-01]; European Community [242095, 223601]info:eu-repo/semantics/publishedVersio

Phenoloxidase activity acts as a mosquito innate immune response against infection with semliki forest virus

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses

Anopheles Imd Pathway Factors and Effectors in Infection Intensity-Dependent Anti-Plasmodium Action

The Anopheles gambiae immune response against Plasmodium falciparum, an etiological agent of human malaria, has been identified as a source of potential anti-Plasmodium genes and mechanisms to be exploited in efforts to control the malaria transmission cycle. One such mechanism is the Imd pathway, a conserved immune signaling pathway that has potent anti-P. falciparum activity. Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes. However, unexplored features of this powerful mechanism that may be essential for the implementation of a malaria control strategy still remain. Using RNA interference to singly or dually silence caspar and other components of the Imd pathway, we have identified genes participating in the anti-Plasmodium signaling module regulated by Caspar, each of which represents a potential target to achieve over-activation of the pathway. We also determined that the Imd pathway is most potent against the parasite's ookinete stage, yet also has reasonable activity against early oocysts and lesser activity against late oocysts. We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota. Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models. Our results highlight the physiological considerations that are integral to a thoughtful implementation of Imd pathway manipulation in A. gambiae as part of an effort to limit the malaria transmission cycle, and they reveal a variety of previously unrecognized nuances in the Imd-directed immune response against P. falciparum

The JAK-STAT Pathway Controls Plasmodium vivax Load in Early Stages of Anopheles aquasalis Infection

Malaria affects 300 million people worldwide every year and 450,000 in Brazil. In coastal areas of Brazil, the main malaria vector is Anopheles aquasalis, and Plasmodium vivax is responsible for the majority of malaria cases in the Americas. Insects possess a powerful immune system to combat infections. Three pathways control the insect immune response: Toll, IMD, and JAK-STAT. Here we analyze the immune role of the A. aquasalis JAK-STAT pathway after P. vivax infection. Three genes, the transcription factor Signal Transducers and Activators of Transcription (STAT), the regulatory Protein Inhibitors of Activated STAT (PIAS) and the Nitric Oxide Synthase enzyme (NOS) were characterized. Expression of STAT and PIAS was higher in males than females and in eggs and first instar larvae when compared to larvae and pupae. RNA levels for STAT and PIAS increased 24 and 36 hours (h) after P. vivax challenge. NOS transcription increased 36 h post infection (hpi) while this protein was already detected in some midgut epithelial cells 24 hpi. Imunocytochemistry experiments using specific antibodies showed that in non-infected insects STAT and PIAS were found mostly in the fat body, while in infected mosquitoes the proteins were found in other body tissues. The knockdown of STAT by RNAi increased the number of oocysts in the midgut of A. aquasalis. This is the first clear evidence for the involvement of a specific immune pathway in the interaction of the Brazilian malaria vector A. aquasalis with P. vivax, delineating a potential target for the future development of disease controlling strategies

Differential Plasmodium falciparum infection of Anopheles gambiae s.s. molecular and chromosomal forms in Mali

BACKGROUND: Anopheles gambiae sensu stricto (s.s.) is a primary vector of Plasmodium falciparum in sub-Saharan Africa. Although some physiological differences among molecular and chromosomal forms of this species have been demonstrated, the relative susceptibility to malaria parasite infection among them has not been unequivocally shown. The objective of this study was to investigate P. falciparum circumsporozoite protein infection (CSP) positivity among An. gambiae s.s. chromosomal and molecular forms. METHODS: Wild An. gambiae from two sites Kela (n = 464) and Sidarebougou (n = 266) in Mali were screened for the presence of P. falciparum CSP using an enzyme-linked immunosorbent assay (ELISA). Samples were then identified to molecular form using multiple PCR diagnostics (n = 713) and chromosomal form using chromosomal karyotyping (n = 419). RESULTS: Of 730 An. gambiae sensu lato (s.l.) mosquitoes, 89 (12.2%) were CSP ELISA positive. The percentage of positive mosquitoes varied by site: 52 (11.2%) in Kela and 37 (13.9%) in Sidarebougou. Eighty-seven of the positive mosquitoes were identified to molecular form and they consisted of nine Anopheles arabiensis (21.4%), 46 S (10.9%), 31 M (12.8%), and one MS hybrid (14.3%). Sixty of the positive mosquitoes were identified to chromosomal form and they consisted of five An. arabiensis (20.0%), 21 Savanna (15.1%), 21 Mopti (30.4%), 11 Bamako (9.2%), and two hybrids (20.0%). DISCUSSION: In this collection, the prevalence of P. falciparum infection in the M form was equivalent to infection in the S form (no molecular form differential infection). There was a significant differential infection by chromosomal form such that, P. falciparum infection was more prevalent in the Mopti chromosomal forms than in the Bamako or Savanna forms; the Mopti form was also the most underrepresented in the collection. Continued research on the differential P. falciparum infection of An. gambiae s.s. chromosomal and molecular forms may suggest that Plasmodium – An. gambiae interactions play a role in malaria transmission

Anopheles gambiae Antiviral Immune Response to Systemic O'nyong-nyong Infection

Mosquito-borne viral diseases are found across the globe and are responsible for numerous severe human infections. In order to develop novel methods for prevention and treatment of these diseases, detailed understanding of the biology of viral infection and transmission is required. Little is known about invertebrate responses to infection in mosquito hosts. In this study we used a model system of Anopheles gambiae mosquitoes and O'nyong-nyong virus to study mosquito immune responses to infection. We examined the global transcriptional responses of A. gambiae to viral infection of the mosquito blood equivalent (the hemolymph) identifying a number of genes with immune functions that are switched on or off in response to infection, including complement-like proteins that circulate in the mosquito hemolymph. The switching on of these genes combined with co-infection experiments with malaria parasites suggests that viral infection inhibits the melanisation pathway. Through silencing the function of a selection of viral responsive genes, we identified four genes that have roles in A. gambiae anti-viral immunity; two putative recognition receptors (a galectin and an MD2-like receptor); two effector lysozymes. These molecules have previously non-described roles in antiviral immunity, and suggest uncharacterised mechanisms for targeting viral infection in A. gambiae mosquitoes

Reciprocal Tripartite Interactions between the Aedes aegypti Midgut Microbiota, Innate Immune System and Dengue Virus Influences Vector Competence

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