Limitations in harnessing oral RNA interference as an antiviral strategy in Aedes aegypti
Ontology highlight
ABSTRACT:
SUBMITTER: Romoli O
PROVIDER: S-EPMC10907830 | biostudies-literature | 2024 Feb
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundRNA interference (RNAi) is an important anti-viral defense mechanism. The Aedes aegypti genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use Ae. aegypti as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family Togaviridae, genus Alphavirus).ResultsSINV (TR339-eGFP) (+) strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate Ago2, Dcr2, or TSN mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP) with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut Ago2, Dcr2, and TSN transcript levels during infection revealed that only TSN transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner.ConclusionWe show that silencing RNAi components in Ae. aegypti results in transient increases in SINV replication. Furthermore, Ae. aegypti RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in Ae. aegypti.
| S-EPMC2278134 | biostudies-literature
Project description:Mosquitoes rely on RNA interference (RNAi) as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV) infection in C6/36 (Aedes albopictus) cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses). Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae), Sindbis virus (SINV, Togaviridae) and La Crosse virus (LACV, Bunyaviridae) and total RNA recovered from cell lysates. Small RNA (sRNA) libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs) from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26-27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand) and distribution (position along viral genome) of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney) cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level.
| S-EPMC2964293 | biostudies-literature
Project description:Mosquitoes (Aedes aegypti) were genetically modified to exhibit impaired vector competence for dengue type 2 viruses (DENV-2). We exploited the natural antiviral RNA interference (RNAi) pathway in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA genome. The A. aegypti carboxypeptidase A promoter was used to express the IR RNA in midgut epithelial cells after ingestion of a bloodmeal. The promoter and effector gene were inserted into the genome of a white-eye Puerto Rico Rexville D (Higgs' white eye) strain by using the nonautonomous mariner MosI transformation system. A transgenic family, Carb77, expressed IR RNA in the midgut after a bloodmeal. Carb77 mosquitoes ingesting an artificial bloodmeal containing DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. DENV-2 titration of individual mosquitoes showed that most Carb77 mosquitoes poorly supported virus replication. Transmission in vitro of virus from the Carb77 line was significantly diminished when compared to control mosquitoes. The presence of DENV-2-derived siRNAs in RNA extracts from midguts of Carb77 and the loss of the resistance phenotype when the RNAi pathway was interrupted proved that DENV-2 resistance was caused by a RNAi response. Engineering of transgenic A. aegypti that show a high level of resistance against DENV-2 provides a powerful tool for developing population replacement strategies to control transmission of dengue viruses.
| S-EPMC1449670 | biostudies-literature
Project description:Alphaviruses are globally distributed and predominately transmitted by mosquitoes. Aedes species are common vectors for the clinically important alphaviruses-Chikungunya, Sindbis, and Ross River (RRV) viruses-with Aedes aegypti also being a vector for the flaviviruses dengue, Yellow Fever, and Zika viruses. Ae. aegypti was putatively implicated in the large 1979-1980 South Pacific Islands outbreak of RRV-the leading cause of arboviral disease in Australia today. The RNA interference (RNAi) defense response in mosquitoes involves a number of small RNAs, with their kinetics induced by alphaviruses being poorly understood, particularly at the tissue level. We compared the small RNA profiles between RRV-infected and noninfected Ae. aegypti midgut and fat body tissues at 2, 6, and 12 days post-inoculation (dpi). RRV induced an incremental RNAi response, yielding short interfering and P-element-induced-wimpy-testis (PIWI)-interacting RNAs. Fourteen host microRNAs were differentially expressed due to RRV with the majority in the fat body at 2 dpi. The largely congruent pattern of microRNA regulation with previous reports for alphaviruses and divergence from those for flaviviruses suggests a degree of conservation, whereas patterns of microRNA expression unique to this study provide novel insights into the tissuespecific hostvirus attributes of Ae. aegypti responses to this previously unexplored oldworld alphavirus.
| S-EPMC7412335 | biostudies-literature
Project description:The resurgence of arbovirus outbreaks across the globe, including the recent Zika virus (ZIKV) epidemic in 2015-2016, emphasizes the need for innovative vector control methods. In this study, we investigated ZIKV susceptibility to transgenic Aedes aegypti engineered to target the virus by means of the antiviral small-interfering RNA (siRNA) pathway. The robustness of antiviral effector expression in transgenic mosquitoes is strongly influenced by the genomic insertion locus and transgene copy number; we therefore used CRISPR/Cas9 to re-target a previously characterized locus (Chr2:321382225) and engineered mosquitoes expressing an inverted repeat (IR) dsRNA against the NS3/4A region of the ZIKV genome. Small RNA analysis revealed that the IR effector triggered the mosquito's siRNA antiviral pathway in bloodfed females. Nearly complete (90%) inhibition of ZIKV replication was found in vivo in both midguts and carcasses at 7 or 14 days post-infection (dpi). Furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that Ae. aegypti innate immunity can be co-opted to engineer mosquitoes resistant to ZIKV.
| S-EPMC7692394 | biostudies-literature
Project description:The yellow fever mosquito, Aedes aegypti, serves as the primary vector for epidemic transmission of yellow fever, dengue, Zika (ZIKV), and chikungunya viruses to humans. Control of Ae. aegypti is currently limited to insecticide applications and larval habitat management; however, to combat growing challenges with insecticide resistance, novel genetic approaches for vector population reduction or transmission interruption are being aggressively pursued. The objectives of this study were to assess the ability of the Ae. aegypti antiviral exogenous-small interfering RNA (exo-siRNA) response to inhibit ZIKV infection and transmission, and to identify the optimal RNA interference (RNAi) target region in the ZIKV genome. We accomplished these objectives by in vitro transcription of five long double-stranded RNAs (dsRNAs) from the genome region spanning the NS2B-NS3-NS4A genes, which were the most highly conserved among ZIKV RNA sequences representing both East and West African and Asian-American clades, and evaluation of the ability of these dsRNAs to trigger an effective antiviral exo-siRNA response after intrathoracic injection into Ae. aegypti. In a pilot study, five ZIKV dsRNAs were tested by intrathoracic inoculation of 250 ng dsRNA into groups of approximately 5-day-old mosquitoes. Three days post-inoculation, mosquitoes were provided an infectious blood-meal containing ZIKV strain PRVABC59 (Puerto Rico), MR766 (Uganda), or 41525 (Senegal). On days 7 and 14 post-infection individual whole mosquito bodies were assessed for ZIKV infectious titer by plaque assays. Based on the results of this initial assessment, three dsRNAs were selected for further evaluation of viral loads of matched body and saliva expectorants using a standardized infectious dose of 1 × 107 PFU/mL of each ZIKV strain. Fourteen days post-exposure to ZIKV, paired saliva and carcass samples were harvested from individual mosquitoes and assessed for ZIKV RNA load by qRT-PCR. Injection of each of the three dsRNAs resulted in significant inhibition of replication of all three strains of ZIKV in mosquito bodies and saliva. This study lays critical groundwork for pursuing ZIKV transmission-blocking strategies that exploit the Ae. aegypti exo-siRNA response for arbovirus suppression in natural populations.
| S-EPMC7012353 | biostudies-literature
Project description: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.
| S-EPMC2633610 | biostudies-literature
Project description:RNA interference (RNAi) techniques are being developed for a range of pest insect control technologies, including the sterile insect technique (SIT) and double-stranded RNA (dsRNA)-based insecticides. In SIT applications, where >99% of the released males should be sterile to meet industry standards, the efficiency of RNAi will need to be improved for many insect species if this technology is to be adopted. Endogenous dsRNases can impede dsRNA delivery in some insects, and, here, we investigated whether dsRNases in the midgut could limit RNAi efficacy in the mosquito Aedes aegypti. Ten putative dsRNases were identified in the Ae. aegypti genome, with two highly expressed in the midguts of larvae. Using an ex vivo assay, we observed that dsRNA was rapidly degraded within the mosquito larva's gut. Double-stranded RNA targeting these two dsRNases, when fed to the larvae, effectively reduced gut dsRNase activity. When these dsRNase-specific dsRNAs were co-delivered with dsRNA targeting a cyan fluorescent protein (CFP) reporter gene, greater knockdown of CFP fluorescence was observed. These results suggest that inhibiting dsRNase activity could enable the implementation of RNAi-based mosquito control methods.
| S-EPMC7349537 | biostudies-literature
Project description:The recent global Zika epidemics have revealed the significant threat that mosquito-borne viruses pose. There are currently no effective vaccines or prophylactics to prevent Zika virus (ZIKV) infection. Limiting exposure to infected mosquitoes is the best way to reduce disease incidence. Recent studies have focused on targeting mosquito reproduction and immune responses to reduce transmission. Previous work has evaluated the effect of insulin signaling on antiviral JAK/STAT and RNAi in vector mosquitoes. Specifically, insulin-fed mosquitoes resulted in reduced virus replication in an RNAi-independent, ERK-mediated JAK/STAT-dependent mechanism. In this work, we demonstrate that targeting insulin signaling through the repurposing of small molecule drugs results in the activation of both RNAi and JAK/STAT antiviral pathways. ZIKV-infected Aedes aegypti were fed blood containing demethylasterriquinone B1 (DMAQ-B1), a potent insulin mimetic, in combination with AKT inhibitor VIII. Activation of this coordinated response additively reduced ZIKV levels in Aedes aegypti. This effect included a quantitatively greater reduction in salivary gland ZIKV levels up to 11 d post-bloodmeal ingestion, relative to single pathway activation. Together, our study indicates the potential for field delivery of these small molecules to substantially reduce virus transmission from mosquito to human. As infections like Zika virus are becoming more burdensome and prevalent, understanding how to control this family of viruses in the insect vector is an important issue in public health.
| S-EPMC9017935 | biostudies-literature
Project description:Vector ecology is integral to understanding the transmission of vector-borne diseases, with processes such as reproduction and competition pivotal in determining vector presence and abundance. The arbovirus vectors Aedes aegypti and Aedes albopictus compete as larvae, but this mechanism is insufficient to explain patterns of coexistence and exclusion. Inviable interspecies matings-known as reproductive interference-is another candidate mechanism. Here, we analyse mathematical models of mosquito population dynamics and epidemiology which include two Aedes-specific features of reproductive interference. First, as these mosquitoes use hosts to find mates, reproductive interference will only occur if the same host is visited. Host choice will, in turn, be determined by behavioural responses to host availability. Second, females can become sterilized after mis-mating with heterospecifics. We find that a species with an affinity for a shared host will suffer more from reproductive interference than a less selective competitor. Costs from reproductive interference can be 'traded-off' against costs from larval competition, leading to competitive outcomes that are difficult to predict from empirical evidence. Sterilizations of a self-limiting species can counterintuitively lead to higher densities than a competitor suffering less sterilization. We identify that behavioural responses and reproductive interference mediate a concomitant relationship between vector ecological dynamics and epidemiology. Competitors with opposite behavioural responses can maintain disease where human hosts are rare, due to vector coexistence facilitated by a reduced cost from reproductive interference. Our work elucidates the relative roles of the competitive mechanisms governing Aedes populations and the associated epidemiological consequences.
| S-EPMC6685014 | biostudies-literature