Cytoskeleton-associated gelsolin responds to the midgut distention process in saline meal-fed Aedes aegypti and affects arbovirus dissemination from the midgut.

The mosquito, Aedes aegypti, is the principal vector for several arboviruses. The mosquito midgut is the initial tissue that gets infected with an arbovirus acquired along with a blood meal from a vertebrate host. Blood meal ingestion leads to midgut tissue distention thereby increasing the pore size of the surrounding basal lamina. This allows newly synthesized virions to exit the midgut by traversing the distended basal lamina to infect secondary tissues of the mosquito. We conducted a quantitative label-free proteomic time course analysis with saline meal-fed Ae. aegypti females to identify host factors involved in midgut tissue distention. Around 2000 proteins were detected during each of the seven sampling time points and 164 of those were uniquely expressed. Forty-five of 97 differentially expressed proteins were upregulated during the 96-h time course and most of those were involved in cytoskeleton modulation, metabolic activity, and vesicle/vacuole formation. The F-actin-modulating Ae. aegypti (Aa)-gelsolin was selected for further functional studies. Stable knockout of Aa-gelsolin resulted in a mosquito line, which showed distorted actin filaments in midgut-associated tissues likely due to diminished F-actin processing by gelsolin. Zika virus dissemination from the midgut of these mosquitoes was diminished and delayed. The loss of Aa-gelsolin function was associated with an increased induction of apoptosis in midgut tissue indicating an involvement of Aa-gelsolin in apoptotic signaling in mosquitoes. Here, we used proteomics to discover a novel host factor, Aa-gelsolin, which affects the midgut escape barrier for arboviruses in mosquitoes and apoptotic signaling in the midgut.

Cellular diversity and gene expression profiles in the male and female brain of Aedes aegypti.

BACKGROUND: Aedes aegypti is a medically-important mosquito vector that transmits arboviruses including yellow fever, dengue, chikungunya, and Zika viruses to humans. The mosquito exhibits typical sexually dimorphic behaviors such as courtship, mating, host seeking, bloodfeeding, and oviposition. All these behaviors are mainly regulated by the brain; however, little is known about the function and neuron composition of the mosquito brain. In this study, we generated an initial atlas of the adult male and female brain of Ae. aegypti using 10xGenomics based single-nucleus RNA sequencing. RESULTS: We identified 35 brain cell clusters in male and female brains, and 15 of those clusters were assigned to known cell types. Identified cell types include glia (astrocytes), Kenyon cells, (ventral) projection neurons, monoaminergic neurons, medulla neurons, and proximal medulla neurons. In addition, the cell type compositions of male and female brains were compared to each other showing that they were quantitatively distinct, as 17 out of 35 cell clusters varied significantly in their cell type proportions. Overall, the transcriptomes from each cell cluster looked very similar between the male and female brain as only up to 25 genes were differentially expressed in these clusters. The sex determination factor Nix was highly expressed in neurons and glia of the male brain, whereas doublesex (dsx) was expressed in all neuron and glia cell clusters of the male and female brain. CONCLUSIONS: An initial cell atlas of the brain of the mosquito Ae. aegypti has been generated showing that the cellular compositions of the male and female brains of this hematophagous insect differ significantly from each other. Although some of the rare brain cell types have not been detected in our single biological replicate, this study provides an important basis for the further development of a complete brain cell atlas as well as a better understanding of the neurobiology of the brains of male and female mosquitoes and their sexually dimorphic behaviors.

Quantitative Proteomic Analysis of Chikungunya Virus-Infected Aedes aegypti Reveals Proteome Modulations Indicative of Persistent Infection.

The mosquito-borne chikungunya virus (CHIKV) poses a threat to human health in tropical countries throughout the world. The molecular interactions of CHIKV with its mosquito vector Aedes aegypti are not fully understood. Following oral acquisition of CHIKV via salinemeals, we analyzed changes in the proteome of Ae. aegypti in 12 h intervals by label-free quantification using a timsTOF Pro mass spectrometer. For each of the seven time points, between 2647 and 3167 proteins were identified among CHIKV-infected and noninfected mosquito samples, and fewer than 6% of those identified proteins were affected by the virus. Functional enrichment analysis revealed that the three pathways, Endocytosis, Oxidative phosphorylation, and Ribosome biogenesis, were enriched during CHIKV infection. On the other hand, three pathways of the cellular RNA machinery and five metabolism related pathways were significantly attenuated in the CHIKV-infected samples. Furthermore, proteins associated with cytoskeleton and vesicular transport, as well as various serine-type endopeptidases and metallo-proteinases, were modulated in the presence of CHIKV. Our study reveals biological pathways and novel proteins interacting with CHIKV in the mosquito. Overall, CHIKV infection caused minor changes to the mosquito proteome demonstrating a high level of adaption between the vector and the virus, essentially coexisting in a nonpathogenic relationship. The mass spectrometry data have been deposited to the MassIVE repository (https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=abfd14f7015243c69854731998d55df1) with the data set identifier MSV000085115.

Vulnerability of primitive human placental trophoblast to Zika virus.

Infection of pregnant women by Asian lineage strains of Zika virus (ZIKV) has been linked to brain abnormalities in their infants, yet it is uncertain when during pregnancy the human conceptus is most vulnerable to the virus. We have examined two models to study susceptibility of human placental trophoblast to ZIKV: cytotrophoblast and syncytiotrophoblast derived from placental villi at term and colonies of trophoblast differentiated from embryonic stem cells (ESC). The latter appear to be analogous to the primitive placenta formed during implantation. The cells from term placentas, which resist infection, do not express genes encoding most attachment factors implicated in ZIKV entry but do express many genes associated with antiviral defense. By contrast, the ESC-derived trophoblasts possess a wide range of attachment factors for ZIKV entry and lack components of a robust antiviral response system. These cells, particularly areas of syncytiotrophoblast within the colonies, quickly become infected, produce infectious virus and undergo lysis within 48 h after exposure to low titers (multiplicity of infection > 0.07) of an African lineage strain (MR766 Uganda: ZIKVU) considered to be benign with regards to effects on fetal development. Unexpectedly, lytic effects required significantly higher titers of the presumed more virulent FSS13025 Cambodia (ZIKVC). Our data suggest that the developing fetus might be most vulnerable to ZIKV early in the first trimester before a protective zone of mature villous trophoblast has been established. Additionally, MR766 is highly trophic toward primitive trophoblast, which may put the early conceptus of an infected mother at high risk for destruction.

The midgut transcriptome of Aedes aegypti fed with saline or protein meals containing chikungunya virus reveals genes potentially involved in viral midgut escape.

BACKGROUND: The mosquito Aedes aegypti is the primary vector for medically important arthropod-borne viruses, including chikungunya virus (CHIKV). Following oral acquisition, an arbovirus has to persistently infect several organs in the mosquito before becoming transmissible to another vertebrate host. A major obstacle an arbovirus has to overcome during its infection cycle inside the mosquito is the midgut escape barrier, representing the exit mechanism arboviruses utilize when disseminating from the midgut. To understand the transcriptomic basis of midgut escape and to reveal genes involved in the process, we conducted a comparative transcriptomic analysis of midgut samples from mosquitoes which had received a saline meal (SM) or a protein meal (PM) (not) containing CHIKV. RESULTS: CHIKV which was orally acquired by a mosquito along with a SM or PM productively infected the midgut epithelium and disseminated to secondary tissues. A total of 27 RNA-Seq libraries from midguts of mosquitoes that had received PM or SM (not) containing CHIKV at 1 and 2 days post-feeding were generated and sequenced. Fewer than 80 genes responded differentially to the presence of CHIKV in midguts of mosquitoes that had acquired the virus along with SM or PM. SM feeding induced differential expression (DE) of 479 genes at day 1 and 314 genes at day 2 when compared to midguts of sugarfed mosquitoes. By comparison, PM feeding induced 6029 DE genes at day 1 and 7368 genes at day 2. Twenty-three DE genes encoding trypsins, metalloproteinases, and serine-type endopeptidases were significantly upregulated in midguts of mosquitoes at day 1 following SM or PM ingestion. Two of these genes were Ae. aegypti late trypsin (AeLT) and serine collagenase 1 precursor (AeSP1). In vitro, recombinant AeLT showed strong matrix metalloproteinase activity whereas recombinant AeSP1 did not. CONCLUSIONS: By substituting a bloodmeal for SM, we identified midgut-expressed genes not involved in blood or protein digestion. These included genes coding for trypsins, metalloproteinases, and serine-type endopeptidases, which could be involved in facilitating midgut escape for arboviruses in Ae. aegypti. The presence of CHIKV in any of the ingested meals had relatively minor effects on the overall gene expression profiles in midguts.

A safe insect-based Chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques.

Chikungunya virus (CHIKV), which induces chikungunya fever and chronic arthralgia, is an emerging public health concern. Safe and efficient vaccination strategies are needed to prevent or mitigate virus-associated acute and chronic morbidities for preparation of future outbreaks. Eilat (EILV)/CHIKV, a chimeric alphavirus which contains the structural proteins of CHIKV and the non-structural proteins of EILV, does not replicate in vertebrate cells. The chimeric virus was previously reported to induce protective adaptive immunity in mice. Here, we assessed the capacity of the virus to induce quick and durable protection in cynomolgus macaques. EILV/CHIKV protected macaques from wild-type (WT) CHIKV infection one year after a single dose vaccination. Transcriptome and in vitro functional analyses reveal that the chimeric virus triggered toll-like receptor signaling and T cell, memory B cell and antibody responses in a dose-dependent manner. Notably, EILV/CHIKV preferentially induced more durable, robust, and broader repertoire of CHIKV-specific T cell responses, compared to a live attenuated CHIKV 181/25 vaccine strain. The insect-based chimeric virus did not cause skin hypersensitivity reactions in guinea pigs sensitized to mosquito bites. Furthermore, EILV/CHIKV induced strong neutralization antibodies and protected cynomolgus macaques from WT CHIKV infection within six days post vaccination. Transcriptome analysis also suggest that the chimeric virus induction of multiple innate immune pathways, including Toll-like receptor signaling, type I IFN and IL-12 signaling, antigen presenting cell activation, and NK receptor signaling. Our findings suggest that EILV/CHIKV is a safe, highly efficacious vaccine, and provides both rapid and long-lasting protection in cynomolgus macaques.

Isolation of midgut escape mutants of two American genotype dengue 2 viruses from Aedes aegypti.

BACKGROUND: Several studies have shown that American genotype dengue 2 viruses (DENV2) have reduced viral fitness in the mosquito vector, Aedes aegypti, compared to other DENV2 genotypes. Diminished replication efficiency or inability to efficiently traverse membrane barriers encompassing organs such as the midgut or salivary glands are considered major factors negatively impacting viral fitness in the mosquito. RESULTS: We analyzed the vector competence of Ae. aegypti for two American DENV2 strains, QR94 and PR159 originating from Mexico and Puerto-Rico, respectively. Both strains infected mosquito midguts following acquisition of infectious bloodmeals. However, DENV2-QR94 and DENV2-PR159 poorly disseminated from the midgut at 7 or 14 days post-bloodmeal (pbm). We detected one virus isolate, EM33, among 31 DENV2-QR94 infected mosquitoes, and one isolate, EM41, among 121 DENV2-PR159 infected mosquitoes, generating high virus titers in mosquito carcasses at 7 days pbm. In oral challenge experiments, EM33 and EM41 showed midgut dissemination rates of 40-50%. Replication efficiency of EM41 in secondary mosquito tissue was similar to that of a dissemination-competent control strain, whereas the replication efficiency of EM33 was significantly lower than that of the control virus. The genome sequence of DENV2-QR94 encoded seven unique amino acids (aa), which were not found in 100 of the most closely related DENV2 strains. EM33 had one additional aa change, E202K, in the E protein. DENV2-PR159 encoded four unique aa residues, one of them E202K, whereas EM41 had two additional aa substitutions, Q77E in the E protein and E93D in NS3. CONCLUSIONS: Our results indicate that the midgut of Ae. aegypti acts as a selective sieve for DENV2 in which genetically distinct, dissemination-competent virus variants are rapidly selected from the viral quasispecies to be transmitted to vertebrates.

Detection of La Crosse Virus In Situ and in Individual Progeny to Assess the Vertical Transmission Potential in Aedes albopictus and Aedes aegypti.

La Crosse virus (LACV) is circulating in the midwestern and southeastern states of the United States and can cause human encephalitis. The main vector of the virus is the eastern tree-hole mosquito, Aedes triseriatus. Ae. albopictus has been also described as a natural LACV vector, while Ae. aegypti has been infected with the virus under laboratory conditions. Here, we compare the vertical transmission potential of LACV in Ae. albopictus and Ae. aegypti, with emphasis given to the ovarian infection patterns that the virus generates in both species. Both mosquito species received artificial bloodmeals containing LACV. At defined time points post-infection/bloodmeal, midguts, head tissue, and ovaries were analyzed for the presence of virus. Viral infection patterns in the ovaries were visualized via immunofluorescence confocal microscopy and immunohistopathology assays using an LACV-specific monoclonal antibody. In Ae. aegypti, LACV was confronted with midgut infection and escape barriers, which were much less pronounced in Ae. albopictus, resulting in a significantly higher prevalence of infection in the latter. Following the ingestion of a single virus-containing bloodmeal, no progeny larvae were found to be virus-infected. Regardless, females of both species showed the presence of LACV antigen in their ovariole sheaths. Furthermore, in a single Ae. albopictus female, viral antigen was associated with the nurse cells inside the primary follicles. Following the ingestion of a second non-infectious bloodmeal at 7- or 10-days post-ingestion of an LACV-containing bloodmeal, more progeny larvae of Ae. albopictus than of Ae. aegypti were virus-infected. LACV antigen was detected in the egg chambers and ovariole sheaths of both mosquito species. Traces of viral antigen were also detected in a few oocytes from Ae. albopictus. The low level of vertical transmission and the majority of the ovarian infection patterns suggested the transovum rather than transovarial transmission (TOT) of the virus in both vector species. However, based on the detection of LACV antigen in follicular tissue and oocytes, there was the potential for TOT among several Ae. albopictus females. Thus, TOT is not a general feature of LACV infection in mosquitoes. Instead, the TOT of LACV seems to be dependent on its particular interaction with the reproductive tissues of a female.

Transgene-induced cell death following dengue-2 virus infection in Aedes aegypti.

Dengue viruses (DENVs) are mosquito-borne flaviviruses causing millions of human infections each year and pose a challenge for public health systems worldwide. Aedes aegypti is the principal vector species transmitting DENVs to humans. Controlling Ae. aegypti is difficult due to the abundance of breeding sites and increasing insecticide resistance in the vector populations. Developing new vector control strategies is critical for decreasing the disease burden. One potential approach is genetically replacing Ae. aegypti populations with vector populations highly resistant to DENV transmission. Here, we focus on an alternative strategy for generating dengue 2 virus (DENV-2) resistance in genetically-modified Ae. aegypti in which the mosquitoes express an inactive form of Michelob_x (Mx), an antagonist of the Inhibitor of Apoptosis (IAP), to induce apoptosis in those cells in which actively replicating DENV-2 is present. The inactive form of Mx was flanked by the RRRRSAG cleavage motif, which was recognized by the NS2B/NS3 protease of the infecting DENV-2 thereby releasing and activating Mx which then induced apoptosis. Our transgenic strain exhibited a significantly higher mortality rate than the non-transgenic control when infected with DENV-2. We also transfected a DNA construct containing inactive Mx fused to eGFP into C6/36 mosquito cells and indirectly observed Mx activation on days 3 and 6 post-DENV-2 infections. There were clear signs that the viral NS2B/NS3 protease cleaved the transgene, thereby releasing Mx protein into the cytoplasm, as was confirmed by the detection of eGFP expression in infected cells. The present study represents proof of the concept that virus infection can be used to induce apoptosis in infected mosquito cells.

Identification of the extracellular metallo-endopeptidases ADAM and ADAMTS in the yellow fever mosquito Aedes aegypti.

The mosquito Aedes aegypti is a major vector for dengue, Zika, yellow fever, and chikungunya (CHIKV) viruses, which cause significant morbidity and mortality among human populations in the tropical regions of the world. Following ingestion of a viremic bloodmeal from a vertebrate host, an arbovirus needs to productively infect the midgut epithelium of the mosquito. De novo synthesized virions then exit the midgut by traversing the surrounding basal lamina (BL) in order to disseminate to secondary tissues and infect those. Once the salivary glands are infected, the virus is transmitted to a vertebrate host along with saliva released during probing of the mosquito. Midgut tissue distention due to bloodmeal ingestion leads to remodeling of the midgut structure and facilitates virus dissemination from the organ. Previously, we described the matrix-metalloproteinases (MMP) of Ae. aegypti as zinc ion dependent endopeptidases (Metzincins) and showed MMP activity during midgut BL rearrangement as a consequence of bloodmeal ingestion and subsequent digestion thereby affecting arbovirus dissemination from the midgut. Here we investigate the ADAM/ADAMTS of Ae. aegypti, which form another major group of multi-domain proteinases within the Metzincin superfamily and are active during extra-cellular matrix (ECM) remodeling. Seven different ADAM and five ADAMTS were identified in Ae. aegypti. The functional protein domain structures of the identified mosquito ADAM resembled those of human ADAM10, ADAM12, and ADAM17, while two of the five mosquito ADAMTS had human orthologs. Expression profiling of Ae. aegypti ADAM/ADAMTS in immature forms, whole body-females, midguts, and ovarian tissues showed transcriptional activity of the proteinases during metamorphosis, bloodmeal ingestion/digestion, and female reproduction. Custom-made antibodies to ADAM10a and ADAM12c showed that both were strongly expressed in midgut and ovarian tissues. Furthermore, transient silencing of ADAM12c significantly reduced the carcass infection rate with CHIKV at 24 h post-infection, while silencing of ADAM12a significantly increased viral titers in secondary tissues at the same time point. Our results indicate a functional specificity for several ADAM/ADAMTS in those selected mosquito tissues.

Assessing single-locus CRISPR/Cas9-based gene drive variants in the mosquito Aedes aegypti via single-generation crosses and modeling.

The yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika viruses. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus is amenable to both gene drive and appropriate expression of the antiviral effector. In our study, we used reverse engineering to target 2 intergenic genomic loci, which had previously shown to be highly permissive for antiviral effector gene expression, and we further investigated the use of 3 promoters (nanos, ß2-tubulin, or zpg) for Cas9 expression. We then quantified the accrual of insertions or deletions (indels) after single-generation crossings, measured maternal effects, and assessed fitness costs associated with various transgenic lines to model the rate of gene drive fixation. Overall, MGDrivE modeling suggested that when an autonomous gene drive is placed into an intergenic locus, the gene drive system will eventually be blocked by the accrual of gene drive blocking resistance alleles and ultimately be lost in the population. Moreover, while genomic locus and promoter selection were critically important for the initial establishment of the autonomous gene drive, it was the fitness of the gene drive line that most strongly influenced the persistence of the gene drive in the simulated population. As such, we propose that when autonomous CRISPR/Cas9-based gene drive systems are anchored in an intergenic locus, they temporarily result in a strong population replacement effect, but as gene drive-blocking indels accrue, the gene drive becomes exhausted due to the fixation of CRISPR resistance alleles.

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.

Comparison of transgene expression in Aedes aegypti generated by mariner Mos1 transposition and ΦC31 site-directed recombination.

Transgenic mosquitoes generated by transposable elements (TEs) often poorly express transgenes owing to position effects. To avoid these effects, the ΦC31 site-directed recombination system was used to insert transgenes into a locus favourable for gene expression in Aedes aegypti. We describe phenotypes of mariner Mos1 TE and ΦC31 transgenic mosquitoes expressing the enhanced green fluorescent protein (EGFP) reporter in midguts of blood-fed females. Mosquitoes of nine TE-generated lines [estimated transformation frequency (TF): 9.3%] clearly expressed the eye-specific selection marker but only 2/9 lines robustly expressed the EGFP reporter. The piggyBac TE-generated ΦC31 docking strain, attP26, supported recombination with attB site containing donors at an estimated TF of 1.7-4.9%. Using a codon-optimized ΦC31 integrase mutant instead of the 'wild-type' enzyme did not affect TF. Site-directed recombination of line attP26 with an attB-containing donor expressing EGFP from the Ae. aegypti carboxypeptidase promoter produced one transgenic line with blood-fed females expressing the reporter in midgut tissue. Docking strain attP26 also supported robust expression of Flock House virus B2 from the Ae. aegypti polyubiquitin promoter. Our data confirm that eye-specific selection marker expression alone is not a reliable indicator for robust gene-of-interest expression in Ae. aegypti and that the ΦC31 system can ensure predictable transgene expression in this mosquito species.

Current Effector and Gene-Drive Developments to Engineer Arbovirus-Resistant Aedes aegypti (Diptera: Culicidae) for a Sustainable Population Replacement Strategy in the Field.

Arthropod-borne viruses (arboviruses) such as dengue, Zika, and chikungunya viruses cause morbidity and mortality among human populations living in the tropical regions of the world. Conventional mosquito control efforts based on insecticide treatments and/or the use of bednets and window curtains are currently insufficient to reduce arbovirus prevalence in affected regions. Novel, genetic strategies that are being developed involve the genetic manipulation of mosquitoes for population reduction and population replacement purposes. Population replacement aims at replacing arbovirus-susceptible wild-type mosquitoes in a target region with those that carry a laboratory-engineered antiviral effector to interrupt arboviral transmission in the field. The strategy has been primarily developed for Aedes aegypti (L.), the most important urban arbovirus vector. Antiviral effectors based on long dsRNAs, miRNAs, or ribozymes destroy viral RNA genomes and need to be linked to a robust gene drive to ensure their fixation in the target population. Synthetic gene-drive concepts are based on toxin/antidote, genetic incompatibility, and selfish genetic element principles. The CRISPR/Cas9 gene editing system can be configurated as a homing endonuclease gene (HEG) and HEG-based drives became the preferred choice for mosquitoes. HEGs are highly allele and nucleotide sequence-specific and therefore sensitive to single-nucleotide polymorphisms/resistant allele formation. Current research efforts test new HEG-based gene-drive designs that promise to be less sensitive to resistant allele formation. Safety aspects in conjunction with gene drives are being addressed by developing procedures that would allow a recall or overwriting of gene-drive transgenes once they have been released.

Intrathoracic Inoculation of Zika Virus in Aedes aegypti.

Aedes aegypti mosquitoes are the main vectors of many medically relevant arthropod-borne (arbo) viruses, including Zika (ZIKV), dengue (DENV), and yellow fever (YFV). Vector competence studies with Ae. aegypti often involve challenging mosquitoes with an artificial bloodmeal containing virus and later quantifying viral titer or infectious plaque-forming units (PFU) in various mosquito tissues at relevant time points post-infection. However, Ae. aegypti mosquitoes are known to exhibit midgut infection and escape barriers (MIB and MEB, respectively), which influence the prevalence and titer of a disseminated infection and can introduce unwanted variability into studies analyzing tissues such as the salivary glands. To surmount this challenge, we describe herein a protocol for the intrathoracic inoculation of ZIKV in Ae. aegypti. This method bypasses the midgut, which leads to a more rapid and higher proportion of disseminated infections in comparison to oral challenge, and mosquitoes become infected with a consistent dose of virus. Our protocol is advantageous for studies that need a large sample size of infected mosquitoes, need to bypass the midgut, or are analyzing salivary gland infection or escape barriers. Graphic abstract: Cartoon depiction of Aedes aegypti intrathoracic inoculation. Figure made with Biorender.com.

Starvation at the larval stage increases the vector competence of Aedes aegypti females for Zika virus.

Aedes aegypti is the primary vector of Zika virus (ZIKV), a flavivirus which typically presents itself as febrile-like symptoms in humans but can also cause neurological and pregnancy complications. The transmission cycle of mosquito-borne arboviruses such as ZIKV requires that various key tissues in the female mosquito get productively infected with the virus before the mosquito can transmit the virus to another vertebrate host. Following ingestion of a viremic blood-meal from a vertebrate, ZIKV initially infects the midgut epithelium before exiting the midgut after blood-meal digestion to disseminate to secondary tissues including the salivary glands. Here we investigated whether smaller Ae. aegypti females resulting from food deprivation as larvae exhibited an altered vector competence for blood-meal acquired ZIKV relative to larger mosquitoes. Midguts from small 'Starve' and large 'Control' Ae. aegypti were dissected to visualize by transmission electron microscopy (TEM) the midgut basal lamina (BL) as physical evidence for the midgut escape barrier showing Starve mosquitoes with a significantly thinner midgut BL than Control mosquitoes at two timepoints. ZIKV replication was inhibited in Starve mosquitoes following intrathoracic injection of virus, however, Starve mosquitoes exhibited a significantly higher midgut escape and population dissemination rate at 9 days post-infection (dpi) via blood-meal, with more virus present in saliva and head tissue than Control by 10 dpi and 14 dpi, respectively. These results indicate that Ae. aegypti developing under stressful conditions potentially exhibit higher midgut infection and dissemination rates for ZIKV as adults, Thus, variation in food intake as larvae is potentially a source for variable vector competence levels of the emerged adults for the virus.

The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti.

BACKGROUND: The RNA interference (RNAi) pathway acts as an innate antiviral immune response in Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus) is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity. RESULTS: As a novel tool for studying arbovirus-mosquito interactions, we engineered a transgenic mosquito line with an impaired RNAi pathway in the midgut of bloodfed females by silencing expression of the Aa-dcr2 gene. In midgut tissue of the transgenic Carb/dcr16 line, Aa-dcr2 expression was reduced approximately 50% between 1-7 days post-bloodmeal (pbm) when compared to the recipient mosquito strain. After infection with SINV-TR339EGFP, Aa-dcr2 expression levels were enhanced in both mosquito strains. In the RNAi pathway impaired mosquito strain SINV titers and midgut infection rates were significantly higher at 7 days pbm. There was also a strong tendency for increased virus dissemination rates among the transgenic mosquitoes. Between 7-14 days pbm, SINV was diminished in midgut tissue of the transgenic mosquitoes. Transgenic impairment of the RNAi pathway and/or SINV infection did not affect longevity of the mosquitoes. CONCLUSIONS: We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level. Following ingestion by Ae. aegypti, the recombinant SINV-TR339EGFP was confronted with both MEB and a midgut infection barrier (MIB). Impairment of the RNAi pathway in the midgut strongly reduced both midgut barriers for the virus. This confirms that the endogenous RNAi pathway of Ae. aegypti modulates vector competence for SINV in the midgut. The RNAi pathway acts as a gatekeeper to the incoming virus by affecting infection rate of the midgut, intensity of infection, and dissemination from the midgut to secondary tissues.

Heterogeneity of midgut cells and their differential responses to blood meal ingestion by the mosquito, Aedes aegypti.

Mosquitoes are the most notorious hematophagous insects and due to their blood feeding behavior and genetic compatibility, numerous mosquito species are highly efficient vectors for certain human pathogenic parasites and viruses. The mosquito midgut is the principal organ of blood meal digestion and nutrient absorption. It is also the initial site of infection with blood meal acquired parasites and viruses. We conducted an analysis based on single-nucleus RNA sequencing (snRNA-Seq) to assess the cellular diversity of the midgut and how individual cells respond to blood meal ingestion to facilitate its digestion. Our study revealed the presence of 20 distinguishable cell-type clusters in the female midgut of Aedes aegypti. The identified cell types included intestinal stem cells (ISC), enteroblasts (EB), differentiating EB (dEB), enteroendocrine cells (EE), enterocytes (EC), EC-like cells, cardia cells, and visceral muscle (VM) cells. Blood meal ingestion dramatically changed the overall midgut cell type composition, profoundly increasing the proportions of ISC and three EC/EC-like clusters. In addition, transcriptional profiles of all cell types were strongly affected while genes involved in various metabolic processes were significantly upregulated. Our study provides a basis for further physiological and molecular studies on blood digestion, nutrient absorption, and cellular homeostasis in the mosquito midgut.

Antiviral Effectors and Gene Drive Strategies for Mosquito Population Suppression or Replacement to Mitigate Arbovirus Transmission by Aedes aegypti.

The mosquito vector Aedes aegypti transmits arthropod-borne viruses (arboviruses) of medical importance, including Zika, dengue, and yellow fever viruses. Controlling mosquito populations remains the method of choice to prevent disease transmission. Novel mosquito control strategies based on genetically manipulating mosquitoes are being developed as additional tools to combat arbovirus transmission. Genetic control of mosquitoes includes two basic strategies: population suppression and population replacement. The former aims to eliminate mosquito populations while the latter aims to replace wild populations with engineered, pathogen-resistant mosquitoes. In this review, we outline suppression strategies being applied in the field, as well as current antiviral effector genes that have been characterized and expressed in transgenic Ae. aegypti for population replacement. We discuss cutting-edge gene drive technologies that can be used to enhance the inheritance of effector genes, while highlighting the challenges and opportunities associated with gene drives. Finally, we present currently available models that can estimate mosquito release numbers and time to transgene fixation for several gene drive systems. Based on the recent advances in genetic engineering, we anticipate that antiviral transgenic Ae. aegypti exhibiting gene drive will soon emerge; however, close monitoring in simulated field conditions will be required to demonstrate the efficacy and utility of such transgenic mosquitoes.

The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic Aedes aegypti.

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.