Aag2 Cells Research Articles

Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Mosquito E-20-Monooxygenase Gene Knockout Increases Dengue Virus Replication in Aedes aegypti Cells.

E-20-monooxygenase (E20MO) is an enzymatic product of the shade (shd) locus (cytochrome p450, E20MO). Initially discovered in Drosophila, E20MO facilitates the conversion of ecdysone (E) into 20-hydroxyecdysone (20E) and is crucial for oogenesis. Prior research has implicated 20E in growth, development, and insecticide resistance. However, little attention has been given to the association between the E20MO gene and DENV2 infection. The transcriptome of Ae. aegypti cells (Aag2 cells) infected with DENV2 revealed the presence of the E20MO gene. The subsequent quantification of E20MO gene expression levels in Aag2 cells post-DENV infection was carried out. A CRISPR/Cas9 system was utilized to create an E20MO gene knockout cell line (KO), which was then subjected to DENV infection. Analyses of DENV2 copies in KO and wild-type (WT) cells were conducted at different days post-infection (dpi). Plasmids containing E20MO were constructed and transfected into KO cells, with pre- and post-transfection viral copy comparisons. Gene expression levels of E20MO increased after DENV infection. Subsequently, a successful generation of an E20MO gene knockout cell line and the verification of code-shifting mutations at both DNA and RNA levels were achieved. Furthermore, significantly elevated DENV2 RNA copies were observed in the mid-infection phase for the KO cell line. Viral RNA copies were lower in cells transfected with plasmids containing E20MO, compared to KO cells. Through knockout and plasmid complementation experiments in Aag2 cells, the role of E20MO in controlling DENV2 replication was demonstrated. These findings contribute to our understanding of the intricate biological interactions between mosquitoes and arboviruses.

Oxidative stress governs mosquito innate immune signalling to reduce chikungunya virus infection in Aedes-derived cells.

Arboviruses such as chikungunya, dengue and zika viruses cause debilitating diseases in humans. The principal vector species that transmits these viruses is the Aedes mosquito. Lack of substantial knowledge of the vector species hinders the advancement of strategies for controlling the spread of arboviruses. To supplement our information on mosquitoes' responses to virus infection, we utilized Aedes aegypti-derived Aag2 cells to study changes at the transcriptional level during infection with chikungunya virus (CHIKV). We observed that genes belonging to the redox pathway were significantly differentially regulated. Upon quantifying reactive oxygen species (ROS) in the cells during viral infection, we further discovered that ROS levels are considerably higher during the early hours of infection; however, as the infection progresses, an increase in antioxidant gene expression suppresses the oxidative stress in cells. Our study also suggests that ROS is a critical regulator of viral replication in cells and inhibits intracellular and extracellular viral replication by promoting the Rel2-mediated Imd immune signalling pathway. In conclusion, our study provides evidence for a regulatory role of oxidative stress in infected Aedes-derived cells.

Aedes aegypti Beta-1,3-Glucan-Binding Protein Inhibits Dengue and ZIKA Virus Replication.

GNBPB6, a beta-1,3-glucan-binding protein, was identified in the transcriptome of Aedes aegypti (A. aegypti) with dengue (DENV), Zika (ZIKV), and chikungunya viruses (CHIKV). In this study, we not only clarified that DENV2 and ZIKV regulate the changes in GNBPB6 expression but also identified the relationship of this gene with viral infections. The changes in GNBPB6 expression were quantified and showed a decrease in A. aegypti cells (Aag2 cells) at 2 dpi and 3 dpi and an increase at 4 dpi and 5 dpi (p < 0.05). A significant increase was observed only at 5 dpi after DENV2 infection. Subsequently, a GNBPB6 knockout (KO) cell line was constructed using the CRISPR/Cas9 system, and the DENV2 and ZIKV RNA copies, along with cell densities, were quantified and compared between the KO and wild type (WT) cells at different dpi. The result showed that DENV2 and ZIKV RNA copies were significantly increased in the KO cell line with no significant change in cell growth. Finally, DENV2 copies decreased after GNBPB6 was complemented in the KO. In conclusion, GNBPB6 knockout and complementation in Aag2 cells revealed that GNBPB6 can inhibit the replication of both DENV2 and ZIKV. These results contribute to subsequent research on mosquito-virus interactions.

642. The impact of novel E1:M407I mutation on replicative fitness of the Chikungunya virus ECSA-IOL strain during infection of mammalian and mosquito cells,

Abstract Background Chikungunya virus (CHIKV) is an Alpha virus which is transmitted by Aedes genus of mosquitoes. It causes febrile illness with severe arthralgia, myalgia and rashes. Since 2000, the re-emergence of this pathogen have made it a global health security threat as it has caused severe form of diseases with epidemics of large magnitude and also spread to European and American continent. Studies have identified different point mutations in E1 and E2 region associated with virulence and infectivity, which is behind these severe epidemics. Pakistan reported first CHIKV outbreak with more than 30,000 infections from its largest city in 2016 which is already endemic for Dengue virus (DENV). Methods 32 CHIKV PCR-positive serum samples were collected from molecular pathology section of DUHS followed by RNA extraction and amplifcation of E1 region by PCR. The amplified products were prurified and sanger sequenced by BigDye termination method. Mutation analyses &amp; Bayesian phylogeny was done by bioinformatic tools. CHIKV E1:M407I mutation was cloned in to CHIKV wild type infectious cDNA clone plasmid (SL-CK-1) by site directed mutagenesis (SDS). In vitro viral RNA was synthesized after plasmid linearization, which was electroporated into VERO-CCL-81 cells. M407I mutant virus was recovered from the culture, titrated by plaque assay and sanger sequenced to confirmed the M407I mutation. Viral competition assay was performed in mammalian and mosquitoes cell lines to determine the relative replicative fitness of mutant CHIKV virus over wild type. Results Sequence analysis revealed 7 unique mutations in E1 including one mutation (M407I) in more than half of the sequenced samples. Phylogenetic analyses revealed this outbreak was caused by CHIKV-IOL genotype with possible inception from India. Virus competition assay showed relative replicative fitness for CHIKV E1:M407I mutant in Vero-CLL-81 and AAG2 cells have greater advantage while in C6/36, it has slight advantage over wild type virus. Conclusion This study reports a novel mutations in E1 protein of CHIKV with fitness advantage over wild type strain. Phylogeny revealed the outbreak was caused by A226-IOL strain with possible origin from ancestral strain of Indian outbreak. Disclosures All Authors: No reported disclosures

Cell fusing agent virus isolated from Aag2 cells does not vertically transmit in Aedes aegypti via artificial infection

BackgroundCell fusing agent virus (CFAV) was the first insect-specific virus to be characterized, and has been reported to negatively influence the growth of arboviruses such as dengue, Zika, and La Cross, making it a promising biocontrol agent for mosquito-borne disease prevention. Aedes aegypti Aag2 cells were naturally infected with CFAV. However, the ability of this virus to stably colonize an Ae. aegypti population via artificial infection and how it influences the vector competence of this mosquito have yet to be demonstrated.MethodsCFAV used in this study was harvested from Aag2 cells and its complete genome sequence was obtained by polymerase chain reaction and rapid amplification of complementary DNA ends, followed by Sanger sequencing. Phylogenetic analysis of newly identified CFAV sequences and other sequences retrieved from GenBank was performed. CFAV stock was inoculated into Ae. aegypti by intrathoracic injection, the survival of parental mosquitoes was monitored and CFAV copies in the whole bodies, ovaries, and carcasses of the injected F0 generation and in the whole bodies of the F1 generation on different days were examined by reverse transcription-quantitative polymerase chain reaction.ResultsThe virus harvested from Aag2 cells comprised a mixture of three CFAV strains. All genome sequences of CFAV derived from Aag2 cells clustered into one clade but were far from those isolated or identified from Ae. aegypti. Aag2-derived CFAV efficiently replicated in the mosquito body and did not attenuate the survival of Ae. aegypti. However, the viral load in the ovarian tissues was much lower than that in other tissues and the virus could not passage to the offspring by vertical transmission.ConclusionsThe results of this study demonstrate that Aag2-derived CFAV was not vertically transmitted in Ae. aegypti and provide valuable information on the colonization of mosquitoes by this virus.Graphical abstract

Mosquito CYP4C21 knockout reduces dengue virus and Zika virus replication in Aedes aegypti cells

Aedes aegypti (Ae. aegypti) is a major vector of dengue virus (DENV) and Zika virus (ZIKV). Understanding the complex interaction mechanisms between mosquito vectors and arboviruses is essential to interrupt virus transmission. This study constructed CYP4C21 knockout (KO) Aag2 cells (Ae. aegypti cells) and confirmed that CYP4C21 KO reduced DENV2 and ZIKV copies in Aag2 cells, which suggests that CYP4C21 may play an important role in mosquito infection with arboviruses. Furthermore, it is the first report of the CYP4 family related to viral infection, which lays the foundation for exploring the role of the CYP4C21 in the interaction of Ae. aegypti and arbovirus and provides novel insights into the function of cytochrome family proteins.

Rift Valley Fever Virus Primes Immune Responses in Aedes aegypti Cells.

The ongoing global emergence of arthropod-borne (arbo) viruses has accelerated research into the interactions of these viruses with the immune systems of their vectors. Only limited information exists on how bunyaviruses, such as Rift Valley fever virus (RVFV), are sensed by mosquito immunity or escape detection. RVFV is a zoonotic phlebovirus (Bunyavirales; Phenuiviridae) of veterinary and human public health and economic importance. We have shown that the infection of mosquitoes with RVFV triggers the activation of RNA interference pathways, which moderately restrict viral replication. Here, we aimed to better understand the interactions between RVFV and other vector immune signaling pathways that might influence RVFV replication and transmission. For this, we used the immunocompetent Aedes aegypti Aag2 cell line as a model. We found that bacteria-induced immune responses restricted RVFV replication. However, virus infection alone did not alter the gene expression levels of immune effectors. Instead, it resulted in the marked enhancement of immune responses to subsequent bacterial stimulation. The gene expression levels of several mosquito immune pattern recognition receptors were altered by RVFV infection, which may contribute to this immune priming. Our findings imply that there is a complex interplay between RVFV and mosquito immunity that could be targeted in disease prevention strategies.

Analysing inhibition of dengue virus in Wolbachia-infected mosquito cells following the removal of Wolbachia

Wolbachia pipientis is known to block replication of positive sense RNA viruses. Previously, we created an Aedes aegypti Aag2 cell line (Aag2.wAlbB) transinfected with the wAlbB strain of Wolbachia and a matching tetracycline-cured Aag2.tet cell line. While dengue virus (DENV) was blocked in Aag2.wAlbB cells, we found significant inhibition of DENV in Aag2.tet cells. RNA-Seq analysis of the cells confirmed removal of Wolbachia and lack of expression of Wolbachia genes that could have been due to lateral gene transfer in Aag2.tet cells. However, we noticed a substantial increase in the abundance of phasi charoen-like virus (PCLV) in Aag2.tet cells. When RNAi was used to reduce the PCLV levels, DENV replication was significantly increased. Further, we found significant changes in the expression of antiviral and proviral genes in Aag2.tet cells. Overall, the results reveal an antagonistic interaction between DENV and PCLV and how PCLV-induced changes could contribute to DENV inhibition.

The DEAD-box RNA helicase Dhx15 controls glycolysis and arbovirus replication in Aedes aegypti mosquito cells.

Aedes aegypti mosquitoes are responsible for the transmission of arthropod-borne (arbo)viruses including dengue and chikungunya virus (CHIKV) but in contrast to human hosts, arbovirus-infected mosquitoes are able to efficiently control virus replication to sub-pathological levels. Yet, our knowledge of the molecular interactions of arboviruses with their mosquito hosts is incomplete. Here, we aimed to identify and characterize novel host genes that control arbovirus replication in Aedes mosquitoes. RNA binding proteins (RBPs) are well-known to regulate immune signaling pathways in all kingdoms of life. We therefore performed a knockdown screen targeting 461 genes encoding predicted RBPs in Aedes aegypti Aag2 cells and identified 15 genes with antiviral activity against Sindbis virus. Amongst these, the three DEAD-box RNA helicases AAEL004419/Dhx15, AAEL008728, and AAEL004859 also acted as antiviral factors in dengue and CHIKV infections. Here, we explored the mechanism of Dhx15 in regulating an antiviral transcriptional response in mosquitoes by silencing Dhx15 in Aag2 cells followed by deep-sequencing of poly-A enriched RNAs. Dhx15 knockdown in uninfected and CHIKV-infected cells resulted in differential expression of 856 and 372 genes, respectively. Interestingly, amongst the consistently downregulated genes, glycolytic process was the most enriched gene ontology (GO) term as the expression of all core enzymes of the glycolytic pathway was reduced, suggesting that Dhx15 regulates glycolytic function. A decrease in lactate production indicated that Dhx15 silencing indeed functionally impaired glycolysis. Modified rates of glycolytic metabolism have been implicated in controlling the replication of several classes of viruses and strikingly, infection of Aag2 cells with CHIKV by itself also resulted in the decrease of several glycolytic genes. Our data suggests that Dhx15 regulates replication of CHIKV, and possibly other arboviruses, by controlling glycolysis in mosquito cells.

Identification of species-specific juvenile hormone response elements in the fall armyworm, Spodoptera frugiperda

Juvenile hormones (JH) regulate insect development and reproduction. The JH analogs (JHA) are used as insecticides. However, JHAs are rarely used in managing pests such as the fall armyworm, Spodoptera frugiperda that cause damage during larval stages. The insecticides that antagonize JH action and induce stoppage of feeding and precocious metamorphosis might work better to control these pests. Treating insects with JHA insecticides induces the expression of an early JH response gene, Krüppel homolog 1 (Kr-h1) by working through JH response elements (JHRE) present in its promoter. In this study, we identified JHREs present in the promoter of Kr-h1 gene of a global pest, S. frugiperda, and used them to develop a JHRE-reporter cell platform to screen for JH analogs. JHA, methoprene induced the expression of SfKr-h1 both in vitro and in vivo. JHRE present in the promoters of two SfKr-h1 isoforms, SfKr-h1α and SfKr-h1β were identified. In Sf9 cells, the knockout of isoform-specific JHRE affected JH response in an isoform-specific manner. We also found that S. frugiperda JHRE (SfJHRE) did not function in the mosquito Aedes aegypti Aag2 cells and Tribolium castaneum TcA cells. Similarly, Ae. aegypti AaJHRE and T. castaneum TcJHRE were only functional in cells derived from these insects. The nucleotide sequence at the 3'end to the conserved core JHRE E-box sequence seems to be responsible for the species specificity observed. Two stable cell lines expressing the luciferase and enhanced green fluorescent protein genes under the control of SfJHRE were established. These cell lines responded well to JHA; these two JHRE-reporter cell lines could be used in screening assays to identify insecticides to manage S. frugiperda and other major pests.

Sublethal exposure to spinetoram impacts life history traits and dengue virus replication in Aedes aegypti.

Insecticides are anthropogenic environmental stressors and also a common stressor for mosquito vectors. However, the use of insecticides is often guided by short-term efficacy, and the sublethal effect on their target or nontarget species has long been ignored. Here, we analyzed how sublethal exposure of the promising vector-control bioinsecticide spinetoram to Aedes aegypti larvae alter adult performance and susceptibility to dengue virus (DENV) infection. We found that the surviving adult mosquitoes were significantly smaller and exhibited weaker blood-feeding capacity than control females, apart from the extended immature development period. In terms of reproductive potential, although the F0 generation produced a similar number of eggs and offspring during the first gonotrophic cycle, the survival rates of the F1 generations were significantly lower as compared to the control group, suggesting transgenerational sublethal effects on the F1 generation. Notably, surviving adult females had higher DENV-2 viral loads than the control group after spinetoram sublethal exposure. Mechanistically, transcriptomic analysis showed that inhibition of oxidative phosphorylation may function in stimulating DENV production in adult Ae. aegypti. In Aag2 cells, significant accumulation of apoptosis, mitochondrial reactive oxygen species production, and DENV-2 replication by spinetoram exposure consistently support our conclusion. Our study highlights the threat of sublethal spinetoram exposure on outbreaks of mosquito-borne viruses.

The Involvement of Atlastin in Dengue Virus and Wolbachia Infection in Aedes aegypti and Its Regulation by aae-miR-989.

ABSTRACTEndoplasmic reticulum (ER)-shaping atlastin proteins (ATLs) have been demonstrated to play a functional role during flavivirus replication in mammalian cells. For dengue virus (DENV), atlastin is required in the formation of the replication organelles and RNA replication, virion assembly, production of the infectious virus particles, and trafficking or directing the association of vesicle packets with furin. Here, we investigated the involvement of atlastin in DENV replication in the mosquito Aedes aegypti and explored the possibility of its manipulation by the endosymbiotic bacterium Wolbachia to interfere with DENV replication. Results showed the expression of Ae. aegypti atlastin gene (AaATL) was upregulated in DENV-infected Aag2 cells, and its silencing led to reduced DENV replication. Contrary to our assumption that AaATL could be downregulated by Wolbachia, we did not find evidence for that in Wolbachia-infected cell lines, but this was the case in mosquitoes. Further, silencing AaATL did not have any effect on Wolbachia density. Our results also suggest that aae-miR-989 miRNA negatively regulates AaATL. The oversupply of the miRNA mimic led to reduced DENV replication consistent with the positive role of AaATL in DENV replication. Overall, the results favor AaATL’s involvement in DENV replication; however, there is no support that the protein is involved in Wolbachia-mediated DENV inhibition. In addition, the results contribute to discerning further possible overlapping functions of ATLs in mosquitoes and mammalian cells.IMPORTANCE Atlastin is a protein associated with the endoplasmic reticulum and has been shown to play a role in replication of flaviviruses in mammalian cells. This study aimed to investigate the role of mosquito Aedes aegypti atlastin (AaATL) in dengue virus replication and maintenance of Wolbachia, an endosymbiotic bacterium, in the mosquito. Our results suggest that AaATL facilitates dengue virus replication in mosquito cells, considering silencing the gene led to reductions in virus replication and virion production. Further, AaATL was found to be regulated by a mosquito microRNA, aae-miR-989. Despite an effect on dengue virus, AaATL silencing did not affect Wolbachia replication and maintenance in mosquito cells. The results shed light on the role of atlastins in mosquito-pathogen interactions and their overlapping roles in mosquito and mammalian cells.

Expression of fatty acid synthase genes and their role in development and arboviral infection of Aedes aegypti

BackgroundFatty acids are the building blocks of complex lipids essential for living organisms. In mosquitoes, fatty acids are involved in cell membrane production, energy conservation and expenditure, innate immunity, development and reproduction. Fatty acids are synthesized by a multifunctional enzyme complex called fatty acid synthase (FAS). Several paralogues of FAS were found in the Aedes aegypti mosquito. However, the molecular characteristics and expression of some of these paralogues have not been investigated.MethodsGenome assemblies of Ae. aegypti were analyzed, and orthologues of human FAS was identified. Phylogenetic analysis and in silico molecular characterization were performed to identify the functional domains of the Ae. aegypti FAS (AaFAS). Quantitative analysis and loss-of-function experiments were performed to determine the significance of different AaFAS transcripts in various stages of development, expression following different diets and the impact of AaFAS on dengue virus, serotype 2 (DENV2) infection and transmission.ResultsWe identified seven putative FAS genes in the Ae. aegypti genome assembly, based on nucleotide similarity to the FAS proteins (tBLASTn) of humans, other mosquitoes and invertebrates. Bioinformatics and molecular analyses suggested that only five of the AaFAS genes produce mRNA and therefore represent complete gene models. Expression levels of AaFAS varied among developmental stages and between male and female Ae. aegypti. Quantitative analyses revealed that expression of AaFAS1, the putative orthologue of the human FAS, was highest in adult females. Transient knockdown (KD) of AaFAS1 did not induce a complete compensation by other AaFAS genes but limited DENV2 infection of Aag2 cells in culture and the midgut of the mosquito.ConclusionAaFAS1 is the predominant AaFAS in adult mosquitoes. It has the highest amino acid similarity to human FAS and contains all enzymatic domains typical of human FAS. AaFAS1 also facilitated DENV2 replication in both cell culture and in mosquito midguts. Our data suggest that AaFAS1 may play a role in transmission of dengue viruses and could represent a target for intervention strategies.Graphical

N6-methyladenosine modification of the Aedes aegypti transcriptome and its alteration upon dengue virus infection in Aag2 cell line

The N6-methyladenosine (m6A) modification of RNA has been reported to affect viral infections. Studies have confirmed the role of m6A in replication of several vector-borne flaviviruses, including dengue virus (DENV), in mammalian cells. Here, we explored the role of m6A in DENV replication in the mosquito Aedes aegypti Aag2 cell line. We first determined the presence of m6A on the RNAs from mosquito cells and using methylated RNA immunoprecipitation and sequencing (MeRIP-Seq) identified m6A modification of the mosquito transcriptome and those that changed upon DENV infection. Depletion of m6A methyltransferases and the m6A binding protein YTHDF3 RNAs decreased the replication of DENV. In particular, we found that the Ae. aegypti ubiquitin carrier protein 9 (Ubc9) is m6A modified and its expression increases after DENV infection. Silencing of the gene and ectopic expression of Ubc9 led to reduced and increased DENV replication, respectively. The abundance of Ubc9 mRNA and its stability were reduced with the inhibition of m6A modification, implying that m6A modification of Ubc9 might enhance expression of the gene. We also show that the genome of DENV is m6A modified at five sites in mosquito cells. Altogether, this work reveals the involvement of m6A modification in Ae. aegypti-DENV interaction.

Aedes aegypti Aag-2 Cell Proteome Modulation in Response to Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is a single-stranded positive RNA virus that belongs to the genus Alphavirus and is transmitted to humans by infected Aedes aegypti and Aedes albopictus bites. In humans, CHIKV usually causes painful symptoms during acute and chronic stages of infection. Conversely, virus–vector interaction does not disturb the mosquito’s fitness, allowing a persistent infection. Herein, we studied CHIKV infection of Ae. aegypti Aag-2 cells (multiplicity of infection (MOI) of 0.1) for 48 h through label-free quantitative proteomic analysis and transmission electron microscopy (TEM). TEM images showed a high load of intracellular viral cargo at 48 h postinfection (hpi), as well as an unusual elongated mitochondria morphology that might indicate a mitochondrial imbalance. Proteome analysis revealed 196 regulated protein groups upon infection, which are related to protein synthesis, energy metabolism, signaling pathways, and apoptosis. These Aag-2 proteins regulated during CHIKV infection might have roles in antiviral and/or proviral mechanisms and the balance between viral propagation and the survival of host cells, possibly leading to the persistent infection.

Engineered anti-viral shRNAs are more effective than lhRNAs in transgenic Aedes aegypti

The Aedes aegypti mosquito is a major vector of chikungunya virus (CHIKV), which has no licensed vaccine. Engineered mosquitoes expressing long RNA hairpins (lhRNA) or small RNAs against selected arboviruses have been developed to limit virus replication, but their silencing efficiency has not been compared. We developed lhRNA and short hairpin RNA (shRNA) arrays against CHIKV non-structural protein nsP2. We used a Tet-response element (TRE) and a tTA transactivator to control expression of lhRNAs (TRE-lhRNA) and shRNAs (TRE-shRNA). Constitutive expression in Aag2 cells was assessed with a PUb-tTA driverand midgut specific expression in transgenic mosquitoes was assessed using Carboxypeptidase A (AeCPA)-tTA as driver. In vitrointerference ability was determined with a CHIKV split replication system, and a synthetic luciferase reporter with mRNA containing the targeted CHIKV sequence (CHIK-FF). In vivo interference was tested by inserting the TRE-lhRNA and TRE-shRNA constructs into a AeCPA-tTA line so both constructs expressed from the same locus, and a TRE reporter line that expressed an AmCyan reporter with an N terminal CHIKV fusion (CHIK-AmC). In Aag2 cells, shRNAs were more effective than lhRNA in silencing both the CHIKV split replication system (99.7%, S. D. ± 0.47 and 72.8%, S. D. ± 9.50, respectively, P&lt;0.001) and CHIK-FF (98.8%, S. D. ± 0.71 and 50.9%, S. D. ± 7.92, respectively, P&lt;0.05). Similar results were observed in transgenic mosquitoes when comparing AmC expression in the midgut. This study demonstrates that, in mosquitoes, effectively chosen shRNAs can induce greater interference of the desired viral target than the corresponding lhRNA.

An E3 Ubiquitin Ligase Scaffolding Protein Is Proviral during Chikungunya Virus Infection in Aedes aegypti.

ABSTRACTChikungunya virus (CHIKV) is a reemerging alphavirus causing chikungunya disease (CHIKD) and is transmitted to humans by Aedes mosquitoes. The virus establishes an intricate balance of cellular interactions that ultimately helps in its replication and dodges cellular immune response. In an attempt to identify cellular host factors required during CHIKV replication in Aag2 cells, we performed global transcriptomics of CHIKV-infected Aag2 cells, and further, we compared this library with the Drosophila RNAi Screening Center (DRSC) database and identified transcripts that were regulated in Aedes aegypti during CHIKV infection. These analyses revealed specific pathways, such as ubiquitin-related pathways, proteolysis pathways, protein catabolic processes, protein modification, and cellular protein metabolic processes, involved during replication of the virus. Loss-of-function assays of selected candidates revealed their proviral or antiviral characteristics upon CHIKV infection in A. aegypti-derived Aag2 cells. Further validations identified that the ubiquitin proteasomal pathway is required for CHIKV infection in A. aegypti and that an important member of this family of proteins, namely, AeCullin-3 (Aedes ortholog of human cullin-3), is a proviral host factor of CHIKV replication in Aag2 cells.IMPORTANCE Arboviruses cause several diseases in humans and livestock. Vector control is the main strategy for controlling diseases transmitted by mosquitoes. In this context, it becomes paramount to understand how the viruses replicate in the vector for designing better transmission blocking strategies. We obtained the global transcriptome signature of A. aegypti cells during CHIKV infection, and in order to obtain the maximum information from these data sets, we further utilized the well-characterized Drosophila system and arrived upon a set of transcripts and their pathways that affect A. aegypti cells during CHIKV infection. These analyses and further validations reveal that important pathways related to protein degradation are actively involved during CHIKV infection in A. aegypti and are mainly proviral. Targeting these molecules may provide novel approaches for blocking CHIKV replication in A. aegypti.

Biological Characterization of Yellow Fever Viruses Isolated From Non-human Primates in Brazil With Distinct Genomic Landscapes.

Since the beginning of the XXI Century, the yellow fever virus (YFV) has been cyclically spreading from the Amazon basin to Brazil’s South and Southeast regions, culminating in an unprecedented outbreak that started in 2016. In this work, we studied four YFV isolated from non-human primates obtained during outbreaks in the states of Rio Grande do Sul in 2008 (PR4408), Goiás (GO05), and Espírito Santo (ES-504) in 2017, and Rio de Janeiro (RJ 155) in 2019. These isolates have genomic differences mainly distributed in non-structural proteins. We compared the isolates’ rates of infection in mammal and mosquito cells and neurovirulence in adult mice. RJ 155 and PR4408 YFV isolates exhibited higher infectivity in mammalian cells and neurovirulence in mice. In mosquito Aag2 cells, GO05 and PR4408 displayed the lowest proliferation rates. These results suggest that RJ 155 and PR4408 YFV isolates carry some genomic markers that increase infectivity in mammal hosts. From this characterization, it is possible to contribute to discovering new molecular markers for the virulence of YFV.

Wolbachia Utilizes lncRNAs to Activate the Anti-Dengue Toll Pathway and Balance Reactive Oxygen Species Stress in Aedes aegypti Through a Competitive Endogenous RNA Network.

Long non-coding RNAs (lncRNA), a class of RNA molecules without protein coding potential, are more than 200 nucleotides in length and widely present in a variety of species. Although increasing progress in regard to the determination of lncRNA function has been made in vertebrates, Aedes aegypti lncRNAs were only identified recently and the functions of few lncRNAs have been annotated so far. Herein, the genome-wide alteration of the lncRNA expression profile trigged by Wolbachia wAlbB infection was investigated by comparing A. aegypti Aag2 cells and W-Aag2 cells infected with Wolbachia wAlbB. Based on lncRNA sequencing, 3035 differentially expressed lncRNAs (DE lncRNAs) in total were identified upon Wolbachia infection, which were further validated by quantitative PCR. The constructed co-expression network of DE lncRNAs and mRNAs revealed that Wolbachia-induced DE lncRNAs were highly enriched in the oxidative phosphorylation pathway via trans-activity, according to the KEGG pathway enrichment analyses. In addition, the established competitive endogenous RNA (ceRNA) network identifies the DE lncRNAs enriched in cellular oxidant detoxification based on GO enrichment analysis. Furthermore, silencing of aae-lnc-7598, the significantly up-regulated lncRNA with the highest fold change induced by Wolbachia, caused a significant reduction of antioxidant catalase 1B (CAT1B) gene expression as well as the enhancement of mitochondrial reactive oxygen species (ROS) production in living cells. These findings indicate that Wolbachia manipulates lncRNA to balance intracellular ROS stress and ensure its endosymbiosis in host A. aegypti. Notably, the function assay demonstrated that aae-lnc-0165 suppressed by Wolbachia could induce expression of the REL1 gene, the key regulator of downstream Toll pathway, through the sequence-specific binding of aae-miR-980-5p, which contributes to the activation of Toll pathway. Moreover, the depletion of aae-lnc-0165 caused the suppression of mitochondrial ROS levels in living cells. Our data reveal that Wolbachia activates the anti-dengue Toll pathway through a lncRNA-ceRNA pattern. Taken together, our finding suggested that Wolbachia utilizes lncRNAs to activate host anti-dengue Toll pathway via a ceRNA network. Moreover, Wolbachia employs lncRNAs to ensure ROS homeostasis for ROS-based anti-dengue defense through either trans-regulation or the ceRNA network. This study identifies novel potential molecular biomarkers for prevention and control of epidemic dengue.