Project description:We have generated transgenic Ae. aegypti that express a ZIKV-specific inverted repeat RNA intended to trigger the mosquito siRNA anti-viral immune pathway. To confirm that the transgene is expressed and processed, we performed small RNA sequencing on both Higgs White Eye (HWE) & anti-NS3/4A-ZIKV transgenic Ae. aegypti 24 hours post-bloodmeal
Project description:Arthropod-borne viruses (arboviruses) such as dengue virus (DENV) and Zika virus (ZIKV) pose a significant threat to global health. Novel approaches to control arbovirus spread are focused on harnessing the antiviral immune system of their main vector, the Aedes aegypti mosquito. In arthropods, genes of the Vago family are often presented as analogs of mammalian cytokines with potential antiviral functions, but the role of Vago genes upon virus infection in Ae. aegypti is largely unknown. We performed a phylogenetic analysis of the Vago gene family in Diptera that prompted us to focus on a Vago-like gene that we named VLG-1. Using CRISPR/Cas9-mediated gene editing, we generated a VLG-1 mutant line of Ae. aegypti that revealed a proviral effect of this gene upon DENV and ZIKV infection. In the absence of VLG-1, virus dissemination throughout the mosquito’s body was impaired, albeit not altering virus transmission rates. A tissue-specific transcriptome analysis revealed that the loss of VLG-1 impacted numerous biological processes potentially linked to viral replication, such as the oxidative stress response. Our results challenge the conventional understanding of Vago-like genes as antiviral factors and underscores the need for further research to elucidate the molecular mechanisms underlying mosquito-arbovirus interactions.
Project description:Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance among mosquito populations is a consequence of the application of insecticides for mosquito control. We used RNA-sequencing to compare transcriptomes between permethrin resistant and susceptible strains of Florida Ae. aegypti in response to Zika virus infection. A total of 2,459 transcripts were expressed at significantly different levels between resistant and susceptible Ae. aegypti. Gene ontology analysis placed these genes into 7 categories of biological processes. The 863 transcripts were expressed at significantly different levels between two strains (up/down regulated) more than 2-fold. Quantitative real-time PCR analysis validated Zika-infected response, and suggested a highly overexpressed P450, with AAEL014617 and AAEL006798 as potential candidates for the molecular mechanism of permethrin resistance in Ae. aegypti. Our findings indicated that most detoxification enzymes and immune system enzymes altered their gene expression between the two strains of Ae. aegypti in response to Zika virus infection. Understanding the interactions of arboviruses with resistant mosquito vectors at the molecular level allows for the possible development of new approaches in mitigating arbovirus transmission. This information sheds light on Zika-induced changes in the insecticide resistance of Ae. aegypti with implications for mosquito control strategies.
Project description:Aedes aegypti is a vector of many infectious agents, including flaviviruses like Zika virus. We demonstrate that Nest1, a 34kDa mosquito salivary protein, facilitates Zika virus dissemination in human skin explants. Our aim was to analyze the effect of Nest1 at a transcriptomic level (RNAseq) in human skin explants, in the presence and absence of virus (ZIKV), at different timepoints (day 1, 2,3 ,and 4)
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.
Project description:Zika virus (ZIKV) of the Flaviviridae family is a recently emerged mosquito-borne virus that has been implicated in the surge of the number of microcephaly instances in south America. The virus is transmitted mainly by the mosquito Aedes aegypti that also vectors dengue virus. Considering rather recent rapid spread of the virus and its declaration as a global health emergency by the World Health Organization, little is known about the interactions of the virus with the mosquito vector. In this study, we investigated the transcriptome profiles of whole Ae. aegypti mosquitoes in response to ZIKV infection at 2, 7 and 14 days post-infection using deep sequencing. Results showed a large number of transcripts were altered at each time point following infection, but 18 transcripts were commonly changed at the three time points. The outcomes provide a basic understanding of Ae. aegypti responses to ZIKV and help determining host factors involved in replication or anti-viral response against the virus.
Project description:Aedes aegypti mosquitoes infect hundreds of millions of people each year with dangerous viral pathogens including dengue, yellow fever, Zika, and chikungunya. Progress in understanding the biology of this insect, and developing tools to fight it, depends on the availablity of a high-quality genome assembly. Here we use DNA proximity ligaton (Hi-C) and Pacific Biosciences long reads to create AaegL5 - a highly contiguous A. aegypti reference.
Project description:Zika virus is a global public health emergency due to its association with microcephaly, Guillain-Barré syndrome, neuropathy, and myelitis in children and adults. A total of 87 countries have had evidence of autochthonous mosquito-borne transmission of Zika virus, distributed across four continents, and no antivirus therapy or vaccines are available. Therefore, several strategies have been developed to target the main mosquito vector, Aedes aegypti, to reduce the burden of different arboviruses. Among such strategies, the use of the maternally-inherited endosymbiont Wolbachia pipientis has been applied successfully to reduce virus susceptibility and decrease transmission. However, the mechanisms by which Wolbachia orchestrate resistance to ZIKV infection remains to be elucidated. In this study, we apply quantitative isobaric mass spectrometry-based proteomics to quantify proteins and identify pathways altered during ZIKV infection; Wolbachia infection; co-infection with Wolbachia/ZIKV in the Ae. aegypti. We show that Wolbachia regulates proteins involved in ROS production, regulates humoral immune response, and antioxidant production. The reduction of ZIKV polyprotein in the presence of Wolbachia in the mosquitoes was determined by mass spectrometry and corroborates the idea that Wolbachia helps to block ZIKV infections in Ae. aegypti. The present study offers a rich resource of data to help elucidate mechanisms by which Wolbachia orchestrate resistance to ZIKV infection in Ae. aegypti.
Project description:We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway’s antiviral role.