Periodic Reporting for period 1 - MetAeAvIm (The Role of the Metabolism in Mosquito Immunity against Dengue virus in Aedes aegypti)
Reporting period: 2019-05-01 to 2021-04-30
Mosquito-borne diseases remain among the most prevalent diseases in the world. For example, estimates suggest that the Dengue virus (DENV) causes 400 million infections every year. At present, more than half of the world population lives in areas where mosquito vectors are present, and this is predicted to expand as a result of climate change. In that sense, the manipulation of vector competence is a potential tool for the development of new strategies of vector control. However, the incipient understanding of antiviral mechanisms has limited developments in this field. DENV, the causative agent of dengue fever, is a single positive-strand RNA flavivirus primarily transmitted by A. aegypti. Following ingestion by the mosquito in a blood meal, DENV is able to invade midgut epithelial cells. During this process, the mosquito immunity is modulated by its physiological state and nutritional status. By focusing on the analysis of metabolic pathways by gene set enrichment analysis, we identified 17 biological pathways that were enriched in at least 3 out of 4 populations, suggesting that these pathways play a conserved role in the acquisition of resistance against DENV. With this in mind, we proposed to study the metabolism of mosquitoes and to identify metabolic pathways and genes that are determinants for DENV resistance. We additionally proposed to test whether these pathways could be explored in transgenic mosquitoes to modulate vector competence.
Work during the period of the project was focused on the standardization of the conditions needed for project development. Briefly, the researcher focused on increasing the weekly output of wild-type mosquitoes (Aedes aegypti Bangkok strain), adapting the mosquito husbandry routine to obtain a constant biweekly supply of adult females and standardizing the drug treatment (e.g rapamycin) regimens and protocols. The researcher further worked to standardize the cell culture routine and cell line transfection protocols. Specifically, the researcher adapted, expanded, and produced stocks of cell lines for DENV expansion and in vitro assays. Following, work was focused on the development of standard protocols for the main methodologies to be used during the project, including the timing of TOR activation at the midgut following blood-feeding, ROS homeostasis, and subcellular compartmentalization of viral RNA binding proteins. Finally, he obtained training on the techniques for virus expansion, titration, storage, and infections. Soon after starting the project, the researcher was offered an independent investigator position in his home country. For that reason, the project and the fellowship were interrupted on October 15th
Due to the early termination of the project, the project did not provide significant progress beyond the state of the art of the field. Despite the limited period of project development, the researcher contributed toward the implementation and standardization of protocols that will be used during related project development at the host institution. Mosquito routine and cell lines left by the researcher are being maintained and used at the host institution. Initial screening performed by the researchers, such as the initial results on the mobilization of the TOR pathway and immunohistochemistry data of Ago2 and Loqs2 will be integrated into other projects being developed at the host institution. Thus, the material, protocols, and initial data made available by the researcher will be used during future investigations at the host institution.