Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti

By April 8, 2015RNAi, Workgroup 3

Author(s): Alexander W. E. Franz, Irma Sanchez-Vargas, Zach N. Adelman, Carol D. Blair, Barry J. Beaty, Anthony A. James

Publication: PNAS

Abstract: 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) path- way in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premem- brane 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 trans- genic 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 mos- quitoes 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.