The molecular responses of Anopheles gambiae to o'nyong-nyong virus infection
O'nyong-nyong virus (ONNV) is an Alphavirus (family: Togaviridae) transmitted by Anopheles gambiae (An. gambiae) mosquitoes. Alphavirus transmission causes a number of severe diseases in vertebrates. However, in mosquito hosts, infection rarely causes pathology. To date, little is known about the cellular responses that privilege mosquitoes with the ability to cope with infection. This study evaluated the genetic responses of An. gambiae to ONNV infection. We adopted a proteomics approach to identify proteins and molecular pathways that respond to virus infection in the midgut, the primary physical and biological barrier to infection. The sensitive mass spec-based technique, Tandem Mass Tagging (TMT; Thermo Scientific), was used to quantify and compare protein expression changes induced by the virus. Several proteins were identified that mediated virus infection, including an FK506 binding protein (FKBP). Evidence suggests FKBPs are phylogenetically conserved virus resistance factors that may exert their function via the PI3K/Akt/TOR pathway. There are many powerful tools for investigating protein function, such as reverse genetics using RNA interference (RNAi). This technique, however, requires the ability to consistently and efficiently transfect cells, which has proven difficult with regard to mosquito studies; there is currently no acceptable in vitro model for studying gene function in An. gambiae. Therefore, having identified proteins of importance to virus infection, we next created an in vitro model system for future protein function studies. By evaluating different transfection reagents and cell culture methods, we developed a method for transfecting the immune-competent Sua5b (An. gambiae) cell line with very high efficiency. Finally, we evaluated the role of the PI3K/Akt/TOR pathway in o'nyong-nyong virus infection of both mammalian (Vero) and mosquito (Sua5b) cells. This pathway offered a window of understanding into how alphaviruses interact with their hosts, and perhaps more importantly, in what ways those interactions differ. We found that the PI3K/Akt/TOR pathway likely plays distinct, if not opposite roles in infection of the host cells studied.