RNA interference and dengue virus replication in insect cell culture and Aedes aegypti mosquitoes

Abstract

RNA interference, or RNAi, can effectively block dengue virus (DEN) replication in mosquito cell culture. The purpose of this study is to examine the mechanism of the RNAi response to DEN-2 virus in mosquito cell culture and to examine the feasibility of RNAi to DEN-2 in the entire mosquito using germ-line transformation. Mosquito cells (C6/36) transformed with a plasmid designed to express double-stranded RNA (dsRNA) derived from the DEN-2 genome do not allow replication of the virus. These cells do not accumulate viral antigen or viral genomic RNA upon infection. The block in DEN-2 replication in these cells was found to be due to an RNAi response, generated by the expression of dsRNA within these cells. The nature of this RNAi response including the dsRNA trigger, the effector molecules (small interfering RNAs or siRNA) and the degradation products of DEN-2 RNA in mosquitoes are defined here. Upon confirmation that the replication silencing observed in mosquito cells was due to RNAi, the process was examined in the mosquito as a whole using a transgenesis approach. Transgenic mosquitoes were engineered to express DEN-2 dsRNA as well as a selectable eye-specific marker. These mosquitoes were tested for their ability to resist DEN-2 infection. None of the transgenic mosquitoes exhibited resistance to DEN-2 infection. The reasons behind the lack of observable DEN-2 resistance in these mosquitoes are discussed along with the pitfalls encountered in the generation of these transgenic mosquitoes.