Evidence for RNA interference as an antiviral immune response to alphavirus infection in the mosquitoes Anopheles gambiae and Aedes aegypti

Abstract

Mosquito-borne viruses remain a serious threat to public health worldwide. A lack of effective vaccines and vector control programs have led to the geographical expansion of the vectors and their associated agents, thereby increasing the percent of the global population that are affected. The studies described here are designed to determine whether RNA interference occurs in the mosquito and functions to naturally target replicating viruses in the mosquito. The results will provide information that will lead to the development of new strategies to control arbovirus transmission. First, potential genes in the mosquito RNAi pathway were identified in the Anopheles gambiae genome. Genes in the dicer and argonaute gene families were tested for their involvement in the RNAi pathway in An. gambiae cells using a dual-luciferase assay and dsRNA knockdown techniques. These experiments identified the Dcr2, Ago2, and Ago3 genes from An. gambiae as genes involved in the RNAi pathway. To test the hypothesis that the RNAi pathway is a part of the mosquitoe's innate defenses against virus infection, genes in the pathway were targeted using RNAi techniques to reduce gene expression. Simultaneous injection of dsRNA with sequences homologous to dcr and ago genes and infection with o'nyong-nyong virus (ONNV) (Alphavirus; Togaviridae) in An. gambiae resulted in a significant increase in viral titers and increased dissemination of the virus in those mosquitoes. Additionally, injection of the virus along with dsRNA targeting a viral gene resulted in decreased viral replication in the mosquito. These studies confirmed that ONNV replication in An. gambiae is naturally targeted by the RNAi pathway. To test the hypothesis that the RNAi response would interfere with Sindbis virus replication in Aedes aegypti, dsRNA with sequence homologous to a gene from the TR339 strain was injected into mosquitoes three days prior to the infectious bloodfeed. This demonstrated that RNA interference could be used to prevent TR339 infection of the midgut. In addition, dsRNA homologous to Ae. aegypti argonaute-2 injected into the mosquito prior to an infectious bloodfeed resulted in increased virus replication in the midgut, as well as increased dissemination from midgut tissues. This work, in conjunction with previous work based on RNA-mediated interference o virus replication in mosquitoes, provides a basis for additional studies that could lead to generating RNAi-based arbovirus resistance in mosquitoes.