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Background  The RNA interference (RNAi) pathway acts as an innate antiviral immune response in  Aedes aegypti , modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family:  Togaviridae , genus:  Alphavirus ) is an arbovirus that infects  Ae. aegypti  in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of  Ae. aegypti . The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity.    Results  As a novel tool for studying arbovirus-mosquito interactions, we engineered a transgenic mosquito line with an impaired RNAi pathway in the midgut of bloodfed females by silencing expression of the  Aa - dcr2  gene. In midgut tissue of the transgenic Carb/dcr16 line,  Aa - dcr2  expression was reduced ~50% between 1-7 days post-bloodmeal (pbm) when compared to the recipient mosquito strain. After infection with SINV-TR339EGFP,  Aa - dcr2  expression levels were enhanced in both mosquito strains. In the RNAi pathway impaired mosquito strain SINV titers and midgut infection rates were significantly higher at 7 days pbm. There was also a strong tendency for increased virus dissemination rates among the transgenic mosquitoes. Between 7-14 days pbm, SINV was diminished in midgut tissue of the transgenic mosquitoes. Transgenic impairment of the RNAi pathway and/or SINV infection did not affect longevity of the mosquitoes.    Conclusions  We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level. Following ingestion by  Ae. aegypti , the recombinant SINV-TR339EGFP was confronted with both MEB and a midgut infection barrier (MIB). Impairment of the RNAi pathway in the midgut strongly reduced both midgut barriers for the virus. This confirms that the endogenous RNAi pathway of  Ae. aegypti  modulates vector competence for SINV in the midgut. The RNAi pathway acts as a gatekeeper to the incoming virus by affecting infection rate of the midgut, intensity of infection, and dissemination from the midgut to secondary tissues.

The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti