RNA interference acts as a natural antiviral response to O'nyong-nyong virus ( <i>Alphavirus</i> ; Togaviridae) infection of <i>Anopheles gambiae</i>

<jats:p> RNA interference (RNAi) is triggered in eukaryotic organisms by double-stranded RNA (dsRNA), and it destroys any mRNA that has sequence identity with the dsRNA trigger. The RNAi pathway in <jats:italic>Anopheles gambiae</jats:italic> can be silenced by transfecting cells with dsRNA derived from exon sequence of the <jats:italic>A. gambiae Argonaute2</jats:italic> ( <jats:italic>AgAgo2</jats:italic> ) gene. We hypothesized that RNAi may also act as an antagonist to alphavirus replication in <jats:italic>A. gambiae</jats:italic> because RNA viruses form dsRNA during replication. Silencing <jats:italic>AgAgo2</jats:italic> expression would make <jats:italic>A. gambiae</jats:italic> mosquitoes more permissive to virus infection. To determine whether RNAi conditions the vector competence of <jats:italic>A. gambiae</jats:italic> for O'nyong-nyong virus (ONNV), we engineered a genetically modified ONNV that expresses enhanced GFP (eGFP) as a marker. After intrathoracic injection, ONNV-eGFP slowly spread to other <jats:italic>A. gambiae</jats:italic> tissues over a 9-day incubation period. Mosquitoes were then coinjected with virus and either control β- <jats:italic>galactosidase</jats:italic> dsRNA (dsβgal; note that “ds” is used as a prefix to indicate the dsRNA derived from a given gene throughout) or ONNV dsnsP3. Treatment with dsnsP3 inhibited virus spread significantly, as determined by eGFP expression patterns. ONNV-eGFP titers from mosquitoes coinjected with dsnsP3 were significantly lower at 3 and 6 days after injection than in mosquitoes coinjected with dsβgal. Mosquitoes were then coinjected with ONNV-eGFP and dsAgAgo2. Mosquitoes coinjected with virus and <jats:italic>AgAgo2</jats:italic> dsRNA displayed widespread eGFP expression and virus titers 16-fold higher than dsβgal controls after 3 or 6 days after injection. These observations provide direct evidence that RNAi is an antagonist of ONNV replication in <jats:italic>A. gambiae</jats:italic> , and they suggest that the innate immune response conditions vector competence. </jats:p>