RNAi Conditions Arbovirus Infection in Mosquitoes

Studies were conducted to determine if RNAi constitutes a robust innate immune response to arboviruses in vectors in vivo (Keene, et al. 2004). RNAi is triggered in eukaryotic organisms by double-stranded RNA (dsRNA), and it results in destruction of any mRNA that has sequence identity with the dsRNA trigger. Most arboviruses are RNA viruses and form dsRNA during their replication cycles. We hypothesized that RNAi may act as an antagonist to alphavirus replication in Anopheles gambiae. We first demonstrated that the RNAi pathway in An. gam-biae can be silenced by transfecting cells with dsRNA derived from exon sequence of the An. gambiae Argonaute2 (Agago2) gene (Hoa et al 2003). The next step was to determine if RNA silencing of Agago2 expression would make An. gam-biae mosquitoes more permissive (more competent) to arbovirus infection. Studies were conducted to determine whether RNAi conditions the vector competence of An. gambiae for O'nyong-nyong virus (ONNV, genus Alphavirus). We genetically engineered a modified ONNV to express enhanced GFP (eGFP) as a marker. When the ONNV-eGFP was intrathoricacially injected into An. gambiae, the virus first replicated at the portal of entry and then spread slowly over a 9-day period to other tissues. Mosquitoes were then co-injected with ONNV-eGFP and either control dsRNA derived from the fi-galactosidase (dsfigal) gene or from ONNVnsP3 (dsnsP3) gene. Treatment with E. coli dsnsP3 RNA inhibited ONNV-eGFP spread,

Fig. 1 Silencing the RNAi response makes Anopheles gambiae mosquitoes susceptible to O' nyong nynong virus (adapted from Keene et al. 2004)

as determined by eGFP expression patterns, and ONNV-eGFP titers were significantly reduced in the dsnsP3 RNA-injected mosquitoes compared to mosquitoes co-injected with the dsßgal gene sequence RNA (Fig. 1). Thus An. gambiae did mount an RNAi response to suppress the arbovirus infection. To demonstrate unequivocally that RNAi was involved, mosquitoes were then co-injected with ONNV-eGFP and dsAgago2 RNA, the latter of which would inhibit the vector RNAi response. Mosquitoes co-injected with virus and Agago2 dsRNA were much more permissive to virus infection and displayed widespread eGFP expression and virus titers 16-fold higher than dsßgal RNA controls at three or six days after injection. Perturbation of the RNAi response by silencing Argonaute 2, a component of the RNAi-induced silencing complex, changed An. gambiae mosquitoes from ONNV-resistant to permissive phenotypes.

These landmark studies provided direct evidence that RNAi is an antagonist of ONNV replication in An. gambiae, and they suggest that the innate immune response conditions vector competence. Indeed, studies have now demonstrated that RNAi also modulates infection, replication, and transmission of dengue viruses in mosquitoes (Franz et al. 2006). Thus RNAi may be an important determinant of vector competence in all of the major arbovirus families.

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