Once SINV enters the saliva, the virus has completed its extrinsic incubation period

and the mosquito is able to transmit the virus to a new host [9]. The TR339 strain of SINV is based on a consensus sequence derived from the type strain AR339 that has been isolated in Egypt LY3009104 molecular weight [10–12]. For this study, we used a full-length infectious cDNA clone of the virus with the enhanced green fluorescent protein (EGFP) marker gene inserted downstream of a second subgenomic promoter [3]. After ingestion by females of the Ae. aegypti RexD strain, SINV-TR339 has been shown to encounter an escape barrier in the midgut (MEB); whereas reported midgut infection rates were >90%, dissemination rates only reached 40% [9, 13]. Midgut infection barrier (MIB) and/or MEB have

been observed for a number of other alphaviruses and for flaviviruses [14, 15]. MIB prevents ingested arboviruses from entering and replicating in mesenteronal (midgut) cells, whereas MEB prevents virions from escaping from the basal lamina of midgut cells and disseminating to other tissues in the hemocoel. Often these barriers depend on the amount of virus ingested by the mosquito because the virus has to reach a certain threshold to either establish an infection in the midgut or to disseminate to other tissues [9, 14–16]. Furthermore, dose-independent RG7112 cell line MIB or MEB have been reported, implying an incompatibility between arbovirus and vector at the midgut level, thus preventing arboviruses from entering Nutlin-3 in vitro or exiting the epithelial cells [13, 17–20]. Until now, the molecular nature

of MIB and MEB, which appears to depend on specific virus-mosquito strain combinations, is not well understood. Saracatinib solubility dmso However, recent correlation analysis of RNAi pathway genes with MIB and MEB combined with linkage mapping of Aa-dcr2, Aa-r2d2, and Aa-ago2 genes in the genome of Ae. aegypti suggests that MIB and MEB for dengue virus could be RNAi associated phenomena [21]. To investigate the nature of MIB and MEB for SINV-TR339EGFP in Ae. aegypti, we impaired the RNAi pathway in the mosquito midgut at a time point when the ingested virus is replicating in cells of the midgut epithelium. We expected that impairment of the RNAi pathway in the midgut of Ae. aegypti would allow the virus to overcome potential MIB and/or MEB and to increase its overall titer in the insect. We chose a transgenic approach to impair the RNAi pathway in the midgut of Ae. aegypti by generating mosquitoes expressing an inverted-repeat (IR) RNA derived from the RNAi pathway gene Aa-dcr2 under control of the bloodmeal inducible, midgut-specific Ae. aegypti carboxypeptidase A (AeCPA) promoter [22–25]. According to our strategy the midgut-specific IR effector would produce dsRNA in bloodfed females, triggering RNAi against Aa-dcr2 and eventually causing depletion of dicer2 protein in the midgut. This would cause impairment of the RNAi pathway in this tissue.