The goals of this study were to a) characterize changes in viRNA production

and b) to identify host processes that are differentially regulated by RNAi over the course of infection. DENV2 Jamaica 1409 (JAM1409) was used to infect its natural mosquito vector, Aedes aegypti. Most current RNA deep sequencing studies use duplicate technical replicates. By using triplicate biological click here replicates, deep sequencing and rigorous statistical metrics Selleck GDC 0032 similar to those used for microarrays, we identify products of RNAi pathway activity that are altered in DENV2-infected mosquitoes. The resulting data provide a basis for determining cellular pathways important to virus infection. This analysis is unique in that we focus on only those gene targets which are cleaved by post-transcriptional SRRPs producing sRNAs from 13-30 nts. Therefore, targets may be revealed that would not be identified using traditional microarray approaches. Alterations to gene expression levels that are controlled at the transcriptional level or by mechanisms of the de-capping or de-adenylation mRNA decay pathways will not be considered here [23]. Results Virus feeding Ae. aegypti Rexville D-Puerto Rico were fed a blood meal containing DENV2 Jamaica 1409 and negative controls were fed blood with an equivalent volume of un-infected insect cell culture

homogenate. As with previous studies [24], the mosquitoes had an Pevonedistat infection rate of 50% at 9 dpi and geometric mean titers of 2.5 log10 plaque-forming units (pfu) per mosquito. RNAi machinery components We performed a series of experiments to determine how Ae. aegypti RNAi pathway components respond to a blood

feeding or DENV2 infection. Hemocytes are critical to mosquito immunity, circulate in the hemolymph and harbor DENV2 particles [24, 25]. To give an indication of whether RISC complexes are present in hemolymph before blood feeding, thus supporting the hypothesis that mosquitoes mount an anti-viral response upon infection, soluble fractions were collected using two different methods, separated and probed with anti-Ago2 antibody. High molecular weight complexes containing Ago2 are present in cells from hemolymph/fat body fraction prior to a blood meal and depleted at 1 day post-blood feeding (Figure 1A-1B). Y-27632 2HCl Purified hemolymph from sugar-fed and blood-fed females showed a 143 kDa species, and all samples showed the lower molecular bands that are commonly seen in Ae. aegypti (Figure 1A, D) [3]. Figure 1 Antiviral RNAi components are expressed and active in Ae. aegypti. A) Ago2 associates with a high MW complex in hemolymph and fat body prior to a blood-feeding. HWE strain hemolymph (collected through proboscis) or hemolymph collected with fat body before and 1 day following a blood meal. About 30 μg protein was separated on a 3-10% Blue Native gel and subjected to immunoblot analysis using anti-Ago2 antibody. ‘H’, hemolymph, ‘H/F’, hemolymph with fat body.