However, by 4 dpi, mean mapped reads have dropped by half. Because a previous study showed evidence of full-length viral genomes at 4 dpi, we speculate that viral genomes are protected from RNAi-mediated degradation [6]. This time period also marks the prelude to expanded virus infection in the midgut prior to dissemination and therefore could be a critical window wherein the vector competence phenotype is determined for a given individual. BAY 80-6946 nmr Moreover, early host responses
may determine whether a persistent virus infection will be established in susceptible mosquitoes or, alternatively, cleared in resistant individuals. Our host sRNA profile data support this hypothesis. Significant differences in sRNA profiles across mosquito pools are most pronounced at 2 dpi, lessened at 4 dpi and not detectable by 9 dpi. This could be due to increasingly individualized host responses as the infection progresses. This is the first demonstration that viRNAs of 24-30 nts are a product of arbovirus infection using a natural vector/virus combination and important supportive evidence that the piRNA pathway plays a role in anti-viral defense in mosquitoes, as has been postulated previously
[21, 31]. BAY 73-4506 cost viRNAs are most abundant in the 24-30 nt size group at 2 dpi. As infection progresses, the viRNA size range is altered, until at 9 dpi, the predominant population of viRNAs are from 20-23 nts, indicative of a dominant Dicer2-dependent RNAi response. We show that high molecular weight complexes containing Ago2 are present in cells of the mosquito’s open circulatory
system prior to infection. This is the first evidence from mosquitoes showing the presence of these high molecular weight complexes. Multiple anti-Ago2 antibody cross-reacting bands are present in whole mosquitoes, suggesting that several Ago2 isoforms are present [3]. The 116 kDa Ago2 protein previously identified in mosquito midguts was not seen in IPs of whole mosquitoes [3], likely because of preferential binding of smaller molecular weight products. Moreover, a 66 kDa alternate spliceform has been identified and could be represented in Fluorouracil in vitro the 66 kDa IP band (data not shown, CLC). We also immunoprecipitated 20-21 nt sRNAs and usRNAs (13-19 nts) from aedine mosquitoes using anti-Ago2 antibody. The presence of the usRNA size class adds to the complexity of possible regulatory control mediated by Ago2. Gene expression of anti-viral RNAi components is enhanced early in DENV2 infection, in contrast to alphavirus infection, which does not produce significant alteration to either Ago2 or Dicer-2 transcript levels [3]. Total transcriptome-mapped reads grouped by sRNA size group show an overall increase in 24-30 nt size group in DENV-infected libraries (Additional File 1C); although this result is not statistically significant, a similar result was also observed in West Nile Virus-infected Culex pipiens quinquefasciatus (data not shown).