**13. RNA interference in** *Aedes aegypti* **with an insect***-***specific flavivirus**

RNA interference is considered a vital antiviral defense response in mosquitoes as lot of studies reported the production of viral-specific small RNAs for different viruses. However, several reports also confirm the establishment of persistent viral infection only in mosquitoes either infected by pathogenic mosquito-borne viruses or insect-specific viruses that produce these small RNAs. While doing so, viral-derived DNA (vDNA) is produced by reverse transcription during persistent infection and may survive in extrachromosomal or integrated forms. Such vDNAs increase RNAi-mediated antiviral response in mosquitoes to boost mosquito tolerance to arbovirus infection while establishing persistent infection. In one study, Lee et al. detected PCV-specific 21 nucleotide small-RNAs in mosquitoes at different time points of infection (2, 6, and 12 days p.i.) as a potential indicator of active viral replication in the mosquitoes [99]. These findings complied with other studies where the researchers demonstrated the similar phenomenon for other pathogenic mosquito-borne flaviviruses (e.g., DENV) and insect-specific flaviviruses (e.g., cell fusing agent viruses). Their presence as hot spots suggests that either those are the potential targets of Dicer-2, or are more stable or can be reverse-transcribed into vDNA. All these might be the potential cause to enhance RNAi antiviral response. However, the authors are also afraid of bias, which could be due to technical issues such as library preparation. In addition to RNAi, piRNAs (piwi RNAs) have been reported in mosquitoes and knockdown of piRNAs-related specific proteins indicates their endogenous antiviral activity instead of well-established exogenous RNAi activities in mosquitoes. Similarly, virus-specific piRNAs with typical A10 bias in sense RNA and U1 bias in antisense RNA have been reported to be produce by Bunya viruses and alphaviruses. However, the piRNAs expressed by dengue virus and cell fusing agent virus have only A10 bias in sense RNA. Interestingly, the PCV-specific small RNAs do not show either A10 or U1 bias features. In this scenario, it is uncertain that either they are piRNAs or just viral degradation products. This situation is very similar seen in other flavivirus-specific piRNAs, which map to very small number of sequences in the genome where the presence of one copy of genome indicates more specific targeting than random RNA degradation. The production of virus small RNAs and piRNAs through vDNA synthesis is also documented in mosquitoes or in their derived cell lines. It is worth mentioning to note the contribution of vDNA in the production of small RNAs of virus and piRNAs in mosquitoes, although insect-specific viruses like PCV establish persistent infection in their hosts [99].

lincRNA in antiviral defense. The findings might be consistent as the highly overexpressed lincRNA\_1317 expression (2.33 fold) was reported in *Wolbachia-*infected mosquitoes as com-

RNA Association, RNA Interference, and microRNA Pathways in Dengue Fever Virus-Host…

http://dx.doi.org/10.5772/intechopen.80334

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The authors also described lincRNAs potential involvement in mosquito-pathogen interaction by determining its association with host-endogenous small RNAs and its direct interaction with DENV-2 infection. lincRNA\_1317 was not found to be located in any of the known piRNA clusters; however, no differences were found in the mapping pattern and mapped read length distribution when reads from DENV-infected and noninfected small RNA libraries were mapped to lincRNA\_1317. Gene silencing like role of piRNAs on lincRNA\_1317 transcriptome was also speculated. However, a little information is available about piRNAmediated lincRNA although some recent studies predict piRNA-mediated degradation of

The researchers also tested the hypothesis that *Ae. aegypti* lincRNA\_1317 response to microbial challenge could be due to cross regulation between miRNAs and the lincRNAs. For this purpose, the normalized minimum free energy (mfe) of hybridization for each *Ae. aegypti* miRNA and lincRNA\_1317 was calculated by using RNAhybrid core script. The basic objective was to identify *Ae. aegypti* miRNA recognition elements on lincRNA\_1317. The results were quite interesting as binding sites enrichment for a few miRNAs with more than two recognition elements were detected on lincRNA\_1317 (e.g., more than four recognition sites for miR-278-5p and miR-252-3p were predicted on lincRNA\_1317). Furthermore, some hot spots for miRNA recognition sites on lincRNA\_1317 were also identified, which may facilitate multiple miRNAs to bind the same regions. microRNAs contain the capability to shake lincRNA stability by targeting their transcripts similar to targeted mRNAs. Similarly, lincRNAs possessing multiple recognition sites might act as a competitive inhibitor of miRNA by eliminating them to bind their genuine targets by sequestration. The mfe values of hybridization for miRNA-lincRNA recognition sites could be a strong predictor of a binding event between two; however, further investigations and trials are still needed to validate

**15. IsomiRs and their impact on miRNAs in dengue fever vector** 

microRNAs may exist in various lengths and sequence variations, which are known as isomiRs [101]. Earlier, those were considered as sequencing errors; however, some studies predicted to be physiologically relevant and posttranscriptionally modified miRNA variants. IsomiRs may express affinities for different targets than their canonical miRNA counterparts. Furthermore, nucleotide heterogeneity could be found at both ends of miRNA sequence in the form of nucleotide substitutions despite the variations are more frequent at 3′ end. The molecular biology, genome structure, and epigenetics of isomiRs are still poorly understood, and the mechanisms of their biogenesis are also considered very complex and even cell-type specific. Some variations in miRNA sequences are supposed to be the product of template variations, which might be brought by the exonuclease activity of Drosha and Dicer [101].

pared to noninfected [100].

this interface [100].

*Ae. aegypti*

lincRNAs in mouse's late spermatocytes [100].
