**11. Virus-encoded miRNAs**

*9.1.1. Canonical pathway of miRNA biogenesis*

100 Current Topics in Tropical Emerging Diseases and Travel Medicine

*9.1.2. Noncanonical pathway of miRNA biogenesis*

mouse embryonic stem cells [79].

**10. Mode of action of miRNAs**

to mature miRNAs.

So far, a vast majority of reported miRNAs are produced through an RNase III enzyme-controlled canonical pathway. Transcription of primary transcript (i.e., pri-miRNA) harboring one or several stem loop structures by RNA polymerase II or otherwise RNA polymerase III kick start the biogenesis process in nucleus [68, 69]. pri-miRNAs are then further processed at stem loop sites by two RNase III proteins Drosha and DGCR8/Pasha (DiGeorge critical region 8) to produce about 70 nucleotide pre-miRNA [70]. This pre-miRNA is then exported to cytoplasm with the help of exportin-5 and RanGTP proteins [71]. Once in cytoplasm, pre-miRNA is chopped down into 22 nucleotide miRNA duplexes. Apart from above-mentioned proteins, many other pro-

Although, it was thought earlier that there is only one universal mechanism of all mature miRNAs biogenesis; however, multiple recent discoveries have led us to the conclusion that there may be several other miRNA biogenesis pathways as well. Biogenesis of quite a few miRNAs has been demonstrated to be not only Drosha independent, but also Dicer dependent [73]. It has been reported that in Drosophila and *C. elegans* along with some other vertebrates, mature miRNAs called "mirtrons" can be produced after splicing from intron hairpins independently of Drosha processing [74–76]. Recent studies have revealed that mature functional miRNAs can arise from highly conserved small nucleolar RNA (snoRNAs) in human and *Giardia lamblia* [77, 78] as well as DicerII-processed endogenous siRNAs mostly derived from transposable elements and load into AgoI in Drosophila [76]. Furthermore, researchers have identified miRNAs derived from transfer RNA (tRNA) in

Recent studies have led to the discovery of other types of miRNA noncanonical biogenesis pathway that is Dicer independent. One prime example of this is miR-451 biogenesis, which is Dicer independent as pre miR-451 has ~18 nucleotide stem duplex that is too short for Dicer activity. However, pre miR-451 requires AgoII for its maturation [80]. A few examples of other types of noncanonical biogenesis come from virus-encoded miRNAs. For example, several miRNAs are produced by a retrovirus, Bovine leukemia virus (BLV), based on DNA polymerase III transcription *in vivo* as well as *in vitro* [57]. The products of these transcripts were too small to be recognized by Drosha; therefore, these were directly processed by Dicer-1

After the production of mature miRNA by either canonical or noncanonical pathway, the resulting mature miRNA duplex is loaded into miRNA inducing silencing complex (miRISC) having Ago as core component [81]. After degradation of one strand, other strands of duplex, representing mature miRNA, then guide miRISC complex to target mRNA to determine its

teins also work as cofactors to influence the outcome of miRNA biogenesis [65, 72].

As most viruses need host genes to facilitate their replication, one cannot rule out the possibility of viruses encoding miRNAs in order to manipulate host gene expression or keep viral copy number under check to ensure persistent infection. Possible benefits of virus-encoded miRNAs are that they usually do not elicit immune response, require less coding ability, and they have ability to constantly evolve to target new transcripts. Till date, more than 200 viral miRNAs have been identified. Most of the well-characterized viral-encoded miRNA comes from DNA viruses as compared to RNA viruses, with herpes viruses as a major contributor having average copy number of more than 10/genome [58]. Different recent studies have identified novel microRNAs from different RNA viruses. Bovine leukemia virus (BLV), a retrovirus, also encodes microRNAs [57]. Hussain et al. has reported microRNA like small RNAs in both West Nile virus and Dengue virus [88]. These viral-encoded miRNAs are produced through canonical as well as noncanonical biogenesis pathways. Based on their function, viral encoded miRNAs can be classified into two broad classes:


Important functions of viral miRNAs, which target host genes, include promoting cell survival through downregulation of apoptotic factors, thus promoting infected cells survival and proliferation, as well as modulating the immune response of the host cell [89]. It is becoming increasingly evident that one mechanism the viruses have evolved to facilitate regulatory control over their hosts is by generating transcripts, which outmatch cellular miR-NAs. Interestingly, Human Kaposi's sarcoma-associated herpes virus (KSHV) and chicken oncogenic Marek's disease virus (MDV) transcribe miR-K12-11 and miR-M4, respectively; these miRNAs appear to be an ortholog of miR-155 to identical seed region [90, 91]. miR-155 has been reported to be involved in many malignancies [92], so exploitation of its targets by viruses may contribute to viral oncogenesis. Recently, it has been reported that a flavivirus named West Nile virus encode an miRNA KUN-miR-1 that specifically induces the expression of GATA4 in *Ae. albopictus* that in turn facilitates the WNV replication [88].

of miRNAs in Palm Creek virus (PCV) infection using the *Ae. aegypti* mosquito model [99]. They proposed differently expressed miRNAs after combining small-RNA sequencing and bioinformatics tools although the results hardly predicted the potential involvement of PCV infection to alter host miRNAs. They revealed that only one miRNA (i.e., aae-miR-2940-5p) out of 101 reported miRNAs of *Ae. aegypti* that had significantly altered expression over the course of PCV infection. The level of aae-miR-2940-5p was induced within 2 days p.i. and suppressed at 12 days p.i.; however, a different pattern of miRNA screening was demonstrated in *Ae. aegypti* Aa20 cell line without displaying any significant change upon PCV infection. Further *in vitro* miRNA inhibition experiments while using aae-miR-2940-5p inhibitors demonstrated that this miRNA did not directly impact on PCV replication and has no significant role in PCV-*Ae. aegypti* interaction. This variable response to virus infection in cell lines and host mosquito might be due to tissue tropism of the virus. At mRNA target level, the study reported the inconsistent expression level of MetP with miR-2940-5p expression although previously it was reported a potential target of aae-miR-2940-5p with positive interaction. It means that there was no correlation of MetP expression to PCV infection because silencing the gene did not significantly affect the virus replication. The pair MetP--aae-miR-2940-5p also positively regulate West Nile virus (WNV) virion production where aae-miR-2940-5p was selectively downregulated upon WNV infection in infected cells. The MetP human ortholog like M41 ftsh, YME1L, plays an important role as an antiapoptotic factor. It is assumed that the induction of aae-miR-2940-5p in PCV-infected cells during infection boosts MetP transcript levels and thus protects infected cells undergoing apoptosis under stress responses. However, the MetP physiological role is still uncertain in insects and requires further investigation [99].

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

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

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

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

**flavivirus**

Interestingly, virus-encoded miRNAs can regulate their own replication as well their own benefit. For example, simian virus 40 (SV40)-encoded miRNA miR-S1, expressed during late infection, has been shown to downregulate viral T-antigen, which is crucial to evade cytotoxic T cell response [93]. Another example is HvAV-miR-1 encoded by *Heliothis virescens* ascovirus (HvAV), an insect with dsDNA virus that downregulate viral replication by targeting viral DNA polymerase I [94]. Recently, it has been reported that Dengue virus (DENV)-encoded miRNA like viral small RNA DENV-vsRNA-5 plays an important role in the autoregulation of DENV replication by directly targeting dengue virus NS1 protein during late infection [30].
