**6. RNA interference (RNAi)**

RNA interference (RNAi) is a major component of innate defense of invertebrates against pathogens. Most arboviruses cause persistent infections within their arthropod vectors. However, how viruses maintain persistent infection in the face of a robust RNAi response is still not fully understood. Many plants and animal viruses have evolved molecular mechanisms for subverting the host RNAi response. For example, Flock house virus (FHV) encodes B2 protein, which directly binds to dsRNA to inhibit DCR2 activity, to inert siRNA pathway [52]. In another study, researchers have found that plasmid-expressed La Crosse virus (LACV) NSs protein successfully inhibits interferon (IFN) and RNAi pathway in mammalian cells and mice, while fails to show any RNAi-suppressive effect in transfected U4.4 cells and C6/36 cells infected with LACV [53]. A recent publication has revealed that DENV NS4b functions as an RNAi suppressor in human Huh-7 cells via inhibition of dsRNA processing by Dicer [8]. However, whether NS4b behaves similarly in mosquitoes has not been investigated, and to date, no RNAi suppressor activity has been described for an arbovirus protein during mosquito infection. Given that DENV successfully develops persistent infection in mosquitoes despite their RNAi response, the precise mechanism of RNAi evasion is definitely a significant point of interest.

viruses lack nuclear access [55]. However, recent discovery of noncanonical miRNA biogenesis pathways that utilize Argonaute (Ago) 2 for processing of pre-miRNA to mature miRNA has highlighted the fact that RNA viruses may opt similar pathway to generate functional miRNAs. Similar noncanonical pathways might be used to generate functional miRNAs derived from RNA viruses that replicate in the cytoplasm. Several examples up to now have been accumulated like cellular miRNAs, miR-124, is expressed from a cytoplasmic RNA virus, Sindbis virus (SINV) [56]. Several miRNAs were found to be produced by a retrovirus, Bovine leukemia virus (BLV), based on DNA polymerase III transcription *in vivo* as well as *in vitro* [57]. Recently, Hussain et al. have identified six DENV-encoded miRNA like small RNAs in which one of miRNAs directly targets DENV NS1 gene during late infection to autoregulate its replication. Functional analysis has revealed that viral miRNAs can target both cellular and viral mRNAs to regulate viral replication leading to a successful infection [58]. Although, very few virus-encoded miRNAs have been fully characterized, it is understandable that they can target both viral and host genes.

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

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

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Flaviviruses are well reported to manipulate host miRNA machinery to facilitate their replication. It has been well reported that DENV induce differential expression of miRNAs in human peripheral blood mononuclear cells (PBMCs) [59]. It has been previously reported that miR-124a, -128a, -218, and -let-7c may be important for neurological symptoms caused by a chimeric tickborne encephalitis/dengue virus [60]. Additional studies have shown that miR-122 and miR-142 of the host cells are involved in restricting dengue virus [61, 62]. However, most of the above work has been done in human. Very little is known about the differential miRNA expression of *Ae. aegypti* influenced by DENV infection, leaving a significant gap in our understanding of DENV-*Aedes aegypti* interaction. Although a recent study has shown aberrant host small RNA (sRNA) profiles in *Ae. aegypti* during DENV infection, it mainly focused on piRNAs [63] leaving a significant gap in understanding the differential expression

miRNAs are usually encoded by different regions of genome ranging from coding as well as noncoding [64]. Seventy percent of mammalian miRNAs are the intron products of protein coding genes, while rest comes from the noncoding transcription units. miRNAs have been found to be originated from both senses and antisense strands of DNA [65]. Studies have shown that about 30% of *C. elegans* miRNA genes are on the antisense strand overlapping protein coding region [66]. In Drosophila too, many miRNAs are originated from antisense strand overlapping protein coding region [67]. Multiple miRNAs can be transcribed as one

Majority of miRNAs are transcribed by RNA polymerase II; however, some miRNAs usually located near Alu repeats are transcribed by RNA polymerase III, from independent genes having their own promoter or represent introns of protein-coding genes [18]. Nearly, all the independent miRNA promoters have particular features of Pol II promoters, including initia-

of *Ae. aegypti* miRNAs during DENV replication.

**9.1. miRNAs genomic arrangement and transcription**

**9. miRNA biogenesis**

long transcript named clusters [18].

tor elements and TATA boxes [18].

#### **7. RNA activation (RNAa)**

RNA activation (RNAa) is one of the nascent phenomena around the scientific field. RNAa is considered to be a potent emerging therapeutic strategy for the disease that can be cured by particular gene activation [54]. However, most of the studies relating to RNAa research have been carried out in human cell line. Till date, proper mechanism of RNAa is still not clear. However, it has been demonstrated that it is mostly a nuclear event that leads to chromatin restructuring [17]. RNA activation is still in its infancy, so its exact mechanism is still elusive. Recently, Portnoy et al. has proposed a model for the promoter-directed-small-activating-RNA (saRNAs)-mediated RNA activation. In this model, authors proposed that an exogenously introduced saRNA or endogenously produced small noncoding RNA, (e.g., miRNA) is loaded into an Ago protein in the cytoplasm. The Ago protein processes the saRNA by discarding its passenger strand to form an active Ago-RNA complex. Ago-RNA complex enters the nucleus through active transport/passive diffusion cell division. Guide strand directs Ago-RNA complex to complementary genomic DNA sequences usually in promoter region or to noncoding RNA (ncRNA) sequences, which tethers to the DNA. After that, the Ago-RNA complex initiates a process that differs from RNAi, which alter chromatin structure and epigenetic states of target gene via two different potential mechanistic models. In model A, the saRNA guide strand leads the Ago protein to its DNA target by constituting an RNA–DNA duplex or triplex structure, while Ago protein then serves as a docking platform to attract histone-modifying activities, like opening of chromatin structure and active transcription. On the other hand, in model B, the RNA guide strand binds to cognate promoter transcripts making saRNA-RNA complex and then Ago protein recruits histone modifiers to introduce active chromatin marks on local chromosome, resulting in activation of transcription [17].

#### **8. MicroRNAs (miRNAs)**

As the molecular biology, bioinformatics approaches and protocols of small RNA sequencing continue to grow and sophisticate, so is the number of miRNAs discovered in different species. Virus-encoded miRNAs have been known to be crucial for the viral replication. miRNAs encoded by DNA viruses have been well studied in comparison with those by RNA viruses because RNA viruses lack nuclear access [55]. However, recent discovery of noncanonical miRNA biogenesis pathways that utilize Argonaute (Ago) 2 for processing of pre-miRNA to mature miRNA has highlighted the fact that RNA viruses may opt similar pathway to generate functional miRNAs. Similar noncanonical pathways might be used to generate functional miRNAs derived from RNA viruses that replicate in the cytoplasm. Several examples up to now have been accumulated like cellular miRNAs, miR-124, is expressed from a cytoplasmic RNA virus, Sindbis virus (SINV) [56]. Several miRNAs were found to be produced by a retrovirus, Bovine leukemia virus (BLV), based on DNA polymerase III transcription *in vivo* as well as *in vitro* [57]. Recently, Hussain et al. have identified six DENV-encoded miRNA like small RNAs in which one of miRNAs directly targets DENV NS1 gene during late infection to autoregulate its replication. Functional analysis has revealed that viral miRNAs can target both cellular and viral mRNAs to regulate viral replication leading to a successful infection [58]. Although, very few virus-encoded miRNAs have been fully characterized, it is understandable that they can target both viral and host genes.

Flaviviruses are well reported to manipulate host miRNA machinery to facilitate their replication. It has been well reported that DENV induce differential expression of miRNAs in human peripheral blood mononuclear cells (PBMCs) [59]. It has been previously reported that miR-124a, -128a, -218, and -let-7c may be important for neurological symptoms caused by a chimeric tickborne encephalitis/dengue virus [60]. Additional studies have shown that miR-122 and miR-142 of the host cells are involved in restricting dengue virus [61, 62]. However, most of the above work has been done in human. Very little is known about the differential miRNA expression of *Ae. aegypti* influenced by DENV infection, leaving a significant gap in our understanding of DENV-*Aedes aegypti* interaction. Although a recent study has shown aberrant host small RNA (sRNA) profiles in *Ae. aegypti* during DENV infection, it mainly focused on piRNAs [63] leaving a significant gap in understanding the differential expression of *Ae. aegypti* miRNAs during DENV replication.
