**1. Introduction**

Dengue virus (DENV) is a major arbovirus that uses *Aedes aegypti* mosquitoes as an intermediate host to be transmitted to human and is the causative agent of most serious mosquito-borne viral disease (i.e., dengue fever) afflicting around more than 390 million people worldwide [1]. Despite substantial efforts to control dengue virus vector, it is still emerging rapidly [2]. Therapeutic

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

options are limited as the available treatments are just supportive, and there is no specific therapy or approved vaccine available. DENV belongs to *Flaviviridae* family, which comprises lipid-enveloped, positive-sense single-stranded RNA viruses [3]. DENV is classified into four closely related serotypes based on antigen distinction and represented as DENV-1 to DENV-4. The length of DENV genome is 10.7 kilobases comprising a single open reading frame (ORF) flanked by highly conserved 5′ and 3′ nontranslated regions. Single ORF encodes a polyprotein of approximately 3391 amino acids, which is further processed by host (furin and signalase) and viral proteases (NS2B/NS3 protease complex) to generate three structural (C, prM, and E) and seven nonstructural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [4, 5].

microRNAs (miRNAs) represent one of the shortest functional classes of noncoding RNAs, depicting hottest spot of gene regulation research. They were first reported in 1993 in small round worms *Caenorhabditis elegans* [18]. Since then, miRNAs have been implicated in regulating all cellular pathways in eukaryotes ranging from development to oncogenesis. These 20–24 nucleotide-long noncoding RNAs have been demonstrated to be encoded by a diverse range of organisms including plants, humans, insects, and even viruses [19]. The miRNA shows a very high degree of evolutionary sequence conservation among species [20] proved by the presence of miRNA biogenesis complex in archaea and eubacteria [21]. Historically, these microRNAs were thought to regulate different gene expressions either by degradation of particular mRNA or inhibition of translation [22, 23]. However, recent studies have revealed that these small noncoding RNAs can also activate the target gene expression through mRNA stabilization [15, 24]. 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. It is becoming more and more apparent that miRNAs not only constitute a fundamental part of regulatory machinery but also may act as molecular decoys to interfere with the function of regulatory RNA-binding proteins [25]. miRNA is involved in fundamental physiological and pathological processes like cancer development and host-pathogen interactions [26–29]. The pathogens have evolved to use to host machinery by altering the expression level of host miRNAs for their benefit [30]. Furthermore, microorganism-encoded miRNAs have also been reported to vary the host gene expression to their advantage [30]. Despite recent advances, there are still significant gaps in our understanding of miRNAs role in host-pathogen interactions, which is probably

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

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This pragmatic book chapter overviews the possibility of RNAi suppressor activity of DENV in the context of different viral genes. By characterizing the role of different DENV genes in RNAi suppression, we will review to gather valuable lead into the mechanism that governs DENV RNAi evasion in mosquitoes and the viral genes that are responsible for it. We will also explicit the possibility of RNA activation (RNAa) in insect, especially mosquitos. It is interesting to characterize whether RNAa has any role in host pathogen interaction because, despite the difference in their mode of action, RNAa use same RNAi machinery to induce gene activation. Although very few virus-encoded miRNAs have been fully characterized, it is understandable that they can target both viral and host genes. The information will produce a better understanding of microRNAs role in DENV replication and further deeper insight into

characterization of DENV-mediated differential expression of *Ae. aegypti* miRNAs.

The dengue virus (DENV) genome comprises a single open reading frame (ORF) of approximately 10.7 kilobases flanked by 5′ and 3′ nontranslated regions (NTRs) [30]. The nontranslated regions contain highly conserved secondary structures and coding regions in ORF, which play integral roles in viral life cycle. The composition of secondary structures is based on the sequencing of DENV-2 (a southeast Asian strain) strain, which is similar to other

due to lack of miRNA characterization.

**2. Dengue virus genome organization**

RNA interference (RNAi) is evolutionarily conserved phenomenon, constituting a major component of mosquito innate immune response to virus infections [6]. It has been demonstrated that dengue virus elicits RNAi response in *Ae. aegypti* but is unable to completely repress viral replication instead it may just modulate virus replication to maintain persistent viral infection to ensure long-term survival of infected mosquitoes [7]. It has been explored that DENV employs two different mechanisms to inhibit RNAi pathway, involving RNAi suppression protein NS4B and subgenomic flavivirus RNA (sfRNA). Dengue virus encoded nonstructural protein, NS4B, has been reported to suppress RNAi pathway in both human Huh-7 cells by the mechanism still not fully understood [8]. Furthermore, dengue virus infection produces 3' NTR degradation product sfRNA that is thought to incapacitate RNAi pathway by inhibiting dicer-mediated cleavage of dsRNA [9]. Recently, it has been ferreted out that in Culex mosquitoes, West Nile virus (WNV) replication is repressed by cytokine-like secreted antiviral protein "vago" in a dicer-2-dependent manner [10]. However, the possible effect of vago protein against dengue virus replication in *Ae. aegypti* research area is not explored yet. Such evidence clearly indicates that dengue virus opts variety of mechanisms to evade mosquito immune response to successfully replicate in the mosquitoes.

Apart from small interference RNAs (siRNA) reputation to inhibit gene expression through a phenomenon called RNAi, the target gene can also be induced by a mechanism called RNA activation (RNAa). The term RNAa was first coined by Li, who named them after he surprisingly found up regulation of human E-cadherin, p21, and VEGF genes by synthetic dsRNAs designed to target promoters of these genes [11]. He further named those promoters targeting small RNAs as short activating RNAs (saRNAs). Recent studies in which authors evaluated RNAa of above-said genes in monkey, rat, and mouse found that RNAa is highly conserved among all mammals [12]. However, the term RNA activation is not necessarily limited to transcriptional activation by saRNA, targeting promoter region only, rather it should be used to describe all small RNA-mediated gene and epigenome activation mechanisms, including transcriptional activation by targeting 3′ terminal regions of genes with saRNAs [13], piwi-interacting RNA (piRNAs)-mediated epigenetic activation [14], and microRNA (miRNA)-mediated translation activation [15]. One of the key differences between RNAi and RNAa is their kinetics. RNAi effect is known to occur within a couple of hours and disappears 5–7 days following siRNA transfection, while RNAa effect does not appear until about 48 h posttransfection and lasts much longer (at least 2 weeks) [16]. This delay may be due to the more complex mechanism of RNAa involving nuclear access and chromatin restructuring as compared to RNAi [17].

microRNAs (miRNAs) represent one of the shortest functional classes of noncoding RNAs, depicting hottest spot of gene regulation research. They were first reported in 1993 in small round worms *Caenorhabditis elegans* [18]. Since then, miRNAs have been implicated in regulating all cellular pathways in eukaryotes ranging from development to oncogenesis. These 20–24 nucleotide-long noncoding RNAs have been demonstrated to be encoded by a diverse range of organisms including plants, humans, insects, and even viruses [19]. The miRNA shows a very high degree of evolutionary sequence conservation among species [20] proved by the presence of miRNA biogenesis complex in archaea and eubacteria [21]. Historically, these microRNAs were thought to regulate different gene expressions either by degradation of particular mRNA or inhibition of translation [22, 23]. However, recent studies have revealed that these small noncoding RNAs can also activate the target gene expression through mRNA stabilization [15, 24]. 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. It is becoming more and more apparent that miRNAs not only constitute a fundamental part of regulatory machinery but also may act as molecular decoys to interfere with the function of regulatory RNA-binding proteins [25]. miRNA is involved in fundamental physiological and pathological processes like cancer development and host-pathogen interactions [26–29]. The pathogens have evolved to use to host machinery by altering the expression level of host miRNAs for their benefit [30]. Furthermore, microorganism-encoded miRNAs have also been reported to vary the host gene expression to their advantage [30]. Despite recent advances, there are still significant gaps in our understanding of miRNAs role in host-pathogen interactions, which is probably due to lack of miRNA characterization.

This pragmatic book chapter overviews the possibility of RNAi suppressor activity of DENV in the context of different viral genes. By characterizing the role of different DENV genes in RNAi suppression, we will review to gather valuable lead into the mechanism that governs DENV RNAi evasion in mosquitoes and the viral genes that are responsible for it. We will also explicit the possibility of RNA activation (RNAa) in insect, especially mosquitos. It is interesting to characterize whether RNAa has any role in host pathogen interaction because, despite the difference in their mode of action, RNAa use same RNAi machinery to induce gene activation. Although very few virus-encoded miRNAs have been fully characterized, it is understandable that they can target both viral and host genes. The information will produce a better understanding of microRNAs role in DENV replication and further deeper insight into characterization of DENV-mediated differential expression of *Ae. aegypti* miRNAs.
