**2. Dengue virus genome organization**

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

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

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

seven nonstructural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [4, 5].

94 Current Topics in Tropical Emerging Diseases and Travel Medicine

immune response to successfully replicate in the mosquitoes.

compared to RNAi [17].

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


**4. Viral replication**

**5. Virus-host interactions**

**6. RNA interference (RNAi)**

DENV like other flaviviruses, after adsorption to receptors, is endocytosed by cells in clathrindependent manner and then is further transported to endosomes [42]. However, according to one study, DENV can adopt clathrin-independent pathways as well to gain entry into mammalian cells in particular [43]. DENV nucleocapsid is released into cytosol after acidic pH triggered rearrangements of envelope protein in late endosomes leading to fusion of viral envelope and cellular endosome membrane [44, 45]. The nucleocapsid disassembles, and viral genome is translated by endoplasmic reticulum (ER)-located ribosomes generating numerous copies of viral proteins. NS5 and other viral nonstructural proteins along with various host proteins establish replication complexes [46]. At replication complex, the viral polymerase (NS5) transcribes negative viral RNA, which serves as a template for synthesis of subsequent positive viral RNA copies. Viral replication is regulated by 5′-3' UTR sequences (upstream of AUG region) present in 5′-3' UTRs, which are actively involved in the circularization of DENV RNA [47]. These newly synthesized positive DENV RNAs interact with core proteins to assemble into nucleocapsids [48]. The nucleocapsid buds into ER lumen, thereby getting enveloped in a membrane bilayer carrying the viral prM and E proteins [46]. From ER lumen, immature viral particles are transported through cellular secretary pathways, furin protease cleaves prM, and

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

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results in the formation of mature viral particles capable of infecting naïve cells [49].

replication without interfering too much with normal growth of mosquitos.

In nature, almost every organism engages in ecological or molecular interactions whether antagonistic or mutualistic with other organisms of different species to thrive and excel. These interactions are major drivers of diversification and adaptive evolution. Among these interactions, most fascinating examples are those involved in invertebrate susceptibility to pathogens [50]. According to a recent study, the susceptibility of *Ae. aegypti* to dengue virus has a genetic basis for genotype-by-genotype interactions [51]. It is evident by the fact that despite pathogenic nature of dengue virus in human, its interaction with *Ae. aegypti* mosquito is somehow mutualistic. DENV has somehow improvised to overcome mosquito defenses and manipulate its cellular machinery in a way still not well known that emancipates its

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

**Table 1.** Functions of DENV genome structural and nonstructural proteins.

different viral strains and serotypes. Although most of the DENV proteins are with known functions but some remains to be explored (**Table 1**) [31].
