**Abstract**

Dengue is an acute febrile disease caused by a virus of the genus *Flavivirus*, family Flaviviridae, endemic in tropical regions of the globe. The agent is a virus with single-stranded RNA, classified into four distinct dengue virus (DENV) serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. The host's innate and adaptive immune responses play an essential role in determining the natural history of viral infections, especially in dengue. In this context, it has observed in recent years that the presence of RNA interference (RNAi) in viral infection processes is increasing, as well as immune defense. The context microRNAs (miRNAs) go for stood out, as their presence during viral infection, both in the replication of the virus and in the defense against these infections, becomes increasingly noticeable, therefore, making it increasingly necessary to better understand the role of these small RNAs within viral infection by DENV and what their consequences are in aggravating the consequences of patients affected by this disease.

**Keywords:** dengue, miRNA, genetics, immunology, *Flavivirus*

### **1. Introduction**

RNA interference (RNAi) is understood as the mechanism of gene silencing through transcription or post-transcription. Post-transcription gene silencing (PTGS) operates through translational repression induced by microRNAs (miRNAs), from precursors transcribed in the nucleus [1].

The silencing machinery by microRNAs directs mRNA to the P bodies present in the cytoplasm. They are deprived of the translation machinery and conserve proteins involved in the degradation of the target mRNA [2].

The natural functions of RNAi and their related processes appear to be the protection of the genome against invasion by mobile genetic elements, such as viruses and transposons, as well as the functioning of eukaryotic organism development programs [3, 4].

These analyses led to the identification of proteins encoded by the host involved in gene silencing. Also, they revealed that several enzymes or essential factors are common to these processes. Some components have identified to serve as initiators. In contrast, others serve as effectors, amplifiers, and transmitters for the gene silencing process [5].

The RNAi is widely used to fight viruses, due to the exposure of their genetic material in the intracellular environment at various stages of their replication cycle [6, 7]. Due to the distinction between viral and cellular genomes, the

chances of cross-silencing are low. In contrast, mutational changes in the viral genome allow mechanisms to escape interference pathways [8, 9].

### **2. miRNA**

The microRNAs are defined as small single-stranded RNA molecules with approximately 19–25 nucleotides, not protein encoders, that act as mediators for the regulation of the posttranscriptional gene expression [10].

The first miRNA was described in 1993 and related to the regulation of larval development in *Caenorhabditis elegans*. However, understand that the miRNA class is the largest class of gene regulators, with around 1000 miRNAs. Promoting regulation is necessary to bind the 3'untranslated region of the target mRNA [11].

There are more than 2500 miRNAs identified in the human genome. Although transcriptional targets are predicted, most of these have not been validated, which makes this area for investigation rich [6, 12]. Studies revealed that viruses encode miRNA, for example, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and human immunodeficiency virus type 1 (HIV-1) [13].

miRNAs play essential roles in the cell, such as proliferation, differentiation, apoptosis, stress response, and transcriptional regulation. Studies have shown that miRNAs change their expression in several pathologies, increasing their importance and the need for a better understanding of this process [14].

#### **2.1 miRNA biogenesis and mechanism of action**

The miRNA biogenesis process consists of transcription, export, processing, and maturation, at the cytoplasmic and cellular levels. As a fundamental element of this miRNA transcription, RNA polymerases II and III are found, with the function of transcribing gene encoding proteins [12].

Currently, most of the identified miRNAs are intragenic and processed from introns. Simultaneously, the rest are intergenic and transcribed independently of a host gene and regulated by their promoters [15]. Sometimes, miRNAs are transcribed as a long transcript called "clusters," which can have similar regions, and, in this case, are considered a family. miRNA biogenesis can be classified into two pathways: canonical and noncanonical (**Figure 1**) [5].

What defines the choice of these mechanisms is the complementarity between the bases of miRNA and mRNA. When there is perfect parity between the bases, degradation of the mRNAs will occur [16]. On the other hand, incomplete pairings generate inhibition of the translation of the target mRNA. Since miRNAs are small molecules, there is no need for complete pairing for binding. Thus, a miRNA can act in the regulation of several target mRNAs, or several miRNAs regulate a single mRNA [17].

#### *2.1.1 Canonical pathway*

The canonical pathway of biogenesis is the dominant pathway by which miRNAs are processed. In this way, pri-miRNAs are transcribed from their genes and processed into pre-miRNAs by the microprocessor complex, consisting of a protein that binds RNA to the critical region of DiGeorge syndrome 8 (DGCR8) and a ribonuclease III enzyme, Drosha [18]. DGCR8 recognizes an N6-methyladenylated GGAC and other motifs within the pri-miRNA [19]. At the same time, Drosha cleaves the duplex pri-miRNA based on the characteristic structure of the pri-miRNA, resulting in the formation of an excess of 2 nt 3′ in the pre-miRNA [20]. Once pre-miRNAs

**135**

**Figure 1.**

sively unwound and degraded [23].

*Dengue Virus and the Relationship with MicroRNAs DOI: http://dx.doi.org/10.5772/intechopen.92453*

are generated, they are exported to the cytoplasm by an exportin 5 (XPO5)/RanGTP

*MicroRNA biogenesis and mechanism of action. The canonical miRNA biogenesis begins with the generation of the pri-miRNA transcript passing through the microprocessor complex, composed of the critical region 8 of Drosha and DiGeorge syndrome (DGCR8), and cleaves the pri-miRNA to produce the precursor miRNA (pre-miRNA). The mature miRNA is associated with the Argonaute (AGO) protein family forming a miRNAinduced silencing complex (miRISC). In noncanonical pathways, small hairpin RNA (shRNA) is initially cleaved by the microprocessor complex and exported to the cytoplasm via exportin 5/RanGT, cleaved by AGO2,* 

This processing involves removing the loop from the terminal, resulting in a mature miRNA duplex. The directionality of the miRNA chain determines the name of the mature miRNA form. The 5p chain emerges from the 5′ end of the premiRNA hairpin, while the 3p chain originates from the 3′ end. Both chains derived from the mature miRNA duplex can carry in the Argonaute family of proteins. miRNA chains that do not contain incompatibilities are cleaved by AGO2 and degraded by cellular machinery that can produce a strong chain bias. Otherwise, miRNA duplexes with central mismatches or miRNA not loaded with AGO2 are pas-

complex and processed by the RNase III Dicer endonuclease [5, 22].

*but this action is independent of Dicer (modified by Tanzer et al.) [21].*

*Dengue Fever in a One Health Perspective*

**2. miRNA**

chances of cross-silencing are low. In contrast, mutational changes in the viral

The microRNAs are defined as small single-stranded RNA molecules with approximately 19–25 nucleotides, not protein encoders, that act as mediators for the

The first miRNA was described in 1993 and related to the regulation of larval development in *Caenorhabditis elegans*. However, understand that the miRNA class is the largest class of gene regulators, with around 1000 miRNAs. Promoting regulation is necessary to bind the 3'untranslated region of the target mRNA [11]. There are more than 2500 miRNAs identified in the human genome. Although transcriptional targets are predicted, most of these have not been validated, which makes this area for investigation rich [6, 12]. Studies revealed that viruses encode miRNA, for example, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV),

miRNAs play essential roles in the cell, such as proliferation, differentiation, apoptosis, stress response, and transcriptional regulation. Studies have shown that miRNAs change their expression in several pathologies, increasing their importance

The miRNA biogenesis process consists of transcription, export, processing, and maturation, at the cytoplasmic and cellular levels. As a fundamental element of this miRNA transcription, RNA polymerases II and III are found, with the function of

Currently, most of the identified miRNAs are intragenic and processed from introns. Simultaneously, the rest are intergenic and transcribed independently of a host gene and regulated by their promoters [15]. Sometimes, miRNAs are transcribed as a long transcript called "clusters," which can have similar regions, and, in this case, are considered a family. miRNA biogenesis can be classified into two

What defines the choice of these mechanisms is the complementarity between the bases of miRNA and mRNA. When there is perfect parity between the bases, degradation of the mRNAs will occur [16]. On the other hand, incomplete pairings generate inhibition of the translation of the target mRNA. Since miRNAs are small molecules, there is no need for complete pairing for binding. Thus, a miRNA can act in the regulation of several target mRNAs, or several miRNAs regulate a single

The canonical pathway of biogenesis is the dominant pathway by which miRNAs are processed. In this way, pri-miRNAs are transcribed from their genes and processed into pre-miRNAs by the microprocessor complex, consisting of a protein that binds RNA to the critical region of DiGeorge syndrome 8 (DGCR8) and a ribonuclease III enzyme, Drosha [18]. DGCR8 recognizes an N6-methyladenylated GGAC and other motifs within the pri-miRNA [19]. At the same time, Drosha cleaves the duplex pri-miRNA based on the characteristic structure of the pri-miRNA, resulting in the formation of an excess of 2 nt 3′ in the pre-miRNA [20]. Once pre-miRNAs

genome allow mechanisms to escape interference pathways [8, 9].

regulation of the posttranscriptional gene expression [10].

and human immunodeficiency virus type 1 (HIV-1) [13].

and the need for a better understanding of this process [14].

**2.1 miRNA biogenesis and mechanism of action**

pathways: canonical and noncanonical (**Figure 1**) [5].

transcribing gene encoding proteins [12].

**134**

mRNA [17].

*2.1.1 Canonical pathway*

#### **Figure 1.**

*MicroRNA biogenesis and mechanism of action. The canonical miRNA biogenesis begins with the generation of the pri-miRNA transcript passing through the microprocessor complex, composed of the critical region 8 of Drosha and DiGeorge syndrome (DGCR8), and cleaves the pri-miRNA to produce the precursor miRNA (pre-miRNA). The mature miRNA is associated with the Argonaute (AGO) protein family forming a miRNAinduced silencing complex (miRISC). In noncanonical pathways, small hairpin RNA (shRNA) is initially cleaved by the microprocessor complex and exported to the cytoplasm via exportin 5/RanGT, cleaved by AGO2, but this action is independent of Dicer (modified by Tanzer et al.) [21].*

are generated, they are exported to the cytoplasm by an exportin 5 (XPO5)/RanGTP complex and processed by the RNase III Dicer endonuclease [5, 22].

This processing involves removing the loop from the terminal, resulting in a mature miRNA duplex. The directionality of the miRNA chain determines the name of the mature miRNA form. The 5p chain emerges from the 5′ end of the premiRNA hairpin, while the 3p chain originates from the 3′ end. Both chains derived from the mature miRNA duplex can carry in the Argonaute family of proteins. miRNA chains that do not contain incompatibilities are cleaved by AGO2 and degraded by cellular machinery that can produce a strong chain bias. Otherwise, miRNA duplexes with central mismatches or miRNA not loaded with AGO2 are passively unwound and degraded [23].

#### *2.1.2 Noncanonical pathway*

Several noncanonical biogenesis pathways in miRNA are elucidated (**Figure 1**). These pathways make use of combinations of proteins involved in the canonical pathway, mainly Drosha, Dicer, exportin 5, and AGO2. The noncanonical miRNA can be grouped into Drosha-/DGCR8-independent and Dicer-independent pathways [24, 25].

The pre-miRNAs produced by the Drosha-/DGCR8-independent pathway resemble Dicer products. On the other hand, Dicer-independent miRNAs are processed by Drosha from endogenous RNA transcripts of hairpins. These premiRNAs require AGO2 to complete their maturation in the cytoplasm. They are of insufficient length to be the substrates for Dicer. That, in turn, promotes the loading of the entire pre-miRNA in the AGO2 slicing [26, 27].

#### *2.1.3 Argonaute and TNRC6 proteins*

The proteins of the Argonaute family are related to the RISC complex, as a member of the machinery of the RNAi pathways [28]. The highly conserved between species and several organisms encode several members of the family. Usually found in the cytoplasm are concentrated close to the P bodies [29].

Such proteins, therefore, act with the transcriptional and posttranscriptional silencing pathways. The main stage of the interference mechanism is the cleavage of mRNAs; the Argonaute protein in the RISC complex catalyzes this process [28].

Argonautes are applied in transcriptional and posttranscriptional gene silencing, acting through the modulation of the degradation or inhibition of the translation of specific mRNAs, when associated with miRNAs [18].

The miRNAs associated with Argonaute proteins constitute a more massive complex called the miRNA-induced silencing complex, which will suppress the expression of mRNAs. In addition to interference at the translational level, it shows that miRNAs can induce poly(A) tail deadening [18, 19]. Studies suggest that proteins of the TNRC6 family are essential components when associated with miRISCs, for the location of cytoplasmic P bodies and the gene silencing of mRNAs [19, 20].

#### **3. miRNA and dengue virus**

The dengue virus is a virus composed of a single-stranded positive RNA belonging to the family Flaviviridae. DENV serotypes have been identified (DENV-1 to DENV-4). All serotypes are causing similar diseases and similar symptoms, without significant severity and serious diseases, such as dengue hemorrhagic fever and dengue shock syndrome. The DENV genome is approximately 11 kb in length that encodes a single polyprotein. This polyprotein is cleaved posttranslationally by the host and viral proteases into three structural proteins (capsid C; pre-membrane/ membrane, prM/M; envelope, E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [30, 31].

The DENVs enter their target cells via receptor-mediated endocytosis in a clathrin-dependent manner. An acidified endosomal vesicle, virion, undergoes conformational changes that allow fusion and endosomal membrane and release RNA from the genome into the cytosol. After initial translation and cleavage of polyprotein, DENV triggers the formation of a replication complex in the perinuclear endoplasmic reticulum, and RNA replication and protein translation occur. Protein C then packages the newly synthesized positive RNA assembled into a virion, which

**137**

*Dengue Virus and the Relationship with MicroRNAs DOI: http://dx.doi.org/10.5772/intechopen.92453*

**3.1 Mosquitoes, miRNAs, and dengue**

resistance to insecticides [34].

blood feeding [35–37].

*Ae. albopictus* [36, 37].

*3.1.2 Mosquitoes and dengue infection*

*3.1.1 Development and metabolism*

secreted [32, 33].

is covered with prM/E heterodimers. When the vesicles containing the immature virions move through the Golgi apparatus, the prM is cleaved by a furin protease. Finally, immature virions become mature or partially mature virions, which are

The functions that miRNA is involved in mosquitoes are related to posttranscriptional regulation of gene expression in physiological and immunological pathways and affect development, metabolism, host-pathogen interactions, and

The specific expression of the miRNA stage in the four stages of development (eggs, larvae, pupae, and adults) was confirmed using sequencing. Understand the role of regulated miRNAs in the development of the mosquito and the action of knocking down the expressed miRNAs in a specific way carried out in *Ae. albopictus*. The knockdown of aal-miR-286b and aal-miR-2942 decreased the hatching of embryos and the hatching rate of larvae, respectively, compared to the knock-in groups. Reduced longevity and fertility (aal-miR-1891) were observed in the knockdown groups for miR-1891 compared to the knock-in and control groups in adults. Female mosquitoes require sugar for energy metabolism and a blood meal for egg development. Recent studies have indicated that blood supply leads to the differential expression of many genes, proteins, and miRNAs. The abundance of miRNA differs under sugar-fed and blood-fed conditions; ast-miR-2796-5p was observed exclusively in sugar-fed. The depletion of aae-miR-275 in *Ae. aegypti* females led to severe defects in blood digestion, fluid excretion, and egg development. aaemiR-1890 is induced after blood feeding and reaches a peak of 24 PMB. The systemic depletion of aae-miR-1890 resulted in less egg development and deposition, suggesting that miR-1890 may be the key to mosquito blood digestion. In contrast to the upregulated miRNAs after blood feeding, some miRNAs were downregulated. For example, reduced ast-miR-989 was observed 72 h after a blood meal. aga-let7 decreased in the midgut and other parts/leftovers, but most miRNAs increased after

The viruses of the *Flavivirus* genus are transmitted by mosquitoes and cause diseases, including dengue. It is observed that *Cx. quinquefasciatus* mosquitoes with West Nile virus (WNV) showed altered miR-92 and miR-989 expressions. In *Aedes albopictus*, the aae-miR-2940 miRNA is downregulated in response to WNV infection to restrict viral replication. In studies, the expressions of 35 miRNAs of mosquitoes modulating DENV infection in *Aedes aegypti* and more than 66 miRNAs were reported. *Ae. albopictus* is differentially expressed after DENV-2 infection. Therefore, aal-miR-34-5p and aal-miR-87 contribute to antipathogenic and immunological responses during DENV-2 infection. ae-miR-375 is the key to DENV replication, which can improve DENV-2 infection in an *Ae. aegypti* cell line. aae-miR-252 is three times more expressed after DENV-2 infection in *Ae. albopictus* cell line (C6/36); this inhibited DENV replication by suppressing the expression of the envelope protein of DENV. Regarding aal-miR-281, an abundant miRNA specific to the midgut, it was found that it facilitates the replication of DENV-2 in

*Dengue Fever in a One Health Perspective*

*2.1.3 Argonaute and TNRC6 proteins*

Several noncanonical biogenesis pathways in miRNA are elucidated (**Figure 1**). These pathways make use of combinations of proteins involved in the canonical pathway, mainly Drosha, Dicer, exportin 5, and AGO2. The noncanonical miRNA can be grouped into Drosha-/DGCR8-independent and Dicer-independent path-

The pre-miRNAs produced by the Drosha-/DGCR8-independent pathway resemble Dicer products. On the other hand, Dicer-independent miRNAs are processed by Drosha from endogenous RNA transcripts of hairpins. These premiRNAs require AGO2 to complete their maturation in the cytoplasm. They are of insufficient length to be the substrates for Dicer. That, in turn, promotes the loading

The proteins of the Argonaute family are related to the RISC complex, as a member of the machinery of the RNAi pathways [28]. The highly conserved between species and several organisms encode several members of the family. Usually found

Such proteins, therefore, act with the transcriptional and posttranscriptional silencing pathways. The main stage of the interference mechanism is the cleavage of mRNAs; the Argonaute protein in the RISC complex catalyzes this process [28].

The miRNAs associated with Argonaute proteins constitute a more massive complex called the miRNA-induced silencing complex, which will suppress the expression of mRNAs. In addition to interference at the translational level, it shows that miRNAs can induce poly(A) tail deadening [18, 19]. Studies suggest that proteins of the TNRC6 family are essential components when associated with miRISCs, for the location of cytoplasmic P bodies and the gene silencing of

The dengue virus is a virus composed of a single-stranded positive RNA belonging to the family Flaviviridae. DENV serotypes have been identified (DENV-1 to DENV-4). All serotypes are causing similar diseases and similar symptoms, without significant severity and serious diseases, such as dengue hemorrhagic fever and dengue shock syndrome. The DENV genome is approximately 11 kb in length that encodes a single polyprotein. This polyprotein is cleaved posttranslationally by the host and viral proteases into three structural proteins (capsid C; pre-membrane/ membrane, prM/M; envelope, E) and seven nonstructural proteins (NS1, NS2A,

The DENVs enter their target cells via receptor-mediated endocytosis in a clathrin-dependent manner. An acidified endosomal vesicle, virion, undergoes conformational changes that allow fusion and endosomal membrane and release RNA from the genome into the cytosol. After initial translation and cleavage of polyprotein, DENV triggers the formation of a replication complex in the perinuclear endoplasmic reticulum, and RNA replication and protein translation occur. Protein C then packages the newly synthesized positive RNA assembled into a virion, which

Argonautes are applied in transcriptional and posttranscriptional gene silencing, acting through the modulation of the degradation or inhibition of the translation of

of the entire pre-miRNA in the AGO2 slicing [26, 27].

in the cytoplasm are concentrated close to the P bodies [29].

specific mRNAs, when associated with miRNAs [18].

*2.1.2 Noncanonical pathway*

ways [24, 25].

mRNAs [19, 20].

**3. miRNA and dengue virus**

NS2B, NS3, NS4A, NS4B, and NS5) [30, 31].

**136**

is covered with prM/E heterodimers. When the vesicles containing the immature virions move through the Golgi apparatus, the prM is cleaved by a furin protease. Finally, immature virions become mature or partially mature virions, which are secreted [32, 33].
