**Abstract**

Dengue is the most prevalent emerging mosquito-borne viral disease, affecting more than 40% of the human population worldwide. Many symptomatic dengue virus (DENV) infections result in a relatively benign disease course known as dengue fever (DF). However, a small proportion of patients develop severe clinical manifestations, englobed in two main categories known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Secondary infection with any of the four dengue virus serotypes (DENV1, -2, -3, and -4) is a risk factor to develop severe forms of dengue disease. DSS is primarily characterized by sudden and abrupt endothelial dysfunction, resulting in vascular leak and organ impairment, which may progress to hypovolemic shock and death. Severe DENV disease (DHF/ DSS) is thought to follow a complex relationship between distinct immunopathogenic processes involving host and viral factors, such as the serotype cross-reactive antibody-dependent enhancement (ADE), the activation of T cells and complement pathways, the phenomenon of the *cytokine storm*, and the newly described viral toxin activity of the nonstructural protein 1 (NS1), which together play critical roles in inducing vascular leak and virus pathogenesis. In this chapter that is divided in two parts, we will outline the recent advances in our understanding of DENV pathogenesis, highlighting key viral-host interactions and discussing how these interactions may contribute to DENV immunopathology and the development of vascular leak, a hallmark of severe dengue. *Part I* will address the general features of the DENV complex, including the virus structure and genome, epidemiology, and clinical outcomes, followed by an updated review of the literature describing the host innate immune strategies as well as the viral mechanisms acting against and in favor of the DENV replication cycle and infection.

**Keywords:** dengue, immunopathogenesis, dengue shock syndrome, severe dengue, virus replication, cell tropism, innate immune response, antiviral response, immune evasion, complement, endothelial dysfunction, vascular leak

## **1. Introduction**

Dengue is still considered the most prevalent viral disease transmitted by arthropod mosquitoes (e.g., *Aedes* mosquitoes), with 50–100 million dengue infections occurring annually, and a global incidence of 30-fold increase observed over the past 50 years [1–3]. Most of the dengue infections with any of the four dengue virus (DENV) serotypes [1–4] result in inapparent, subclinical illness, or mild disease symptoms known as dengue fever (DF). However, some DENV infections can potentially evolve into more severe and fatal disease outcomes known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [2]. DHF and DSS are mainly characterized by low numbers of circulating platelets (thrombocytopenia) associated with hemorrhagic manifestations and increased vascular permeability associated with endothelium hyperpermeability, resulting in plasma leakage, low blood pressure, and shock that can lead to death [2]. DENV infections frequently occur in the context of preexisting immunity, where the immune responses to prior DENV infection play an important role in determining the outcome of dengue epidemics and disease severity via antibody-dependent enhancement (ADE) and potentially harmful T cell responses in an *original antigenic sin*-dependent manner [4–8]. Both mechanisms lead to increased activation of immune cells, resulting in exacerbated immune responses or *cytokine storms* that cause endothelium dysfunction and vascular leak [9, 10]. Collectively, these host immunological responses are thought to create a physiological environment that promotes vascular permeability. However, the exact mechanisms underlying the capillary leak are probably more complex than a *cytokine storm*, and the risk of severe disease upon DENV infection cannot be explained completely by a misdirected host immune response to a prior infecting serotype; rather, disease severity appears to be determined by a combination of multiple host and viral factors leading to favorable and unfavorable interactions that regulate viral pathogenesis.

Despite considerable advances in understanding the immunological mechanisms activated during DENV infection, the pathogenic mechanisms underlying the alterations in permeability of the microvasculature remain unclear. The absence of a good animal model faithful to human disease and the limited knowledge of the factors regulating the intrinsic microvascular permeability in health have seriously hampered the research progress in this area. However, in the last decades, significant progress has been made regarding viral and host cellular components involved in DENV infection and disease [8]. The nonstructural protein 1 (NS1) protein of DENV and other related flaviviruses has been described as an essential cofactor in virus replication and assembly [11, 12]. Interestingly, the secreted form of NS1 is also implicated in immune evasion strategies via interaction with several proteins of the complement pathways that protect the virus-infected cells from the immune system processing [12–14]. Contrary, NS1 and anti-NS1 antibodies can also mediate complement activation that may alter capillary permeability [15]. Additionally, the soluble NS1 from DENV can interact with the surface of endothelial cells, immune cells, and platelets to cause endothelial barrier dysfunction and vascular leakage, and potentially hampers the coagulation cascades leading to hemorrhagic manifestations during DENV infection. These phenomena occur via activation of endothelialintrinsic mechanisms leading to the disruption of the EGL and the integrity of the cell-to-cell contacts and/or induction of pro-inflammatory cytokines, chemokines,

**61**

*Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease…*

and proteases via the TLR4 activation of monocytes/macrophages that may act also on endothelial cells leading to endothelial hyperpermeability and vascular leak [16–22]. Furthermore, NS1 is highly immunogenic and conserved between the *Flavivirus* genus; thus, NS1 from other flaviviruses have been also reported to activate endothelial-intrinsic mechanisms causing vascular leakage in a tissuedependent manner that mimics each flavivirus disease pathophysiology [17, 23–25]. Additionally, NS1 immunization using mouse models and DENV vaccination or natural DENV infection in humans can elicit antibodies' responses that have been implicated in the contradictory roles of protection and pathogenesis in the infected host [25–42]. Today, no specific and effective vaccine candidate, antiviral therapy, or anti-inflammatory therapeutics have been licensed to combat dengue disease. An effective dengue vaccine is surely needed to avert the millions of dengue cases that occur around the world, continuously threatening with fatal outcomes. For decades, numerous experts in infectious diseases including clinicians, epidemiologists, basic scientists, and vaccine and drug developers have been trying to elucidate the ultimate mechanism of DENV pathogenesis leading to severe dengue disease. The NS1 protein of flaviviruses constitutes a unique "viral toxin" that seems to connect many of the already described DENV immunopathogenic mechanisms leading to severe dengue disease; thus, NS1 might represent the corner piece that completes the elusive dengue pathogenesis puzzle. Therapeutic approaches and vaccine development targeting NS1 may provide different opportunities for the future defeating of the global dengue disease. A better understanding of DENV immunopathogenesis will assist not only in the development of therapeutic interventions but also in the understanding of dengue vaccine efficacy or vaccine adverse events. This chapter briefly summaries the key clinical, virological, and epidemiological facts about DENV innate and humoral responses and gives an extensive update of insights about the viral and host factors that contribute to DENV pathogenesis leading to the

development of severe dengue manifestations during DENV infection.

**2. Dengue virus features: genomic organization, structure, and life cycle**

Dengue virus (DENV) belongs to the genus *Flavivirus* (family *Flaviviridae*), a group of small (50-nm virion diameter) viruses containing a single-positivestranded RNA genome [5′ capped, not 3′ poly(A) tail] which encodes for three structural proteins: capsid (C), membrane (M), and envelope (E) and seven nonstructural (NS) proteins named NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (**Figure 1A**, **B**) [43]. The DENV group is comprised of four evolutionary distinct but antigenically and genetically related viruses better known as DENV serotypes −1, −2, −3, and −4 (DENV-1-4), [44, 45]. These four established serotypes (DENV 1–4) share a high degree of sequence similarity between the genomes (~65–70%) [46], with average sequence identity between proteomes of 39–79% [47]. DENV is transmitted to humans by *Aedes* mosquitoes, mainly *Aedes aegypti*. However, the global distribution of *Aedes albopictus* (the Asian tiger mosquito), considered a secondary vector for DENV transmission, is changing rapidly and it is now becoming an increasingly important vector and a common cause of epidemics in *Aedes aegypti*-free countries [48–50]. Human-to-mosquito transmission occurs once the mosquito takes a blood meal from DENV-infected people who are viremic, which is normally up to 2 days before someone shows symptoms of the illness or up to 2 days after the fever has resolved. High viremia and high fever in patients are positively associated with a high rate of DENV transmission from humans to mosquitoes [51]. After feeding on a DENV-infected person, depending mainly on temperature, the virus rapidly replicates in the mosquito's midgut, and within an average of 5.9 days,

*DOI: http://dx.doi.org/10.5772/intechopen.93140*

*Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease… DOI: http://dx.doi.org/10.5772/intechopen.93140*

and proteases via the TLR4 activation of monocytes/macrophages that may act also on endothelial cells leading to endothelial hyperpermeability and vascular leak [16–22]. Furthermore, NS1 is highly immunogenic and conserved between the *Flavivirus* genus; thus, NS1 from other flaviviruses have been also reported to activate endothelial-intrinsic mechanisms causing vascular leakage in a tissuedependent manner that mimics each flavivirus disease pathophysiology [17, 23–25]. Additionally, NS1 immunization using mouse models and DENV vaccination or natural DENV infection in humans can elicit antibodies' responses that have been implicated in the contradictory roles of protection and pathogenesis in the infected host [25–42]. Today, no specific and effective vaccine candidate, antiviral therapy, or anti-inflammatory therapeutics have been licensed to combat dengue disease. An effective dengue vaccine is surely needed to avert the millions of dengue cases that occur around the world, continuously threatening with fatal outcomes. For decades, numerous experts in infectious diseases including clinicians, epidemiologists, basic scientists, and vaccine and drug developers have been trying to elucidate the ultimate mechanism of DENV pathogenesis leading to severe dengue disease. The NS1 protein of flaviviruses constitutes a unique "viral toxin" that seems to connect many of the already described DENV immunopathogenic mechanisms leading to severe dengue disease; thus, NS1 might represent the corner piece that completes the elusive dengue pathogenesis puzzle. Therapeutic approaches and vaccine development targeting NS1 may provide different opportunities for the future defeating of the global dengue disease. A better understanding of DENV immunopathogenesis will assist not only in the development of therapeutic interventions but also in the understanding of dengue vaccine efficacy or vaccine adverse events. This chapter briefly summaries the key clinical, virological, and epidemiological facts about DENV innate and humoral responses and gives an extensive update of insights about the viral and host factors that contribute to DENV pathogenesis leading to the development of severe dengue manifestations during DENV infection.

### **2. Dengue virus features: genomic organization, structure, and life cycle**

Dengue virus (DENV) belongs to the genus *Flavivirus* (family *Flaviviridae*), a group of small (50-nm virion diameter) viruses containing a single-positivestranded RNA genome [5′ capped, not 3′ poly(A) tail] which encodes for three structural proteins: capsid (C), membrane (M), and envelope (E) and seven nonstructural (NS) proteins named NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (**Figure 1A**, **B**) [43]. The DENV group is comprised of four evolutionary distinct but antigenically and genetically related viruses better known as DENV serotypes −1, −2, −3, and −4 (DENV-1-4), [44, 45]. These four established serotypes (DENV 1–4) share a high degree of sequence similarity between the genomes (~65–70%) [46], with average sequence identity between proteomes of 39–79% [47]. DENV is transmitted to humans by *Aedes* mosquitoes, mainly *Aedes aegypti*. However, the global distribution of *Aedes albopictus* (the Asian tiger mosquito), considered a secondary vector for DENV transmission, is changing rapidly and it is now becoming an increasingly important vector and a common cause of epidemics in *Aedes aegypti*-free countries [48–50]. Human-to-mosquito transmission occurs once the mosquito takes a blood meal from DENV-infected people who are viremic, which is normally up to 2 days before someone shows symptoms of the illness or up to 2 days after the fever has resolved. High viremia and high fever in patients are positively associated with a high rate of DENV transmission from humans to mosquitoes [51]. After feeding on a DENV-infected person, depending mainly on temperature, the virus rapidly replicates in the mosquito's midgut, and within an average of 5.9 days,

*Dengue Fever in a One Health Perspective*

tions that regulate viral pathogenesis.

**1. Introduction**

**Keywords:** dengue, immunopathogenesis, dengue shock syndrome, severe dengue, virus replication, cell tropism, innate immune response, antiviral response, immune

Dengue is still considered the most prevalent viral disease transmitted by arthropod mosquitoes (e.g., *Aedes* mosquitoes), with 50–100 million dengue infections occurring annually, and a global incidence of 30-fold increase observed over the past 50 years [1–3]. Most of the dengue infections with any of the four dengue virus (DENV) serotypes [1–4] result in inapparent, subclinical illness, or mild disease symptoms known as dengue fever (DF). However, some DENV infections can potentially evolve into more severe and fatal disease outcomes known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [2]. DHF and DSS are mainly characterized by low numbers of circulating platelets (thrombocytopenia) associated with hemorrhagic manifestations and increased vascular permeability associated with endothelium hyperpermeability, resulting in plasma leakage, low blood pressure, and shock that can lead to death [2]. DENV infections frequently occur in the context of preexisting immunity, where the immune responses to prior DENV infection play an important role in determining the outcome of dengue epidemics and disease severity via antibody-dependent enhancement (ADE) and potentially harmful T cell responses in an *original antigenic sin*-dependent manner [4–8]. Both mechanisms lead to increased activation of immune cells, resulting in exacerbated immune responses or *cytokine storms* that cause endothelium dysfunction and vascular leak [9, 10]. Collectively, these host immunological responses are thought to create a physiological environment that promotes vascular permeability. However, the exact mechanisms underlying the capillary leak are probably more complex than a *cytokine storm*, and the risk of severe disease upon DENV infection cannot be explained completely by a misdirected host immune response to a prior infecting serotype; rather, disease severity appears to be determined by a combination of multiple host and viral factors leading to favorable and unfavorable interac-

Despite considerable advances in understanding the immunological mechanisms

activated during DENV infection, the pathogenic mechanisms underlying the alterations in permeability of the microvasculature remain unclear. The absence of a good animal model faithful to human disease and the limited knowledge of the factors regulating the intrinsic microvascular permeability in health have seriously hampered the research progress in this area. However, in the last decades, significant progress has been made regarding viral and host cellular components involved in DENV infection and disease [8]. The nonstructural protein 1 (NS1) protein of DENV and other related flaviviruses has been described as an essential cofactor in virus replication and assembly [11, 12]. Interestingly, the secreted form of NS1 is also implicated in immune evasion strategies via interaction with several proteins of the complement pathways that protect the virus-infected cells from the immune system processing [12–14]. Contrary, NS1 and anti-NS1 antibodies can also mediate complement activation that may alter capillary permeability [15]. Additionally, the soluble NS1 from DENV can interact with the surface of endothelial cells, immune cells, and platelets to cause endothelial barrier dysfunction and vascular leakage, and potentially hampers the coagulation cascades leading to hemorrhagic manifestations during DENV infection. These phenomena occur via activation of endothelialintrinsic mechanisms leading to the disruption of the EGL and the integrity of the cell-to-cell contacts and/or induction of pro-inflammatory cytokines, chemokines,

evasion, complement, endothelial dysfunction, vascular leak

**60**

#### *Dengue Fever in a One Health Perspective*

it disseminates to secondary tissues, including the salivary glands where the virus can be transmitted to the new host (extrinsic incubation period) [52]. Once infectious, the mosquito is capable of transmitting the virus for the rest of its life [53].

After a mosquito bites a human, DENV is delivered into the dermis where it can infect/replicate in dendritic cells (DCs) (Langerhans cells) and keratinocytes residing in the basal and suprabasal layers of the epidermis [54–56] (**Figure 2**). Virus dissemination to the local lymph nodes occurs in association with infected migratory dendritic cells or as free viruses of the lymphatic fluid leading to viremia [57]. At this stage, mosquito saliva has shown to enhance the replication and

**63**

*PyMOL molecular graphics.*

*Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease…*

*Dengue virus genome organization, NS1 structure, and dengue epidemiology and disease outcome. (A) Schematic representation of the DENV genome and polyprotein. Dengue virus (DENV; genus Flavivirus, family Flaviviridae) is a positive-sense, single-stranded (*∼*11-kb length), and RNA-enveloped virus with an icosahedral capsid protecting the virus genome, which is transmitted by mosquitoes of the Aedes genus (Aedes aegypti, Ae. albopictus) and affects more than 40% of the human population worldwide living in tropical and subtropical areas. The viral RNA genome poses one single open reading frame encoding for one single polyprotein, which after being processed by cellular and viral proteases generates three structural proteins known as the capsid (C), the membrane (M), and the envelope (E) and seven nonstructural (NS) proteins known as NS1, NS2A, NS2B, NS3, NS4A, NS4B, and N5. The viral RNA contains a cap in the 5*′*-end, and it has no poly-a tail in the 3*′*-end. Several secondary structures or UTRs (untranslated regions) are found in both ends which have been shown to participate in viral replication as well as host adaptation. Of the structural proteins, the envelope (E) is the major protein on the virion, which participates in cellular receptor recognition to infect the host cells and the main target for adaptive immune responses in humans. On the other hand, the NS proteins play critical roles in the virus replication cycle and the subversion of the host antiviral responses, particularly those triggered by the innate immune response against DENV infection. (B) Of these NS proteins, NS1 is the only viral protein secreted by DENV-infected cells in which the plasma circulating levels are increased during the acute phase of DENV-infected patients undergoing severe disease. The NS1 protein circulates as a lipoprotein-like particle with a hexameric conformational structure containing three domains termed as the wing domain (here in yellow), the β-ladder domain (in blue), and the β-roll domain (in red) (NS1 hexamer is depicted in this figure). NS1 has been demonstrated to play critical roles in the formation of new viral particles, the evasion of the immune system, and very recently, it was implicated in modulating the virus pathogenesis of DENV that is mainly associated to its potential role in acting as a pathogen-associated pattern molecule (PAMP) which activates the production of pro-inflammatory host soluble factors such as cytokines, chemokines, proteases, etc. from the immune cells and directly triggers the barrier dysfunction of endothelial cell cultures in vitro and vascular leak in the mouse models in vivo. Taking these together, NS1 is now considered as a viral toxin not only of the DENV complex but of many of the closely related flaviviruses, including ZIKV, WNV, and YFV among others. (C) The DENV complex is composed of four serologically but antigenically related types of viruses, known as DENV serotypes (1, 2, 3, and 4). The primary infection with any of the four serotypes often cause inapparent, asymptomatic, or mild diseases that prime the immune system for a long-life immunity against the infecting DENV serotype (here DENV-1, in yellow) which is dominated by homotypic immune responses at mostly all levels, including antibody-B cells' and T cells' responses. During secondary infections, things get more complicated as infection with a different DENV serotype as the one from the primary infection; here DENV-2 (blue), DENV-3 (red), and DENV-4 (green) results in cross-reactive and heterologous immune responses that are considered the main risk factor to develop severe manifestations of DENV infections, including dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), which may lead to life-threatening health complications and sometimes, death. In the course of this chapter, we will explain how these epidemiological factors may be associated with the development of severe dengue disease that may be a consequence of a combined interplay set of immunopathogenic mechanisms triggered by the host immune response having DENV infection as the main trigger. The model of NS1 hexamer was built based on the crystal structure deposited in the Protein Data Bank (PDB4O6B). Molecular graphics were performed using the* 

pathogenesis of numerous arthropod-borne viruses, including DENV [58–62]. DENV infection cycle initiates with the virus attachment to the target cells [63]. The current model suggests that DENV uses both attachment factors and primary receptor(s) that facilitate virus recruitment on the cell surface, and later, internalization inside host cells via receptor-mediated endocytosis including clathrinmediated and nonclassical clathrin-independent endocytosis [64, 65]. Despite this, the single definitive receptor mediating this critical step in the DENV replication cycle continues to be elusive. So far, numerous candidates have been described in the mammalian and mosquito cells, including glycosaminoglycans such as heparan sulfate and lectins, the adhesion molecule of dendritic cells (DC-SIGN), the mannose receptor (MR) of macrophages, the lipopolysaccharide (LPS) receptor CD14, and stress-induced proteins such as the heat-shock proteins 70 and 90 and the endoplasmic reticulum (ER) chaperonin GRP78 [64–68]. This suggests that DENV may not use a unique, specific receptor to enter cells, but recognizes diverse molecules, both in the vertebrate and mosquito hosts, which can potentially explain

the broad tissue range that defines DENV tropism and infection.

After the internalization of the virion, a fusion between the viral E protein and the endosomal membrane mediates the access of the viral genome into the cytoplasm [43, 65]. The E protein is a glycosylated viral protein and a member of class II viral membrane fusion protein family [43, 69]. The crystal structure of

*DOI: http://dx.doi.org/10.5772/intechopen.93140*

**Figure 1.**

*Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease… DOI: http://dx.doi.org/10.5772/intechopen.93140*

#### **Figure 1.**

*Dengue Fever in a One Health Perspective*

it disseminates to secondary tissues, including the salivary glands where the virus can be transmitted to the new host (extrinsic incubation period) [52]. Once infectious, the mosquito is capable of transmitting the virus for the rest of its life [53]. After a mosquito bites a human, DENV is delivered into the dermis where it can infect/replicate in dendritic cells (DCs) (Langerhans cells) and keratinocytes residing in the basal and suprabasal layers of the epidermis [54–56] (**Figure 2**). Virus dissemination to the local lymph nodes occurs in association with infected migratory dendritic cells or as free viruses of the lymphatic fluid leading to viremia [57]. At this stage, mosquito saliva has shown to enhance the replication and

**62**

*Dengue virus genome organization, NS1 structure, and dengue epidemiology and disease outcome. (A) Schematic representation of the DENV genome and polyprotein. Dengue virus (DENV; genus Flavivirus, family Flaviviridae) is a positive-sense, single-stranded (*∼*11-kb length), and RNA-enveloped virus with an icosahedral capsid protecting the virus genome, which is transmitted by mosquitoes of the Aedes genus (Aedes aegypti, Ae. albopictus) and affects more than 40% of the human population worldwide living in tropical and subtropical areas. The viral RNA genome poses one single open reading frame encoding for one single polyprotein, which after being processed by cellular and viral proteases generates three structural proteins known as the capsid (C), the membrane (M), and the envelope (E) and seven nonstructural (NS) proteins known as NS1, NS2A, NS2B, NS3, NS4A, NS4B, and N5. The viral RNA contains a cap in the 5*′*-end, and it has no poly-a tail in the 3*′*-end. Several secondary structures or UTRs (untranslated regions) are found in both ends which have been shown to participate in viral replication as well as host adaptation. Of the structural proteins, the envelope (E) is the major protein on the virion, which participates in cellular receptor recognition to infect the host cells and the main target for adaptive immune responses in humans. On the other hand, the NS proteins play critical roles in the virus replication cycle and the subversion of the host antiviral responses, particularly those triggered by the innate immune response against DENV infection. (B) Of these NS proteins, NS1 is the only viral protein secreted by DENV-infected cells in which the plasma circulating levels are increased during the acute phase of DENV-infected patients undergoing severe disease. The NS1 protein circulates as a lipoprotein-like particle with a hexameric conformational structure containing three domains termed as the wing domain (here in yellow), the β-ladder domain (in blue), and the β-roll domain (in red) (NS1 hexamer is depicted in this figure). NS1 has been demonstrated to play critical roles in the formation of new viral particles, the evasion of the immune system, and very recently, it was implicated in modulating the virus pathogenesis of DENV that is mainly associated to its potential role in acting as a pathogen-associated pattern molecule (PAMP) which activates the production of pro-inflammatory host soluble factors such as cytokines, chemokines, proteases, etc. from the immune cells and directly triggers the barrier dysfunction of endothelial cell cultures in vitro and vascular leak in the mouse models in vivo. Taking these together, NS1 is now considered as a viral toxin not only of the DENV complex but of many of the closely related flaviviruses, including ZIKV, WNV, and YFV among others. (C) The DENV complex is composed of four serologically but antigenically related types of viruses, known as DENV serotypes (1, 2, 3, and 4). The primary infection with any of the four serotypes often cause inapparent, asymptomatic, or mild diseases that prime the immune system for a long-life immunity against the infecting DENV serotype (here DENV-1, in yellow) which is dominated by homotypic immune responses at mostly all levels, including antibody-B cells' and T cells' responses. During secondary infections, things get more complicated as infection with a different DENV serotype as the one from the primary infection; here DENV-2 (blue), DENV-3 (red), and DENV-4 (green) results in cross-reactive and heterologous immune responses that are considered the main risk factor to develop severe manifestations of DENV infections, including dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), which may lead to life-threatening health complications and sometimes, death. In the course of this chapter, we will explain how these epidemiological factors may be associated with the development of severe dengue disease that may be a consequence of a combined interplay set of immunopathogenic mechanisms triggered by the host immune response having DENV infection as the main trigger. The model of NS1 hexamer was built based on the crystal structure deposited in the Protein Data Bank (PDB4O6B). Molecular graphics were performed using the PyMOL molecular graphics.*

pathogenesis of numerous arthropod-borne viruses, including DENV [58–62]. DENV infection cycle initiates with the virus attachment to the target cells [63]. The current model suggests that DENV uses both attachment factors and primary receptor(s) that facilitate virus recruitment on the cell surface, and later, internalization inside host cells via receptor-mediated endocytosis including clathrinmediated and nonclassical clathrin-independent endocytosis [64, 65]. Despite this, the single definitive receptor mediating this critical step in the DENV replication cycle continues to be elusive. So far, numerous candidates have been described in the mammalian and mosquito cells, including glycosaminoglycans such as heparan sulfate and lectins, the adhesion molecule of dendritic cells (DC-SIGN), the mannose receptor (MR) of macrophages, the lipopolysaccharide (LPS) receptor CD14, and stress-induced proteins such as the heat-shock proteins 70 and 90 and the endoplasmic reticulum (ER) chaperonin GRP78 [64–68]. This suggests that DENV may not use a unique, specific receptor to enter cells, but recognizes diverse molecules, both in the vertebrate and mosquito hosts, which can potentially explain the broad tissue range that defines DENV tropism and infection.

After the internalization of the virion, a fusion between the viral E protein and the endosomal membrane mediates the access of the viral genome into the cytoplasm [43, 65]. The E protein is a glycosylated viral protein and a member of class II viral membrane fusion protein family [43, 69]. The crystal structure of

E glycoprotein ectodomain revealed three domains contributing to the β-barrel central structure of the protein (domain I, DI), permitting the fusion of viral and cellular membranes during virus entry (domain II, DII, and *fusion loop*), and a structural basis for immune recognition and cellular receptor binding (domain III, DIII) [43, 70]. The low pH of the endosomal compartment induces conformational changes in the E glycoprotein, which allows the fusion of the viral and host membranes [43, 69]. This results in the release of the viral RNA genome into the cytoplasm. The single-stranded positive-sense RNA immediately acts as a messenger RNA, which can be subsequently translated by cellular machinery to generate viral polyproteins, subsequently processed by both cellular and viral proteases to generate mature viral proteins [69, 70]. In this stage, the nonstructural proteins have been shown to induce massive remodeling of ER membranes, manifesting as convoluted membranes and vesicle packets (VPs) to form a dynamic and membrane-bound multi-protein assembly, named the replication complex (RC) where the genome is replicated, and new viral RNA copies are incorporated into nascent particles [71, 72]. Viral RNA synthesis relies on NS5, the RNA-dependent RNA polymerase as well as on critical RNA secondary and tertiary structures [73–75]. NS3 is a

**65**

*Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease…*

*Dengue virus infection, pathogenesis, and immune responses in the skin. The skin represents the first line of defense of the human body against pathogens such as viruses, as it generates early immune responses, aiming to protect humans against cutaneous and systemic infection (1). The human skin constitutes a complex organ nicely structured in three escalated layers known as the epidermis, which is composed of closely packed epithelial cells including keratinocytes, melanocytes, and Langerhans cells (LCs), a specialized type of dendritic cell (DC) that constantly probes for antigen in the most exposed, superficial layer of the skin; the dermis, which is made of dense, irregular connective tissue, blood vessels, and other structures; and the inner hypodermis, which is composed mainly of loose connective and fatty tissues (2). Upon disturbance of the epidermal barrier by mosquito blood-feeding, DENV-infected mosquitoes inoculate newly generated infectious virus particles along with mosquito saliva in which a complex mixture of proteins that exerts profound effects in the human immune system allows the acquisition of the mosquito blood meal from its host by circumventing vasoconstriction, platelet aggregation, coagulation, and inflammation or hemostasis (3). In the skin, major constituents of the innate immune system include phagocytic cells such as macrophages, neutrophils, and DCs as well as innate leukocytes such as natural killer (NK) cells, mast cells (MCs), basophils, and eosinophils. Also, epidermal keratinocytes act as active innate immune cells. In response to sensing pathogen-associated molecular patterns (PAMPs) expressed by microbes and host danger molecules, innate immune receptors present on keratinocytes and APCs become activated, causing the release of inflammatory cytokines and antimicrobial peptides (4). At the site of inoculation in the skin, the key targets of DENV infection are immune cells of the myeloid lineage, including various subsets of DCs, monocytes/macrophages, and MCs (5). Despite this, limited virus particles are thought to be deposited in the epidermis during mosquito blood-feeding (3), and in that location, Langerhans cells as well as keratinocytes are considered target cells (6). In the dermis, DCs and monocytes/macrophages are also prime infection targets (7). MCs are not substantially infected in the skin (8). However, exposure to DENV triggers an augmented activation of MCs leading to degranulation and release of de novo-synthesized inflammatory and vasoactive mediators, including proteases, leukotrienes, and histamine that, along with some vasoactive molecules and maybe the secreted NS1 originated from infected mosquito cells in the salivary glands, promote edema within the site of infection as a consequence of the increased microvascular permeability (9). Activation of MCs also induces the secretion of cytokines and chemokines that leads to the recruitment of NK cells, neutrophils, and monocyte-derived dendritic cells (mDCs) to the site of infection (10). Already in the skin, mDCs can serve as targets of infection, allowing the amplification of the virus in the skin, while natural killer (NK) cells, natural killer T (NKT) cells, and CD8+ T cells can kill DENV-infected cells and promote virus clearance in a cellular cytotoxic-dependent manner (11). Once human skin is infected, DENV-infected DCs take virus into the draining lymph nodes using afferent lymphatic vessels* 

protease-helicase which together with its cofactor NS2B, participates in the processing, efficient RNA synthesis, and capping of the viral polyprotein [72, 76, 77]. NS2A recruits nascent RNA as well as C-pre-M-E [78]. NS1 interacts with structural proteins and NS4A-2 K-4B to facilitate the production of infectious virus particles [11, 79]. Because of their critical roles in the DENV replication cycle, NS2B, NS3, and NS5 along with NS4B are the main focus to design new inhibitors for antiviral therapy against DENV and other related flaviviruses [80–83]. Assembled viruses are transported through the trans-Golgi network (TGN) where under acidic conditions, a cellular protease, *furin*, cleaves pre-M, allowing full maturation of infec-

*which initiates the adaptive immune response (12, 13). In the T cell zones, activated T cells become effectors cells to promote the development of DENV-specific memory B cells and plasma cells in the germinal center of LN. Activated T cells can reenter circulation and potentially return to the skin for virus clearance during subsequent DENV infections, playing an important role in protecting against DENV (14). In the skin, in addition to the mosquito saliva, DENV infection of target cells such as DCs, monocytes/macrophages, and MCs can be also modulated by the presence of preexisting antibody responses against previous infections with distinct DENV serotypes or other closely related flaviviruses, in an antibody-dependent enhancement (ADE) manner (See DENV-ADE in Part II for more details). For MCs and DCs, DENV-ADE is possible through Fc-γ-receptors (FcγRs). Besides, MCs degranulation can be enhanced through cross-linking of FcεRs when bound to DENVspecific IgE, leading to augmented MC activation and presumably immune-mediated vascular injury (15). After skin infection, DENV must achieve systemic infection to complete its transmission cycle by infecting new mosquito hosts. Infection of secondary LNs following infection of the draining LN are considered the amplification centers for DENV that contributes to the systemic infection and virus transmission (16).*

*where they spread DENV infection and most importantly activate antigen-specific CD4+*

 *and CD8+*

 *T cells* 

tious virions that will be finally released via exocytosis [84].

**3. Dengue virus infection, epidemiology, and clinical features**

Dengue is the arboviral infection with the highest disease incidence worldwide, with 2.5 billion people living in dengue-endemic tropical and subtropical regions [1, 85, 86]. In the last four decades, the geographical spread and intensity of dengue

*DOI: http://dx.doi.org/10.5772/intechopen.93140*

**Figure 2.**

*Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease… DOI: http://dx.doi.org/10.5772/intechopen.93140*

#### **Figure 2.**

*Dengue Fever in a One Health Perspective*

E glycoprotein ectodomain revealed three domains contributing to the β-barrel central structure of the protein (domain I, DI), permitting the fusion of viral and cellular membranes during virus entry (domain II, DII, and *fusion loop*), and a structural basis for immune recognition and cellular receptor binding (domain III, DIII) [43, 70]. The low pH of the endosomal compartment induces conformational changes in the E glycoprotein, which allows the fusion of the viral and host membranes [43, 69]. This results in the release of the viral RNA genome into the cytoplasm. The single-stranded positive-sense RNA immediately acts as a messenger RNA, which can be subsequently translated by cellular machinery to generate viral polyproteins, subsequently processed by both cellular and viral proteases to generate mature viral proteins [69, 70]. In this stage, the nonstructural proteins have been shown to induce massive remodeling of ER membranes, manifesting as convoluted membranes and vesicle packets (VPs) to form a dynamic and membrane-bound multi-protein assembly, named the replication complex (RC) where the genome is replicated, and new viral RNA copies are incorporated into nascent particles [71, 72]. Viral RNA synthesis relies on NS5, the RNA-dependent RNA polymerase as well as on critical RNA secondary and tertiary structures [73–75]. NS3 is a

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*Dengue virus infection, pathogenesis, and immune responses in the skin. The skin represents the first line of defense of the human body against pathogens such as viruses, as it generates early immune responses, aiming to protect humans against cutaneous and systemic infection (1). The human skin constitutes a complex organ nicely structured in three escalated layers known as the epidermis, which is composed of closely packed epithelial cells including keratinocytes, melanocytes, and Langerhans cells (LCs), a specialized type of dendritic cell (DC) that constantly probes for antigen in the most exposed, superficial layer of the skin; the dermis, which is made of dense, irregular connective tissue, blood vessels, and other structures; and the inner hypodermis, which is composed mainly of loose connective and fatty tissues (2). Upon disturbance of the epidermal barrier by mosquito blood-feeding, DENV-infected mosquitoes inoculate newly generated infectious virus particles along with mosquito saliva in which a complex mixture of proteins that exerts profound effects in the human immune system allows the acquisition of the mosquito blood meal from its host by circumventing vasoconstriction, platelet aggregation, coagulation, and inflammation or hemostasis (3). In the skin, major constituents of the innate immune system include phagocytic cells such as macrophages, neutrophils, and DCs as well as innate leukocytes such as natural killer (NK) cells, mast cells (MCs), basophils, and eosinophils. Also, epidermal keratinocytes act as active innate immune cells. In response to sensing pathogen-associated molecular patterns (PAMPs) expressed by microbes and host danger molecules, innate immune receptors present on keratinocytes and APCs become activated, causing the release of inflammatory cytokines and antimicrobial peptides (4). At the site of inoculation in the skin, the key targets of DENV infection are immune cells of the myeloid lineage, including various subsets of DCs, monocytes/macrophages, and MCs (5). Despite this, limited virus particles are thought to be deposited in the epidermis during mosquito blood-feeding (3), and in that location, Langerhans cells as well as keratinocytes are considered target cells (6). In the dermis, DCs and monocytes/macrophages are also prime infection targets (7). MCs are not substantially infected in the skin (8). However, exposure to DENV triggers an augmented activation of MCs leading to degranulation and release of de novo-synthesized inflammatory and vasoactive mediators, including proteases, leukotrienes, and histamine that, along with some vasoactive molecules and maybe the secreted NS1 originated from infected mosquito cells in the salivary glands, promote edema within the site of infection as a consequence of the increased microvascular permeability (9). Activation of MCs also induces the secretion of cytokines and chemokines that leads to the recruitment of NK cells, neutrophils, and monocyte-derived dendritic cells (mDCs) to the site of infection (10). Already in the skin, mDCs can serve as targets of infection, allowing the amplification of the virus in the skin, while natural killer (NK) cells, natural killer T (NKT) cells, and CD8+ T cells can kill DENV-infected cells and promote virus clearance in a cellular cytotoxic-dependent manner (11). Once human skin is infected, DENV-infected DCs take virus into the draining lymph nodes using afferent lymphatic vessels where they spread DENV infection and most importantly activate antigen-specific CD4+ and CD8+ T cells which initiates the adaptive immune response (12, 13). In the T cell zones, activated T cells become effectors cells to promote the development of DENV-specific memory B cells and plasma cells in the germinal center of LN. Activated T cells can reenter circulation and potentially return to the skin for virus clearance during subsequent DENV infections, playing an important role in protecting against DENV (14). In the skin, in addition to the mosquito saliva, DENV infection of target cells such as DCs, monocytes/macrophages, and MCs can be also modulated by the presence of preexisting antibody responses against previous infections with distinct DENV serotypes or other closely related flaviviruses, in an antibody-dependent enhancement (ADE) manner (See DENV-ADE in Part II for more details). For MCs and DCs, DENV-ADE is possible through Fc-γ-receptors (FcγRs). Besides, MCs degranulation can be enhanced through cross-linking of FcεRs when bound to DENVspecific IgE, leading to augmented MC activation and presumably immune-mediated vascular injury (15). After skin infection, DENV must achieve systemic infection to complete its transmission cycle by infecting new mosquito hosts. Infection of secondary LNs following infection of the draining LN are considered the amplification centers for DENV that contributes to the systemic infection and virus transmission (16).*

protease-helicase which together with its cofactor NS2B, participates in the processing, efficient RNA synthesis, and capping of the viral polyprotein [72, 76, 77]. NS2A recruits nascent RNA as well as C-pre-M-E [78]. NS1 interacts with structural proteins and NS4A-2 K-4B to facilitate the production of infectious virus particles [11, 79]. Because of their critical roles in the DENV replication cycle, NS2B, NS3, and NS5 along with NS4B are the main focus to design new inhibitors for antiviral therapy against DENV and other related flaviviruses [80–83]. Assembled viruses are transported through the trans-Golgi network (TGN) where under acidic conditions, a cellular protease, *furin*, cleaves pre-M, allowing full maturation of infectious virions that will be finally released via exocytosis [84].

#### **3. Dengue virus infection, epidemiology, and clinical features**

Dengue is the arboviral infection with the highest disease incidence worldwide, with 2.5 billion people living in dengue-endemic tropical and subtropical regions [1, 85, 86]. In the last four decades, the geographical spread and intensity of dengue have grown dramatically around the world accompanied by the wide distribution of the two main vector mosquitoes, *Aedes aegypti* and *Aedes albopictus*, which today are fully adapted to human dwellings creating new opportunities not only for DENV but also for other arthropod-transmitted viruses (arboviruses) transmission, such as Zika virus (ZIKV) and chikungunya virus (CHIKV), within human populations. These features along with the continuous growing of urbanization, globalization, and the lack of effective mosquito control represent some of the critical factors that have contributed to the emergence and reemergence of mosquito-transmitted viruses around the world [48, 87, 88].

Infection with any of the four DENV serotypes results in a diverse range of symptoms going from mild undifferentiated fever to life-threatening manifestations, which are characterized by increased vascular permeability, hemorrhage, and shock [89] (**Figure 1C**). In 1997, the World Health Organization (WHO) classified symptomatic DENV infections into three categories and subcategories known as dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). After an incubation period of 3–7 days, symptoms start suddenly and follow three phases: an initial febrile phase, a critical phase around the time of defervescence, and a spontaneous recovery phase [89]. Classical DF is an incapacitating disease that affects older children, adolescents, and adults, mainly characterized by the abrupt onset of fever (up to 40°C) and severe headache, accompanied by retro-orbital pain, myalgia, arthralgia, gastrointestinal discomfort, and transient rash [89]. In turn, DHF and DSS can rapidly deteriorate, progressing to hemorrhage with or without vascular leak after an early acute-onset febrile period, particularly during defervescence, where the symptoms are similar to those presented during classical DF. DHF and DSS are classified into four subcategories or grades (I–IV), where grades I and II (DHF) are represented by mild cases presenting some bleeding manifestations without shock (petechiae, purpura, ecchymosis, bruising, epistaxis, etc.), whereas III and IV (DSS) are more severe and accompanied by severe hemorrhagic manifestations and thrombocytopenia (platelets counts: <100,000 platelets/μL) and evidence of increased vascular permeability (ascites, pleural effusion, increased hematocrit concentrations, and severe abdominal pain) during a critical period, sometimes accompanied with a profound and prolonged shock that potentially leads to death [90]. In this critical stage, liver failure, myocarditis, and encephalopathy often occur with minimal associated plasma leakage [89]. In 2009, the WHO revised the classification system for dengue and established new guidelines that replaced the more complicated dengue fever/dengue hemorrhagic fever (DF/DHF) system to separate patients enduring severe disease from those with non-severe manifestations. This new guideline defined two new major entities—dengue and severe dengue—which encompasses a set of "warning signs" intended to help clinicians identify the patients likely to develop complications during the critical phase of the illness [89].

Currently, there is no effective and safe vaccine or FDA-approved specific antiviral drug options to combat dengue disease, with treatment being purely supportive [91]. Prevention or reduction of DENV transmission by implementing combined effective control strategies remains as the primary approach to be used to prevent DENV transmission within human populations [92]. With the majority of DENV infections being asymptomatic (70–80%), and most symptomatic infections not progressing to severe disease [3], the global distribution of dengue remains highly uncertain as the actual numbers of dengue cases are underreported and many cases are misclassified. One recent study estimate indicates that 390 million DENV infections occur annually with more than 500,000 cases of hospitalizations and more than 25,000 deaths (2.5% case fatality, annually) [1]. A different study estimated that 3.9 billion people living in 128 countries are at risk of being infected with dengue viruses [85]. These studies

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*Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease…*

**4. Dengue immunopathogenesis and severe disease: host and viral** 

**4.1 Dengue virus tropism and infection of immune cells**

Numerous *in vitro* studies have shown that DENV is able to infect a variety of cell types including epithelial cells, endothelial cells, hepatocytes, muscle cells, dendritic cells, monocytes, B cells, and mast cells [65, 66, 111–117]. Several autopsies and *ex vivo* studies have found the presence of DENV antigens (e.g., envelope protein, NS3) in some tissues such as the skin, liver, spleen, lymph node, kidney, bone marrow, lung, thymus, and brain [56, 67, 68, 118–122]. However, infectious virus particles have not always been isolated from all these organs but only from the liver and peripheral blood mononuclear cells (PBMCs), suggesting that: (a) the presence of DENV antigens such as the structural proteins E, pre-M, and C in several organs may not always be associated with the evidence of productive viral infection and severe organ pathology and (b) the immune cells and liver may be the main targets for DENV replication during the dengue disease [67].

demonstrate the worldwide expansion of the dengue disease and the establishment of an increasingly important infectious disease of global public health significance.

The hallmark of severe dengue is the transient perturbation in the integrity of the endothelium lining the inner side of blood vessels as well as the alteration in the coagulation cascade leading to shock and severe hemorrhage manifestations [9, 89]. Increased vascular permeability in severe dengue results in decreased circulating plasma volume, haemoconcentration, and pleural and peritoneal effusions that result in severe life-threatening shock [93–96]. Numerous epidemiological pieces of evidence indicate that appearance of the life-threatening manifestations during severe dengue occurs shortly after the defervescence stage of dengue disease, when the peak of viremia passed, meaning that host innate and adaptive immune responses have cleared the virus from host tissues [97, 98]. At this time, a transient vascular leakage pathology is observed followed by a rapid recovery in association with the late febrile phase. This association led to the suggestion that the key biological mechanisms such as alterations on the vasculature that leads to the pathogenesis of clinical complications during DENV infection are rather functional than the structural changes in the endothelium and are primarily a consequence of shortlived biological mediators closely linked to the host immune responses [93–96]. Although many severe infections occur upon secondary encounters with heterologous DENV serotypes [9, 99], suggesting an immune-mediated process is involved, the multifactorial immunopathogenic process of DENV infection implies a complex interaction between distinct viral and host processes that sometimes leads to increased virus infection, exacerbated immune responses, and the appearance of life-threatening severe manifestations such as severe plasma leakage, hemorrhage, and organ failure. Higher virus pathogenicity (virulence), preexisting serotype cross-reactive antibodies, activation of DENV-infected immune cells [e.g., monocytes and mast cells (MCs)], T cell responses, activation of complement pathways, the potential infection of endothelial cells, and the new pathogenic roles of the secreted NS1 of DENV may work synergistically to induce the release of vasoactive cytokines which results in increased endothelial permeability causing vascular leakage and pleural effusion, which are still considered pathognomonic features of severe dengue that leads occasionally to shock and death [8, 9, 35, , 96, 99–110]. In this section, we highlight in two parts I and II, the immunological events elicited by DENV infection, which have been suggested to play a key role in the development of severe dengue manifestations.

*DOI: http://dx.doi.org/10.5772/intechopen.93140*

**factors**

*Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease… DOI: http://dx.doi.org/10.5772/intechopen.93140*

demonstrate the worldwide expansion of the dengue disease and the establishment of an increasingly important infectious disease of global public health significance.
