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

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.

#### **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].

*Dengue Fever in a One Health Perspective*

viruses around the world [48, 87, 88].

during the critical phase of the illness [89].

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

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

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

**66**

In animal models such as the alpha/beta (IFN-α/β)-deficient mice (*Ifnar*−/−) and nonhuman primates, DENV has been recovered from the spleen, liver, peripheral lymph nodes, and the central nervous system [123–127]. However, the absence of an appropriate animal disease model has largely hampered with the understanding of the role played by DENV tropism *in vivo*. Sustained viral replication and severe manifestations have been observed in *Ifnar*−/− mice after infection with DENV, which gives a clear advantage to study DENV pathogenesis *in vivo*, but the absence of intact IFN signaling is a limitation that must be considered when interpreting data [128].

The fact that DENV can infect many mammalian and insect cell types *in vitro* and *in vivo* suggest there are different molecules or cellular routes that might be controlling virus attachment and internalization, resulting in productive infection [63]. Numerous studies have shown that C-type lectins including DC-SIGN (CD209) and C-type lectin domain family 5, member A (CLEC5A) expressed on dendritic cells and macrophages act as cellular receptors for DENV [129–131]. Other extensively studied DC receptors are the mannose receptor (MR), Langerins, Fc-receptors, TIM3, TIM4, and AXL [63, 65, 132, 133]. Contrary to the DC-SIGN that may primarily function as a viral attachment factor, DENV binding to CLEC5A (C-type lectin domain family 5, member A), highly expressed by monocytes, macrophages, neutrophils, and dendritic cells, has been shown to induce the production of antiviral and pro-inflammatory cytokines suggesting that this C-type lectin may act as a cognate receptor for dengue virion [131, 134]. These cytokines include type I IFNs and chemotactic factors such as migration inhibition factor (MIF), monocyte chemotactic factor (MCP), and IL-8 [102, 134]. DENV infection of DCs also induces the production of matrix metalloproteinases (MPPs), MMP-2 and MMP-9, which induces migration of DCs to lymph nodes where virus further replicates before it disseminates into the blood circulation [135]. In the skin, DENV also infects mast cells that can be activated leading to degranulation and increased secretion of various inflammatory cytokines (IL-1, IL-6, TNF-α, and IFN-α), chemokines (CCL5, CXCL12, and CX3CL1), and chymase, the latter being a protease found circulating at high levels in the blood of dengue patients, suggesting a potential role in the development of severe dengue that contributes to vascular leakage [115, 136–140]. All these innate immune processes together lead to an antiviral state in nearby cells, generating an inflammatory response and recruitment of natural killer (NK) cells to combat DENV infection [54, 141].

Along with DCs, monocytes and macrophages are also the primary targets of DENV infection [142, 143]. In lymphoid and nonlymphoid tissues, macrophages are considered the primary reservoirs of DENV after its dissemination from the skin [144]. Macrophages susceptible to DENV have been found in different organs in the mouse models or human autopsies, namely, Kupfer cells in the liver, alveolar macrophages in the lungs, dermal macrophages, microglial cells (brain and spinal cord), and monocytes in the peripheral blood [118, 120, 122, 145–147]. Comparable to DENV infection of DCs, DENV can use an array of cell surface receptors to infect monocytes and macrophages, including mannose receptor (CD205), CD14 associated protein, heat shock proteins (HSP70/HSP90), DC-SIGN (CD209), CD300a, AXL, TIM4, PD1, and the Fc receptors, particularly FcγRI (CD64) and FcγRII (CD32, 63). These two Fc-Rs play major roles in enhancing DENV infection of monocytes and macrophages, particularly during secondary infections [148–151].

Other populations of immune cells including NK cells can also be activated during DENV infection, particularly in patients with DHF compared to those with DF [141, 152, 153]. Additionally, B cells and T cells have been studied to test permissiveness to DENV, but these studies have resulted in contradictory results [154–156]. *In vitro* studies using B cell and T cell lines (e.g., *Raji* cells, Daudi, and Jurkat) and primary B cells derived from healthy human peripheral blood mononuclear cells

**69**

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

(PBMCs) have revealed the potential role of these cells in DENV replication, both in presence and absence of heterologous antibodies [67, 155, 157–159]. Additional studies using a humanized mouse model found that DENV infected both B and T cells accompanied by an important production of pro-inflammatory cytokines such as IL-6 and TNF-α, like monocytes and macrophages [160]. Despite this evidence, the role of lymphoid cells such as B and T cells in DENV tropism and replication

Although plasma leakage in severe dengue occurs at the end of the acute illness, there is substantial evidence that the pathophysiologic processes start at the earliest stages of DENV infection [95, 96]. Introduction of DENV particles along with mosquito saliva triggers a variety of host innate immune responses leading to the production of antiviral and pro-inflammatory cytokines mostly from the immune cells exposed to DENV [57, 62, 138]. At this stage, innate immune cells are the first to respond to infection through stimulation of patterns recognition receptors (PRRs) recognizing pathogen-associated molecular patterns (PAMPs) as well as endogenous molecules released from damaged cells, termed damage-associated molecular patterns (DAMPs) [161, 162]. PRR recognition triggers the production of cytokines and chemokines, which induces a local antiviral state [54, 55]. This local innate response could potentially play an important role in modulating local viremia and virus dissemination by recruiting susceptible target cells for DENV infection at the inoculation site [57, 62, 144]. PRRs include transmembrane proteins such as the Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) as well as cytoplasmic proteins such as the retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) and NOD-like receptors (NLRs) [161]. These are an essential part of the innate immune response against the virus, sensing viral replication in the cytoplasm [161, 163]. The PRRs that are associated with DENV recognition after infecting target cells are the cytoplasmic retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated protein 5 (MDA5) and the endosomal Toll-like receptor 3 (TLR3) and TLR7 [164–166]. Recognition of DENV RNA by TLR-3 results in the production of type I IFN and chemokines such as IL-8 via sensing of phosphate-containing RNA and long double-stranded RNA (dsRNA) in the cytoplasm or inside endosomal compartments [167, 168]. DENV infection in nonhuman primates demonstrated that the administration of TLR-3 and TLR7 agonists resulted in significantly decreased viral replication and increased production of pro-inflammatory chemokines as well as increased production of antibodies targeting

DENV [169], indicating a protective role for TLRs during DENV infection.

Additional pathways such as the cyclic GMP-AM synthase (cGAS), a DNA-sensor pathway which triggers the simulator of IFN genes (STING) pathway are also activated during DENV infection leading to the production of type I IFN and activation of TLRs (TLR9), an endosomal PRR that recognizes cytoplasmic DNA originated from mitochondrial damage [170–172]. In addition to type I IFN production, small RNAs such as micro RNAs (miRNA) and the complement system are important components of the innate immune response against viral infections [173, 174]. miR-NAs are processed by and interact with the proteins in the RNA interference (RNAi) pathway, such as Dicer, Drosha, Argo1, and Argo2 [175]. RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates [176]. In DENV infection, knockdown of these components resulted in increased DENV replication in mammalian cells, suggesting that the RNAi pathways may play important roles in the cellular anti-DENV responses [177, 178]. Additional evidence showed that DENV can interfere with RNAi pathways in human hepatocytes cells via NS4B and subgenomic flavivirus RNA (sfRNA) interactions with Dicer's ability to process small

**4.2 DENV infection and the host innate immune responses**

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

needs further exploration.

(PBMCs) have revealed the potential role of these cells in DENV replication, both in presence and absence of heterologous antibodies [67, 155, 157–159]. Additional studies using a humanized mouse model found that DENV infected both B and T cells accompanied by an important production of pro-inflammatory cytokines such as IL-6 and TNF-α, like monocytes and macrophages [160]. Despite this evidence, the role of lymphoid cells such as B and T cells in DENV tropism and replication needs further exploration.
