**5. Concluding remarks (Part I)**

*Dengue Fever in a One Health Perspective*

necessary for viral genome cyclization, viral RNA synthesis, translation, and replication [239]. sfRNAs regulate the pathogenicity in both mammalian and mosquito cells after interacting with proteins such as TRIM25 to inhibit RIG-I signaling and translation of ISGs [73, 240, 241]. Interestingly, reduced IFN responses have been found during DENV outbreaks where the infecting DENV serotype produced greater levels of sfRNA than the less pathogenic strains [100, 240]. Thus, high levels of sfRNAs may cause an epidemiological fitness of DENV, which results in lower stimulation of RIG-IMDA5 RNA sensors and reduced production of IFN, causing higher viremia levels that could be translated in more infections and severe diseases. On the other hand, DENV utilizes the endoplasmic reticulum (ER) of host cells for replication and assembly. In this process, the ER undergoes extensive rearrangements and expansion that requires *de novo* synthesis of viral proteins [71]. Accumulation of unfolded proteins in the ER lumen leads to an unfolded protein response (UPR), a pro-survival cellular reaction induced in response to DENV-mediated ER stress [242, 243]. DENV has evolved to manipulate the UPR to cope with ER stress which hijacks the host cell machinery to evade the host immunity, facilitating viral replication [112]. Distinct *in vitro* and *in vivo* studies have shown that DENV induced ER stress and manipulates the host metabolism and protein production by increasing the autophagy (lipophagy) activity, viral replication, and pathogenesis through UPR signaling pathways [244–247]. Autophagy is the lysosomal degradation of cytoplasmic contents, which results in the recycling of cellular macromolecules as well as the activation of cellular host responses to starvation or stress [248]. Autophagy has been implicated as an innate immune response that would engulf and destroy pathogens by degrading cytosolic contents [249, 250]. In DENV infection, functional autophagy components have been shown to either promote or restrict viral RNA replication and virus production [251–253]. However, DENV has found ways of preventing autophagic processing and degradation of viral components [254, 255]. Several studies have linked DENV induction of autophagy to the regulation of lipid metabolism, leading to increased degradation of lipid droplets that produces more fatty acid material important for viral replication [247]. In this process, the NS3, NS1, and C proteins of DENV have been found to increase fatty acid biosynthesis and recruitment of lipid droplets to the DENV replication complex, facilitating viral particle assembly [256, 257]. Furthermore, several studies have shown that lipids and lipoproteins play a role in modifying DENV infectivity in both mammal and insect cells *in vitro* [258, 259]. Modulation of cholesterol levels in the host cells facilitates viral entry, replication, virus assembly, and control type I IFN response [260, 261]. This modulation involves the regulation of cholesterol levels, expression of cholesterol receptors as well as changes in cholesterol synthesis related to important modifications in the cellular metabolism [114, 262, 263]. Interestingly, clinical studies have found that levels of total serum cholesterol and LDL-C levels are modulated over the course of dengue illness, with generally lower levels associated with increased dengue severity [264–266]. In general, low cholesterol levels have been associated with critical illness related to sepsis and vascular disorders [267]. Thus, the association of cholesterol with severe dengue outcome may be an important indicator of the pathophysiology of DHF/DSS. About the complement pathway, DENV has evolved strategies to limit recognition and activation of the complement cascade [108, 165]. NS1 is the only flavivirus protein that is secreted by infected cells and has been shown to modulate the complement pathway [14, 268]. NS1 promotes efficient degradation of C4 to C4b to protect DENV from complement-dependent neutralization [13, 269]. The NS1 protein of DENV and other flaviviruses such as WNV NS1 interacts with some components of the alternative complement pathway such as the C3bBb convertase, which limits the formation of C5b-9 membrane attack complex (MAC) [268, 270]. Additional studies have found that NS1 proteins from DENV, WNV, and YFV all attenuate classical and

**72**

Dengue is the most prevalent arboviral disease transmitted by mosquitoes, which poses an enormous burden to the public health systems worldwide as more than 40% of the world population is at risk of infection. The infection with any of the four DENV serotypes (DENV1–4) can lead to a wide spectrum of clinical manifestations that range from the asymptomatic or inapparent to moderate flu-like symptoms, known as dengue fever (DF), and life-threatening manifestations identified by the WHO, known as the dengue hemorrhagic fever and dengue shock syndrome (DHF/ DSS), also known as severe dengue, with or without warning signs. In endemic areas where multiple DENV serotypes can seasonally circulate, distinct epidemiological studies have demonstrated that an individual human being can be exposed to sequential infections with distinct DENV serotypes, which poses a risk of developing severe manifestations such as DHF/DSS. This phenomenon has been attributed to the potential enhancement activity that the preexisting antibody response elicited from a previous infection with one serotype (e.g., DENV-1) may have on the infection with a different serotype (e.g., DENV-2). This process leads to an increased viral burden that triggers a series of immunological and cellular events (e.g., ADE, cytokine storm, skewed T cell responses, and complement pathways), which despite being intended to prevent the invasion and infection of the infecting viral pathogens, can induce host tissue damage leading to pathology and disease. The cellular and molecular mechanisms involved in this phenomenon will be explained in more detail in the Part II of this chapter entitled "Adaptive immune response and NS1 pathogenesis."

As an arthropod-transmitted virus (arbovirus), DENV is initially transmitted by an infected vector mosquito in which the virus has already been amplified after replication in its distinct tissues, starting at the midgut to finalize in the salivary glands, where a new transmission cycle begins after blood feeding from a new host (**Figure 2**). Following inoculation from the bite of an infected mosquito, viruses undergo replication in the local tissues such as the skin. In the skin, infectious virus particles along with mosquito saliva components including proteases and immunomodulatory proteins among others are sown in the epidermis and dermis, leading to an activation of a cascade of events including the recruitment of skin resident cells (e.g., Langerhans cells, mast cells, and keratinocytes) and new cells (e.g., T cells and neutrophils) into the site of the infection that later serve as viral targets for viral replication. After infection of target cells, sensing of viral products (e.g., PAMPs and DAMPs) results in the activation of innate immune responses (e.g., type I IFN chemokines), the first line of defense, which establishes inflammatory and antiviral states intended to prevent the virus to colonize and to replicate in the skin; however, DENV has elaborated several pathogenic mechanisms to hijack these responses and escape from the normal immune system processing, which results in its dissemination and seeds into the lymph nodes. There, DENV further replicates in monocyte lineage cells, resulting in a primary viremia after its systemic dissemination through the circulatory bloodstream, which results in the subsequent infection of peripheral tissues such as the liver, spleen, and kidney. Overall, the skin represents not only the first line of

defense against arboviruses but also the main place where viruses have learned to evade the host immune responses leading to invasion and dissemination toward the establishment of systemic host infection, which will potentially assure subsequent virus transmission into a new host. In this *Part I* of the chapter entitled "Dengue virus tropism, host innate immune responses, and subversion of antiviral responses," we discussed the distinct features of DENV as well as the biological and molecular mechanisms that can tilt the balance to either a local viral infection and dissemination through the skin or to the control and prevention of viral infection by the innate and adaptive immune responses at the site of the infection. Thus, the immunopathogenesis of arboviruses such as DENV in the skin is a critical step and must be a focus of future studies intended to reduce/block arthropod-borne transmission into humans.
