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Introduction: Dengue virus (DENV), the etiologic agent of dengue fever illness, represents a global public health concern, mainly in tropical and subtropical areas across the globe. It is well known that this acute viral disease can progress to severe hemorrhagic stages in some individuals, however, the immunopathogenic basis of the development of more severe forms by these patients is yet to be fully understood., Objective: In this context, we investigated and characterized the histopathological features as well as the cytokine profile and cell subpopulations present in liver tissues from three fatal cases of DENV in children., Methods: Hematoxylin and Eosin, Periodic Acid Schiff and Picro Sirius Red staining were utilized for the histopathological analysis. Immunohistochemistry assay was performed to characterize the inflammatory response and cell expression patterns., Results: Vascular dysfunctions such as hemorrhage, vascular congestion and edema associated with a mononuclear infiltrate were observedin all three cases. Liver tissues exhibited increased presence of CD68+ and TCD8+ cells as well as high expression of MMP-9, TNF-a, RANTES, VEGFR-2 mediators. Viral replication was confirmed by the detection of NS3 protein., Conclusion: Taken together, these results evidenced key factors that may be involved in the development of severe alterations in liver tissues of children in response to DENV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Moragas, Alves, Oliveira, Salomão, Azevedo, da Silva, Basílio-de-Oliveira, Basílio-de-Oliveira, Mohana-Borges, de Carvalho, Rosman, Paes and Rabelo.)

Dengue viral (DENV) infections can lead to acute pancreatitis and associated tissue damage. This study examined the pancreas from two fatal cases of DENV for histopathological changes as well as for the detection of cytokines, and other inflammatory mediators. Tissue sections were prepared for examination by ultrastructural and histopathological techniques. Sections from the pancreas of non-infected individuals were prepared in parallel as a control. The presence of viral replication in macrophages was detected by co-staining for the proteins NS3 and CD68 by immunofluorescence. Immunohistochemistry was used to detect cells that expressed cytokines and inflammatory mediators to characterize the inflammatory response. Edema, acinar necrosis and fibrosis areas associated with a mononuclear infiltrate were found in infected tissues. The major site of virus replication appeared to be macrophages based on their exclusive presentation of the viral protein NS3. Pancreatic tissues from the infected individuals also displayed increased levels of high mobility group box-1, caspase-3, gelatinase B and tumor necrosis factor alpha compared to controls. The presence of virus replicating macrophages in the pancreas was associated with multiple changes in tissue structure that included elevated levels of cytokines and inflammatory markers that may differentiate acute pancreatitis due to DENV infections from other causes., Competing Interests: The authors have declared that no competing interests exist.

This chapter describes the methods of propagation and titration for DENV-1 to -4, which are required for most of the experiments using live viruses. DENV does not reach titers as high as those of other viruses or as high as desired for their use in biological assays. Although DENV grows in many different cell lines derived from both vertebrate and invertebrate cells, the most common cell lines used for virus isolation and propagation are mosquito cells C6/36 from Aedes albopictus. Amongst several methods for virus quantification, plaque assay stands out as being very practical and inexpensive. This technique is carried out essentially to estimate virion density in a particular material, being extremely important when evaluating the effect of an antiviral treatment or antibody neutralization capacity, for example. In this assay, viral particles are serially diluted and added onto confluent cell monolayers immersed in a semisolid medium, which is responsible for limiting virus spread throughout the culture. Therefore, regarding the medium consistency, once a virion successfully infects a cell, the newly produced particles can only infect neighboring cells, eventually leading them to death. This phenomenon can be observed as round gaps or plaques in the culture after staining live cells with a crystal violet solution. Then, plaques are counted and used to determine plaque-forming units per milliliter. Here, we describe a protocol established by our group for dengue virus (DENV) titration with porcine kidney (PS) cells., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Emerging evidence identifies major contributions of platelets to inflammatory amplification in dengue, but the mechanisms of infection-driven platelet activation are not completely understood. Dengue virus nonstructural protein-1 (DENV NS1) is a viral protein secreted by infected cells with recognized roles in dengue pathogenesis, but it remains unknown whether NS1 contributes to the inflammatory phenotype of infected platelets. This study shows that recombinant DENV NS1 activated platelets toward an inflammatory phenotype that partially reproduced DENV infection. NS1 stimulation induced translocation of α-granules and release of stored factors, but not of newly synthesized interleukin-1β (IL-1β). Even though both NS1 and DENV were able to induce pro-IL-1β synthesis, only DENV infection triggered caspase-1 activation and IL-1β release by platelets. A more complete thromboinflammatory phenotype was achieved by synergistic activation of NS1 with classic platelet agonists, enhancing α-granule translocation and inducing thromboxane A2 synthesis (thrombin and platelet-activating factor), or activating caspase-1 for IL-1β processing and secretion (adenosine triphosphate). Also, platelet activation by NS1 partially depended on toll-like receptor-4 (TLR-4), but not TLR-2/6. Finally, the platelets sustained viral genome translation and replication, but did not support the release of viral progeny to the extracellular milieu, characterizing an abortive viral infection. Although DENV infection was not productive, translation of the DENV genome led to NS1 expression and release by platelets, contributing to the activation of infected platelets through an autocrine loop. These data reveal distinct, new mechanisms for platelet activation in dengue, involving DENV genome translation and NS1-induced platelet activation via platelet TLR4., (© 2020 by The American Society of Hematology.)

Dengue is an important arboviral infection, causing a broad range symptom that varies from life-threatening mild illness to severe clinical manifestations. Recent studies reported the impairment of the central nervous system (CNS) after dengue infection, a characteristic previously considered as atypical and underreported. However, little is known about the neuropathology associated to dengue. Since animal models are important tools for helping to understand the dengue pathogenesis, including neurological damages, the aim of this work was to investigate the effects of intracerebral inoculation of a neuroadapted dengue serotype 2 virus (DENV2) in immunocompetent BALB/c mice, mimicking some aspects of the viral encephalitis. Mice presented neurological morbidity after the 7 th day post infection. At the same time, histopathological analysis revealed that DENV2 led to damages in the CNS, such as hemorrhage, reactive gliosis, hyperplastic and hypertrophied microglia, astrocyte proliferation, Purkinje neurons retraction and cellular infiltration around vessels in the pia mater and in neuropil. Viral tropism and replication were detected in resident cells of the brain and cerebellum, such as neurons, astrocyte, microglia and oligodendrocytes. Results suggest that this classical mice model might be useful for analyzing the neurotropic effect of DENV with similarities to what occurs in human.

Dengue is an important mosquito-borne disease and a global public health problem. The disease is caused by dengue virus (DENV), which is a member of the Flaviviridae family and contains a positive single-stranded RNA genome that encodes a single precursor polyprotein that is further cleaved into structural and non-structural proteins. Among these proteins, the non-structural 3 (NS3) protein is very important because it forms a non-covalent complex with the NS2B cofactor, thereby forming the functional viral protease. NS3 also contains a C-terminal ATPase/helicase domain that is essential for RNA replication. Here, we identified 47 NS3-interacting partners using the yeast two-hybrid system. Among those partners, we highlight several proteins involved in host energy metabolism, such as apolipoprotein H, aldolase B, cytochrome C oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH directly binds full-length NS3 and its isolated helicase and protease domains. Moreover, we observed an intense colocalization between the GAPDH and NS3 proteins in DENV2-infected Huh7.5.1 cells, in NS3-transfected BHK-21 cells and in hepatic tissue from a fatal dengue case. Taken together, these results suggest that the human GAPDH-DENV NS3 interaction is involved in hepatic metabolic alterations, which may contribute to the appearance of steatosis in dengue-infected patients. The interaction between GAPDH and full-length NS3 or its helicase domain in vitro as well as in NS3-transfected cells resulted in decreased GAPDH glycolytic activity. Reduced GAPDH glycolytic activity may lead to the accumulation of metabolic intermediates, shifting metabolism to alternative, non-glycolytic pathways. This report is the first to identify the interaction of the DENV2 NS3 protein with the GAPDH protein and to demonstrate that this interaction may play an important role in the molecular mechanism that triggers hepatic alterations.

Dengue is an important infectious disease that presents high incidence and yields a relevant number of fatal cases (about 20,000) every year worldwide. Despite its epidemiological relevance, there are many knowledge gaps concerning dengue pathogenesis, especially with regards to the circumstances that drive a mild clinical course to a severe disease. In this work, we investigated the participation of high mobility group box 1 (HMGB1), an important modulator of inflammation, in dengue fatal cases. Histopathological and ultrastructural analyses revealed that liver, lung and heart post-mortem samples were marked by tissue abnormalities, such as necrosis and apoptotic cell death. These observations go in line with an HMGB1-mediated response and raised concerns regarding the participation of this cytokine in promoting/perpetuating inflammation in severe dengue. Further experiments of immunohistochemistry (IHC) showed increased expression of cytoplasmic HMGB1 in dengue-extracted tissues when compared to non-dengue controls. Co-staining of DENV RNA and HMGB1 in the host cell cytoplasm, as found by in situ hybridization and IHC, confirmed the virus specific induction of the HMGB1-mediated response in these peripheral tissues. This report brings the first in-situ evidence of the participation of HMGB1 in severe dengue and highlights novel considerations in the development of dengue immunopathogenesis.

The present report describes a case of splenic rupture due to dengue, a rare complication of dengue that should be considered in any patient with suspected dengue disease who started with left upper quadrant abdominal pain and hypotension. The pathophysiology of this entity is not yet well elucidated, but one of the theories present in the literature is that it is due to a depletion of coagulation factors and platelets leading to intra-splenic hemorrhage and rupture. The RT-PCR technique detected serotype 1 and histopathological studies of the spleen revealed significant atrophy of lymphoid follicles and extensive hemorrhage areas. Besides histopathological observations, virus replication was investigated by detection of dengue antigens, especially the non-structural 3 protein (NS3) in endothelial cells and splenic macrophages. This important complication has serious clinical repercussions and high mortality, due to the diagnostic difficulty and many factors that usually confuse or delay its diagnosis. Therefore, it is of the utmost importance to recognize their manifestations and their management to try to best minimize their consequences and mortality.

Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system., Importance: Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Dengue virus (DENV) represents a major threat to public health worldwide. Early DENV diagnosis should not only detect the infection but also identify patients with a higher likelihood to develop severe cases. Previous studies have suggested the potential for NS1 to serve as a viral marker for dengue severity. However, further studies using different sera panels are required to confirm this hypothesis. In this context, we developed a lab-based ELISA to detect and quantitate NS1 protein from the four DENV serotypes and from primary and secondary cases. This approach was used to calculate the circulating NS1 concentration in positive samples. We also tested the NS1 positivity of DENV-positive samples according to the Platelia Dengue NS1 Ag assay. A total of 128 samples were positive for DENV infection and were classified according to the WHO guidelines. The overall NS1 positivity was 68% according to the Platelia assay, whereas all samples were NS1-positive when analyzed with our lab-based ELISA. Fifty-four samples were positive by PCR, revealing a co-circulation of DENV1 and DENV4, and the NS1 positivity for DENV4 samples was lower than that for DENV1. The circulating NS1 concentration ranged from 7 to 284 ng/mL. Our results support previous data indicating the low efficiency of the Platelia assay to detect DENV4 infection. Moreover, this work is the first to analyze NS1 antigenemia using retrospective samples from a Brazilian outbreak.

Dengue affects millions of people worldwide. No specific treatment is currently available, in part due to an incomplete understanding of the viral components' interactions with host cellular structures. We tested dengue virus (DENV) capsid protein (C) interaction with low- and very low-density lipoproteins (LDL and VLDL, respectively) using atomic force microscopy-based force spectroscopy, dynamic light scattering, NMR and computational analysis. Data reveal a specific DENV C interaction with VLDL, but not LDL. This binding is potassium-dependent and involves the DENV C N-terminal region, as previously observed for the DENV C-lipid droplets (LDs) interaction. A successful inhibition of DENV C-VLDL binding was achieved with a peptide drug lead. The similarities between LDs and VLDL, and between perilipin 3 (DENV C target on LDs) and ApoE, indicate ApoE as the molecular target on VLDL. We hypothesize that DENV may form lipoviroparticles, which would constitute a novel step on DENV life cycle., From the Clinical Editor: Using atomic force microscopy-based force spectroscopy, dynamic light scattering, NMR, and computational analysis, these authors demonstrate that dengue viral capsid proteins (DENV C) bind to very low density lipoprotein surfaces, but not to LDLs, in a potassium-dependent manner. This observation suggests the formation of lipo-viroparticles, which may be a novel step in its life cycle, and may offer potential therapeutic interventions directed to this step., (© 2013.)

Despite the availability of many methods for rapid and early diagnosis of dengue, there is still a need to develop new approaches that not only combine low cost, specificity, and sensitivity, but also are capable of accurately detecting secondary infection in the early stages of the disease. We report the potential of the high mobility group box 1 protein as an auxiliary biomarker for early dengue diagnosis. We tested a 205-sample serum panel that included negative and positive samples from primary and secondary dengue cases, as well as samples from patients with dengue-like symptoms. We observed that high mobility group box 1 protein was generally detected only in dengue-positive samples for persons with primary and secondary infections. These results highlight the possibility of using this endogenous molecule as an auxiliary biomarker to aid in dengue detection and improve current methods for early diagnosis of dengue.

Background: Dengue virus (DENV) is described as the most prevalent arbovirus, infecting at least 50 million people worldwide. During infection, an intricate network of cytokines, a group of substances closely related to disease severity, is released. Recently, it was observed that both DENV-infected epithelial cells undergoing necrosis and dendritic cells (DCs) are able to release a non-classical pro-inflammatory cytokine called high mobility group box 1 (HMGB1)., Objectives: The aim of this study was to evaluate whether HMGB1 levels were altered in DENV-infected patients' sera and whether this augment correlated with disease pathogenesis., Study Design: Samples from DENV-infected patients were collected from different days after the onset of symptoms and from patients experiencing primary or secondary infection. The circulating HMGB1 concentration was measured in healthy blood donors as well as in donors with primary and secondary cases of DENV infection by a quantitative capture ELISA assay., Results: We observed that the HMGB1 concentration in DENV-infected patients was significantly higher than in healthy patients. HMGB1 levels reached the highest concentration in the first day after the onset of symptoms and decreased throughout the course of the infection. Moreover, we observed that the HMGB1 concentration was augmented during secondary infection as well., Conclusion: We hypothesize that HMGB1 levels correlate with disease pathogenesis, specifically with the clinical symptoms and secondary infection, implicating a pro-inflammatory cytokine role for HMGB1 in DENV infection. This is the first report assessing the circulating levels of HMGB1 during DENV infection., (Copyright © 2012. Published by Elsevier B.V.)

Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of -19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at +13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C protein-functionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na(+)/K(+)-ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs.

The non-structural 1 (NS1) protein plays an important role in dengue diagnosis because it has been detected as a soluble serum antigen in both primary and secondary infections. The NS1 protein was expressed in Escherichia coli cells, and the efficiency of four different refolding protocols was tested. All of the protocols generated dimeric NS1 in a conformation similar to that of the protein expressed by eukaryotic cells. A polyclonal antibody produced from the properly folded E. coli recombinant NS1 (rNS1) protein proved to be a useful tool for the diagnosis of Dengue virus because it detected 100% of the Dengue virus 2 (DENV2) in infected patients' sera and 60% of the DENV IgM-positive sera not detected by commercial NS1-based diagnostic kits. These data suggest a high-efficiency method for correctly folding rNS1 that maintains its structural and immunogenic properties. In addition, a detection method using the polyclonal antibody against correctly folded rNS1 seemed to be more sensitive and efficient for NS1 detection in serum, highlighting its usefulness for developing a high-sensitivity diagnostic kit., (Copyright © 2011 Elsevier B.V. All rights reserved.)