Role of mitogen-activated protein kinase signaling in the pathogenesis of dengue virus infection

Dengue virus (DENV) infection is a disease that is endemic to many parts of the world, and its increasing prevalence ranks it among the diseases considered to be a significant threat to public health. The clinical manifestations of DENV infection range from mild dengue fever (DF) to more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increased proinflammatory cytokines and vascular permeability, both of which cause organ injury, are the hallmarks of severe dengue disease. Signs of liver injury were observed in studies using hepatic cell lines, mouse models, and autopsy specimens from DENV-infected patients, and these signs substantiated the effects of inflammatory responses and hepatic cell apoptosis. Mitogen-activated protein kinases (MAPK) are involved in inflammatory responses and cellular stress during viral infections. The roles of MAPK signaling in DENV infection were reviewed, and published data indicate MAPK signaling to be involved in inflammatory responses and hepatic cell apoptosis in both in vitro cultures and in vivo models. Modulation of MAPK signaling ameliorates the inflammatory responses and hepatic cell apoptosis in DENV infection. This accumulation of published data relative to the role of MAPK signaling in inflammatory responses and cell apoptosis in DENV infection is elucidatory, and may help to accelerate the development of novel or repositioned therapies to treat this unpredictable and often debilitating disease.     

Role of cathepsin B in dengue virus-mediated apoptosis

Dengue virus (DENV) infection is one of the most important mosquito-borne viral diseases, which is endemic in the tropical and sub-tropical regions. Patients with dengue hemorrhagic fever (DHF) generally present hemorrhagic tendencies, plasma leakage, thrombocytopenia, and hemoconcentration. Hepatic dysfunction is also a crucial feature of DENV infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show cellular apoptosis, which apparently relate to the pathogenesis. Cathepsins, which are cysteine proteases inside the lysosome, were previously reported to be up-regulated in patients with DHF. However, their functions during DENV infection have not been thoroughly investigated. We show for the first time that DENV induces lysosomal membrane permeabilization. The resulting cytosolic cathepsin B and S contributed to apoptosis via caspase activation. The activity of caspase 3 was significantly reduced in DENV-infected HepG2 cells treatedwith cathepsin B or S inhibitors. Treatment with cathepsin B inhibitor also reduced the activity of caspase 9, suggesting that cathepsin B activates both caspase-9 and caspase-3. Reduced cathepsin B expression, effected by RNA interference, mimicked pharmacological inhibition of the enzyme and confirmed the contribution of cathepsin B to apoptotic events induced by DENV in HepG2 cells.     

Classification of Dengue Fever Patients Based on Gene Expression Data Using Support Vector Machines

Background

Symptomatic infection by dengue virus (DENV) can range from dengue fever (DF) to dengue haemorrhagic fever (DHF), however, the determinants of DF or DHF progression are not completely understood. It is hypothesised that host innate immune response factors are involved in modulating the disease outcome and the expression levels of genes involved in this response could be used as early prognostic markers for disease severity.

Methodology/Principal Findings

mRNA expression levels of genes involved in DENV innate immune responses were measured using quantitative real time PCR (qPCR). Here, we present a novel application of the support vector machines (SVM) algorithm to analyze the expression pattern of 12 genes in peripheral blood mononuclear cells (PBMCs) of 28 dengue patients (13 DHF and 15 DF) during acute viral infection. The SVM model was trained using gene expression data of these genes and achieved the highest accuracy of ∼85% with leave-one-out cross-validation. Through selective removal of gene expression data from the SVM model, we have identified seven genes (MYD88, TLR7, TLR3, MDA5, IRF3, IFN-α and CLEC5A) that may be central in differentiating DF patients from DHF, with MYD88 and TLR7 observed to be the most important. Though the individual removal of expression data of five other genes had no impact on the overall accuracy, a significant combined role was observed when the SVM model of the two main genes (MYD88 and TLR7) was re-trained to include the five genes, increasing the overall accuracy to ∼96%.

Conclusions/Significance

Here, we present a novel use of the SVM algorithm to classify DF and DHF patients, as well as to elucidate the significance of the various genes involved. It was observed that seven genes are critical in classifying DF and DHF patients: TLR3, MDA5, IRF3, IFN-α, CLEC5A, and the two most important MYD88 and TLR7. While these preliminary results are promising, further experimental investigation is necessary to validate their specific roles in dengue disease.     

Re-evaluation of the pathogenic roles of nonstructural protein 1 and its antibodies during dengue virus infection

Dengue virus (DENV) infection can cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage and abnormal hemorrhage are the two major pathogenic changes found in these patients. From previous studies, it is known that both antibodies and cytokines induced in response to DENV infection are involved in the immunopathogenesis of DHF/DSS. However, the role of viral factors during DENV infection remains unclear. Nonstructural protein 1 (NS1), which is secreted in the sera of patients, is a useful diagnostic marker for acute DENV infection. Nevertheless, the roles of NS1 and its antibodies in the pathogenesis of DHF/DSS are unclear. The focus of this review is to evaluate the possible contributions of NS1 and the antibodies it induces to vascular leakage and abnormal hemorrhage during DENV infection, which may provide clues to better understanding the pathogenesis of DHF/DSS.     

Metabolomics-Based Discovery of Small Molecule Biomarkers in Serum Associated with Dengue Virus Infections and Disease Outcomes

Background

Epidemic dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are overwhelming public health capacity for diagnosis and clinical care of dengue patients throughout the tropical and subtropical world. The ability to predict severe dengue disease outcomes (DHF/DSS) using acute phase clinical specimens would be of enormous value to physicians and health care workers for appropriate triaging of patients for clinical management. Advances in the field of metabolomics and analytic software provide new opportunities to identify host small molecule biomarkers (SMBs) in acute phase clinical specimens that differentiate dengue disease outcomes.

Methodology/Principal Findings

Exploratory metabolomic studies were conducted to characterize the serum metabolome of patients who experienced different dengue disease outcomes. Serum samples from dengue patients from Nicaragua and Mexico were retrospectively obtained, and hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry (MS) identified small molecule metabolites that were associated with and statistically differentiated DHF/DSS, DF, and non-dengue (ND) diagnosis groups. In the Nicaraguan samples, 191 metabolites differentiated DF from ND outcomes and 83 differentiated DHF/DSS and DF outcomes. In the Mexican samples, 306 metabolites differentiated DF from ND and 37 differentiated DHF/DSS and DF outcomes. The structural identities of 13 metabolites were confirmed using tandem mass spectrometry (MS/MS). Metabolomic analysis of serum samples from patients diagnosed as DF who progressed to DHF/DSS identified 65 metabolites that predicted dengue disease outcomes. Differential perturbation of the serum metabolome was demonstrated following infection with different DENV serotypes and following primary and secondary DENV infections.

Conclusions/Significance

These results provide proof-of-concept that a metabolomics approach can be used to identify metabolites or SMBs in serum specimens that are associated with distinct DENV infections and disease outcomes. The differentiating metabolites also provide insights into metabolic pathways and pathogenic and immunologic mechanisms associated with dengue disease severity.     

Mechanisms of monocyte cell death triggered by dengue virus infection

Arthropod-borne viral diseases caused by dengue virus (DENV) are major re-emerging public health problem worldwide. In spite of intense research, DENV pathogenesis is not fully understood and remains enigmatic; however, current evidence suggests that dengue progression is associated with an inflammatory response, mainly in patients suffering from a second DENV infection. Monocytes are one of the main target cells of DENV infection and play an important role in pathogenesis since they are known to produce several inflammatory cytokines that can lead to endothelial dysfunction and therefore vascular leak. In addition, monocytes play an important role in antibody dependent enhancement, infection with consequences in viral load and immune response. Despite the physiological functions of monocytes in immune response, their life span in the bloodstream is very short, and activation of monocytes by DENV infection can trigger different types of cell death. For example, DENV can induce apoptosis in monocytes related with the production of Tumor necrosis factor alpha (TNF-α). Additionally, recent studies have shown that DENV-infected monocytes also exhibit a cell death process mediated by caspase-1 activation together with IL-1 production, referred to as pyroptosis. Taken together, the aforementioned studies strongly depict that multiple cell death pathways may be occurring in monocytes upon DENV-2 infection. This review provides insight into mechanisms of DENV-induced death of both monocytes and other cell types for a better understanding of this process. Further knowledge in cell death induced by DENV will help in the developing novel strategies to prevent disease progression.     

Dengue and dengue haemorrhagic fever: implications of host genetics

Little is known of the role of human leucocyte antigen (HLA) alleles or non-HLA alleles in determining resistance, susceptibility or the severity of acute viral infections. Dengue fever (DF) and dengue haemorrhagic fever (DHF) are suitable models for immunogenetic studies, yet only superficial efforts have been made to study dengue disease to date. DF and DHF can be caused by both primary and secondary infection by any of the four serotypes of the dengue virus. Differences in host susceptibility to infectious disease and disease severity cannot be attributed solely to the virus virulence. Variations in immune response, often associated with polymorphism in the human genome, can now be detected. Data on the influence of human genes in DF and DHF are discussed here in relation to (1) associations between HLA polymorphism and dengue disease susceptibility or resistance, (2) protective alleles influencing progression to severe disease, (3) alleles restricting CD4(+) and CD8(+) T lymphocytes, and (4) non-HLA genetic factors that may contribute to DHF evolution. Recent discoveries regarding genetic associations in other viral infections may provide clues to understanding the development of end-stage complications in dengue disease. The scanty positive data presented here indicate a need for detailed genetic studies in different ethnic groups in different countries during the acute phase of DF and DHF on a larger number of patients.     

Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production

Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteins and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.     

DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

Dengue virus (DENV) is a mosquito-borne pathogen that causes a spectrum of diseases including life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage is a common clinical crisis in DHF/DSS patients and highly associated with increased endothelial permeability. The presence of vascular leakage causes hypotension, circulatory failure, and disseminated intravascular coagulation as the disease progresses of DHF/DSS patients, which can lead to the death of patients. However, the mechanisms by which DENV infection caused the vascular leakage are not fully understood. This study reveals a distinct mechanism by which DENV induces endothelial permeability and vascular leakage in human endothelial cells and mice tissues. We initially show that DENV2 promotes the matrix metalloproteinase-9 (MMP-9) expression and secretion in DHF patients’ sera, peripheral blood mononuclear cells (PBMCs), and macrophages. This study further reveals that DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 facilitates the MMP-9 enzymatic activity, which alters the adhesion and tight junction and vascular leakage in human endothelial cells and mouse tissues. Moreover, NS1 recruits MMP-9 to interact with β-catenin and Zona occludens protein-1/2 (ZO-1 and ZO-2) and to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mouse tissues. Thus, we reveal that DENV NS1 and MMP-9 cooperatively induce vascular leakage by impairing endothelial cell adhesion and tight junction, and suggest that MMP-9 may serve as a potential target for the treatment of hypovolemia in DSS/DHF patients.     

Dengue Viral RNA Levels in Peripheral Blood Mononuclear Cells Are Associated with Disease Severity and Preexisting Dengue Immune Status

Background

Infection with dengue viruses (DENV) causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF), to dengue hemorrhagic fever (DHF). The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known.

Method

The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR.

Results

Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells.

Conclusions

B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC). Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.     

Differential Expression of Toll-like Receptors in Dendritic Cells of Patients with Dengue during Early and Late Acute Phases of the Disease

Background

Dengue hemorrhagic fever (DHF) is observed in individuals that have pre-existing heterotypic dengue antibodies and is associated with increased viral load and high levels of pro-inflammatory cytokines early in infection. Interestingly, a recent study showed that dengue virus infection in the presence of antibodies resulted in poor stimulation of Toll-like receptors (TLRs), thereby facilitating virus particle production, and also suggesting that TLRs may contribute to disease pathogenesis.

Methodology/Principal Findings

We evaluated the expression levels of TLR2, 3, 4 and 9 and the co-stimulatory molecules CD80 and CD86 by flow cytometry. This was evaluated in monocytes, in myeloid and plasmacytoid dendritic cells (mDCs and pDCs) from 30 dengue patients with different clinical outcomes and in 20 healthy controls. Increased expression of TLR3 and TLR9 in DCs of patients with dengue fever (DF) early in infection was detected. In DCs from patients with severe manifestations, poor stimulation of TLR3 and TLR9 was observed. In addition, we found a lower expression of TLR2 in patients with DF compared to DHF. Expression levels of TLR4 were not affected. Furthermore, the expression of CD80 and CD86 was altered in mDCs and CD86 in pDCs of severe dengue cases. We show that interferon alpha production decreased in the presence of dengue virus after stimulation of PBMCs with the TLR9 agonist (CpG A). This suggests that the virus can affect the interferon response through this signaling pathway.

Conclusions/Significance

These results show that during dengue disease progression, the expression profile of TLRs changes depending on the severity of the disease. Changes in TLRs expression could play a central role in DC activation, thereby influencing the innate immune response.     

Serum Proteome and Cytokine Analysis in a Longitudinal Cohort of Adults with Primary Dengue Infection Reveals Predictive Markers of DHF

Background

Infections caused by dengue virus are a major cause of morbidity and mortality in tropical and subtropical regions of the world. Factors that control transition from mild forms of disease such as dengue fever (DF) to more life-threatening forms such as dengue hemorrhagic fever (DHF) are poorly understood. Consequently, there are no reliable methods currently available for early triage of DHF patients resulting in significant over-hospitalization.

Methodology/Principal Findings

We have systematically examined the proteome, cytokines and inflammatory markers in sera from 62 adult dengue patients (44 DF; 18 DHF) with primary DENV infection, at three different times of infection representing the early febrile, defervescence and convalescent stages. Using fluorescent bioplex assays, we measured 27 cytokines in these serum samples. Additionally, we used multiple mass spectrometry methods for iTRAQ-based comparative analysis of serum proteome as well as measurements of protein adducts- 3-nitrotyrosine and 3-chlorotyrosine as surrogate measures of free radical activity. Using multiple methods such as OPLS, MRMR and MSVM-RFE for multivariate feature selection and classification, we report molecular markers that allow prediction of primary DHF with sensitivity and specificity of >80%.

Conclusions/Significance

This report constitutes a comprehensive analysis of molecular signatures of dengue disease progression and will help unravel mechanisms of dengue disease progression. Our analysis resulted in the identification of markers that may be useful for early prediction of DHF during the febrile phase. The combination of highly sensitive analytical methods and novel statistical approaches described here forms a robust platform for biomarker discovery.     

Mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice

Dengue viruses (DENV) are transmitted to humans by the bite of Aedes aegypti or Aedes albopictus mosquitoes, with millions of infections annually in over 100 countries. The diseases they produce, which occur exclusively in humans, are dengue fever (DF) and dengue hemorrhagic fever (DHF). We previously developed a humanized mouse model of DF in which mice transplanted with human hematopoietic stem cells produced signs of DENV disease after injection with low-passage, wild-type isolates. Using these mice, but now allowing infected A. aegypti to transmit dengue virus during feeding, we observed signs of more severe disease (higher and more sustained viremia, erythema, and thrombocytopenia). Infected mice mounted innate (gamma interferon [IFN-γ] and soluble interleukin 2 receptor alpha [sIL-2Rα]) and adaptive (anti-DENV antibodies) immune responses that failed to clear viremia until day 56, while a mosquito bite alone induced strong immunomodulators (tumor necrosis factor alpha [TNF-α], IL-4, and IL-10) and thrombocytopenia. This is the first animal model that allows an evaluation of human immunity to DENV infection after mosquito inoculation.     

The role of the unfolded protein response in dengue virus pathogenesis

Symptomatic dengue virus (DENV) infections range from mild fever to severe haemorrhagic disease and death. Host‐viral interactions play a significant role in deciding the fate of the infection. The unfolded protein response (UPR) is a prosurvival cellular reaction induced in response to DENV‐mediated endoplasmic reticulum stress. The UPR has complex interactions with the cellular autophagy machinery, apoptosis, and innate immunity. DENV has evolved to manipulate the UPR to facilitate its replication and to evade host immunity. Our knowledge of this intertwined network of events is continuously developing. A better understanding of the UPR mediated antiviral and proviral effects will shed light on dengue disease pathogenesis and may help development of anti‐DENV therapeutics. This review summarizes the role of the UPR in viral replication, autophagy, and DENV‐induced inflammation to describe how a host response contributes to DENV pathogenesis.     

Kinetics of viremia and NS1 antigenemia are shaped by immune status and virus serotype in adults with dengue

Background

Dengue is a major public health problem in tropical and subtropical countries. Exploring the relationships between virological features of infection with patient immune status and outcome may help to identify predictors of disease severity and enable rational therapeutic strategies.

Methods

Clinical features, antibody responses and virological markers were characterized in Vietnamese adults participating in a randomised controlled treatment trial of chloroquine.

Results

Of the 248 patients with laboratory-confirmed dengue and defined serological and clinical classifications 29 (11.7%) had primary DF, 150 (60.5%) had secondary DF, 4 (1.6%) had primary DHF and 65 (26.2%) had secondary DHF. DENV-1 was the commonest serotype (57.3%), then DENV-2 (20.6%), DENV-3 (15.7%) and DENV-4 (2.8%). DHF was associated with secondary infection (Odds ratio = 3.13, 95% CI 1.04–12.75). DENV-1 infections resulted in significantly higher viremia levels than DENV-2 infections. Early viremia levels were higher in DENV-1 patients with DHF than with DF, even if the peak viremia level was often not observed because it occurred prior to enrolment. Peak viremias were significantly less often observed during secondary infections than primary for all disease severity grades (P = 0.001). The clearance of DENV viremia and NS1 antigenemia occurs earlier and faster in patients with secondary dengue (P<0.0001). The maximum daily rate of viremia clearance was significantly higher in patients with secondary infections than primary (P<0.00001).

Conclusions

Collectively, our findings suggest that the early magnitude of viremia is positively associated with disease severity. The clearance of DENV is associated with immune status, and there are serotype dependent differences in infection kinetics. These findings are relevant for the rational design of randomized controlled trials of therapeutic interventions, especially antivirals.     

Indices of anti‐dengue immunoglobulin G subclasses in adult Mexican patients with febrile and hemorrhagic dengue in the acute phase

Heterologous secondary infections are at increased risk of developing dengue hemorrhagic fever (DHF) because of antibody‐dependent enhancement (ADE). IgG subclasses can fix and activate complement and bind to Fc? receptors. These factors may also play an important role in the development of ADE and thus in the pathogenesis of DHF. The aim of this study was to analyze the indices of anti‐dengue IgG subclasses in adult patients with febrile and hemorrhagic dengue in the acute phase. In 2013, 129 patients with dengue fever (DF) and 57 with DHF in Veracruz, Mexico were recruited for this study and anti‐dengue IgM and IgG determined by capture ELISA. Anti‐dengue IgG subclasses were detected by indirect ELISA. Anti‐dengue IgG2 and IgG3 subclasses were detected in patients with dengue. IgG1 increased significantly in the sera of patients with both primary and secondary infections and DHF, but was higher in patients with secondary infections. The IgG4 subclass index was significantly higher in the sera of patients with DHF than in that of those with DF, who were in the early and late acute phase of both primary and secondary infection. In conclusion, indices of subclasses IgG1 and IgG4 were higher in patients with DHF.

     

Subversion of innate defenses by the interplay between DENV and pre-existing enhancing antibodies: TLRs signaling collapse

Background

The phenomenon of antibody dependent enhancement as a major determinant that exacerbates disease severity in DENV infections is well accepted. While the detailed mechanism of antibody enhanced disease severity is unclear, evidence suggests that it is associated with both increased DENV infectivity and suppression of the type I IFN and pro-inflammatory cytokine responses. Therefore, it is imperative for us to understand the intracellular mechanisms altered during ADE infection to decipher the mechanism of severe pathogenesis.

Methodology/Principal Findings

In this present work, qRT-PCR, immunoblotting and gene array analysis were conducted to determine whether DENV-antibody complex infection exerts a suppressive effect on the expression and/or function of the pathogen recognition patterns, focusing on the TLR-signaling pathway. We show here that FcγRI and FcγRIIa synergistically facilitated entry of DENV-antibody complexes into monocytic THP-1 cells. Ligation between DENV-antibody complexes and FcR not only down regulated TLRs gene expression but also up regulated SARM, TANK, and negative regulators of the NF-κB pathway, resulting in suppression of innate responses but increased viral production. These results were confirmed by blocking with anti-FcγRI or anti-FcγRIIa antibodies which reduced viral production, up-regulated IFN-β synthesis, and increased gene expression in the TLR-dependent signaling pathway. The negative impact of DENV-ADE infection on the TLR-dependent pathway was strongly supported by gene array screening which revealed that both MyD88-dependent and –independent signaling molecules were down regulated during DENV-ADE infection. Importantly, the same phenomenon was seen in PBMC of secondary DHF/DSS patients but not in PBMC of DF patients.

Conclusions/Significance

Our present work demonstrates the mechanism by which DENV uses pre-existing immune mediators to defeat the principal activating pathway of innate defense resulting in suppression of an array of innate immune responses. Interestingly, this phenomenon specifically occurred during the severe form of DENV infection but not in the mild form of disease.