Pathogenesis Of Dengue Disease Research Articles

Platelet derived exosomes disrupt endothelial cell monolayer integrity and enhance vascular inflammation in dengue patients.

Thrombocytopenia is the most notable phenomenon in dengue. Activation status of platelets and interaction of platelets with endothelium contribute towards dengue disease pathogenesis. Platelets are the major cell types known to release extracellular vesicles, especially exosomes in circulation. However, the role of platelet derived exosomes (PLT-EXOs) in endothelial dysfunction during dengue infection remains unknown. In this study, we recruited 28 healthy subjects and 69 dengue patients categorized as WS- (n=31), WS+ (n=29) and SD (n=9). Platelets were isolated from platelet rich plasma of dengue patients and their activation was assessed by flow cytometry. PLT-EXOs were isolated by ultracentrifugation method. Western blot analyses were performed to characterize the exosomes. Exosome uptake experiment was carried out to see the internalization of exosomes inside endothelial cells (HUVECs). To observe the effect of exosomes on endothelial cells, exosomes were added on HUVECs and expression of adherens and tight junctional proteins were examined by immunofluorescence assay and western blot. Expression levels of vascular injury markers were measured in the culture supernatants of Exosome-HUVEC coculture and sera of dengue patients by MSD-multiplex assay. As compared to healthy subjects, CD41/CD61 expression was significantly reduced (p<0.0001) and CD62p expression was significantly increased (p<0.0001) on platelets in dengue patients. PLT-EXOs isolated from the dengue patients showed higher expression of CD63 and CD9 proteins than the healthy subjects. With in-vitro immunofluorescence assays, we illustrated the internalization of PLT-EXOs by the HUVECs and observed disruption of endothelial cell monolayer integrity in the presence of PLT-EXOs from WS+ and SD patients. Furthermore, the significant reduction in the expressions of ZO-2, VE-Cadherin and CD31 in endothelial cells following exposure to PLT-EXOs from the dengue patients provide direct evidence of PLT-EXOs mediated vascular permeability. PLT-EXOs stimulated the release of inflammatory markers CRP, SAA, sVCAM-1 and sICAM-1 in the supernatants of HUVEC cells. Importantly, significantly higher levels of CRP, sVCAM-1 and sICAM-1 in the sera of severe than mild dengue patients (p<0.0001) suggest their role in disease severity. In summary, our data suggest that PLT-EXOs promote vascular leakage via release of proinflammatory mediators and compromise vascular barrier integrity in dengue patients.

Differential critical residues on the overlapped region of the non-structural protein-1 recognized by flavivirus and dengue virus cross-reactive monoclonal antibodies

The non-structural protein-1 (NS1) of dengue virus (DENV) contributes to several functions related to dengue disease pathogenesis as well as diagnostic applications. Antibodies against DENV NS1 can cross-react with other co-circulating flaviviruses, which may lead to incorrect diagnosis. Herein, five anti-DENV NS1 monoclonal antibodies (mAbs) were investigated. Four of them (1F11, 2E3, 1B2, and 4D2) cross-react with NS1 of all four DENV serotypes (pan-DENV mAbs), whereas the other (2E11) also reacts with NS1 of other flaviviruses (flavi-cross-reactive mAb). The binding epitopes recognized by these mAbs were found to overlap a region located on the disordered loop of the NS1 wing domain (amino acid residues 104 to 123). Fine epitope mapping employing phage display technology and alanine-substituted DENV2 NS1 mutants indicates the critical binding residues W115, K116, and K120 for the 2E11 mAb, which are conserved among flaviviruses. In contrast, the critical binding residues of four pan-DENV mAbs include both flavi-conserved residues (W115 to G119) and DENV-conserved flanking residues (K112, Y113, S114 and A121, K122). Our results highlight DENV-conserved residues in cross-reactive epitopes that distinguish pan-DENV antibodies from the flavi-cross-reactive antibody. These antibodies can be potentially applied to differential diagnosis of DENV from other flavivirus infections.

Vitamin D Regulates the Expression of Immune and Stress Response Genes in Dengue Virus-infected Macrophages by Inducing Specific MicroRNAs.

The pathogenesis associated with Dengue virus (DENV) infection is marked by the impairment of host immune response. Consequently, the modulation of immune response has emerged as an important therapeutic target for the control of DENV infection. Vitamin D has been shown to regulate the immune response in DENV infection, although the molecular mechanism remains poorly understood. Post-transcriptional regulation of mRNA by miRNAs offers an opportunity to gain insight into the immunomodulation mediated by vitamin D. Previously, it has been observed that a high dose of vitamin D (4000 IU) decreased DENV-2 infection and inflammatory response in monocyte-derived macrophages (MDMs). Here, we examine whether high or low doses of vitamin D supplements exert differential effect on miRNA expression in DENV-infected macrophages. We analyzed miRNA expression profiles in MDMs isolated from healthy individuals who were given either 1000 or 4000 IU/day of vitamin D for 10 days. MDMs before or after vitamin D supplementation were challenged with DENV-2, and miRNAs profiles were analyzed by qPCR arrays. DENV-2 infected MDMs supplemented with 4000 IU, showed up-regulation of miR-374a-5p, miR-363-3p, miR-101-3p, miR-9-5p, miR-34a-5p, miR-200a-3p, and the family of miRNAs miR-21-5p, and miR-590-p. The miRNA profile and predicted target mRNAs suggested regulatory pathways in MDMs obtained from healthy donors who received higher doses of vitamin D. These DENV-2 infected MDMs expressed a unique set of miRNAs that target immune and cellular stress response genes. The results suggest vitamin D dose-dependent differential expression of miRNAs target key signaling pathways of the pathogenesis of dengue disease.

Tree Shrew as a New Animal Model for the Study of Dengue Virus.

Dengue virus is a significant public health threat worldwide; however, the pathogenesis of dengue disease remains poorly understood due to lack of appropriate small animal models. Tree shrews are an emerging experimental animal model for the study of human diseases due to their resemblance of genetic characteristics to primate animals. Herein we report that dengue infection in tree shrews elicits resemble clinical symptoms as in humans. Dengue fever (△2°C> normal body temperature) developed in ~22% healthy Chinese tree shrews from 2 through 33 days after infection with a low dose (1 ∗ 104 PFU/animal) of dengue virus serotype 2 or 3 intravenously or subcutaneously. The dengue genomic RNA and neutralizing antibodies were detected in ~78% of animals at days 7 and 15 post infection respectively. The serum levels of liver enzymes including aspartate transaminase, alanine aminotransferase and alkaline phosphatase were elevated with peaks at day 7 after infection. Modest thrombocytopenia and a slight decrease in the white blood cell count were observed. Intriguingly, although viral RNA was barely detectable in the liver by 48 days after infection, it was still evident in the brain. The intra-brain bleeding lesions in the intravenous infection group were more severe than those in the subcutaneous infection group. Our data demonstrate that primary dengue virus infection in tree shrews causes resemble clinical disease as in humans and thus tree shrews may be a suitable model for the study of dengue disease pathogenesis.

Levels of 25-hydroxy Vitamin D3 and Vitamin D Receptor Polymorphism in Severe Dengue Cases from New Delhi.

Background: Dengue is the “phoenix” that never went to ashes. First identified in 1943, in Japan, dengue virus has worldwide distribution and is a grave public health concern in developing countries like India; Methods: A cross sectional study was conducted among adults suspected of having dengue fever and attending Lok Nayak Hospital, New Delhi. Restriction Fragment Length Polymorphism was completed for the detection of vitamin D receptor (VDR) gene polymorphism; Results: Serum 25-hydroxy vitamin D3 (vitamin D) levels were found to be 1.6 times elevated in severe dengue cases as compared to healthy controls. Vitamin D levels were significantly higher in secondary infections compared to primary infections as well as secondary severe dengue cases as compared to secondary non-severe cases (p value < 0.05). A significant association of the T allele (rs2228570) was seen in severe dengue cases, while, when comparing the A/A with A/C and C/C genotypes (rs7975232) among dengue cases and healthy controls, the odds ratio was estimated to be 1.24 (0.55–2.75, p > 0.05) and 0.28 (0.08–0.96, p < 0.05) respectively; Conclusions: The present study is an attempt at decoding the role of vitamin D in dengue disease pathogenesis and exploring the role of genetic polymorphism in dengue disease pathogenesis.

Longitudinal Analysis of Memory B and T Cell Responses to Dengue Virus in a 5-Year Prospective Cohort Study in Thailand.

Prior exposure to dengue virus (DENV) has a profound impact on the outcome of infection, which varies according to the interval between infections. Antibodies secreted by B cells and cytokines secreted by T cells are thought to contribute both to protective immunity against DENV and the pathogenesis of dengue disease. We analyzed peripheral blood mononuclear cells (PBMC) collected from Thai children over a 5-year prospective cohort study to define the dynamics of DENV-specific memory B and T cell responses and the impact of symptomatic or subclinical DENV infections. To measure B cell responses, PBMC were stimulated with IL-2 plus R848 and culture supernatants were tested for DENV-binding antibodies by ELISA. To measure T cell responses, PBMC were stimulated in dual-color ELISPOT assays with overlapping peptide pools of structural and non-structural proteins from the four DENV types. B cell responses were low to one or more DENV types prior to symptomatic infection and increased with reactivity to all four types after infection. Subjects who had a subclinical infection or who did not experience a DENV infection during the study period showed strong memory B cell responses to all four DENV types. T cell responses to DENV peptides demonstrated a cytokine hierarchy of IFN-γ > IL-2 > IFN-γ/IL-2. T cell responses were low or absent prior to secondary infections. The trends in T cell responses to DENV peptides over 3 year post-infection were highly variable, but subjects who had experienced a secondary DENV1 infection showed higher cytokine responses compared to subjects who had experienced a secondary DENV2 or subclinical infection. The longitudinal nature of our study demonstrates persistent memory B cell responses over years and a lasting but variable impact of secondary DENV infection on DENV-specific T cell responses.

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.

Characterization of dengue virus 2 growth in megakaryocyte–erythrocyte progenitor cells

Megakaryocyte–erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis.

Role of Oxidative and Nitrosative Stress in Dengue Pathogenesis: A Mini-Review

Dengue is a mosquito–borne acute viral disease with ubiquitous distribution in tropical and subtropical areas of the world. Dengue virus (DENV) infection is transmitted by the bite of a female Aedes aegypti mosquito (the most important vector) infected with DENV. Clinical presentation of this typical arboviral disease varies along a wide spectrum of clinical symptoms. During the course of DENV infection, some individuals develop severe manifestations relates to plasma leakage into tissues caused by increased vascular permeability. The severity of dengue disease may vary considerably according to age, ethnicity, genetic factors, immune status and underlying disease. It may also depend on the co-circulation of DENV serotypes and sequential (secondary) infections with different DENV serotypes. While the exact mechanism of pathogenesis of dengue remains elusive, several lines of evidence demonstrating that DENV infection-derived oxidative stress may trigger the release of proinflammatory cytokines, including TNF-alpha, participating in collective action in dengue disease pathogenesis. In conclusion, we review these findings and discuss about the recent advances that propose a major role of oxidative-nitrosative stress on dengue pathogenesis.

Differential replicative ability of clinical dengue virus isolates in an immunocompetent C57BL/6 mouse model.

BackgroundSeveral experimental animal models have been used to study the pathogenesis of dengue disease; however, most of the studies used laboratory-adapted viruses, which lack the virulence of viruses circulating in humans. The aim of this study was to analyze the ability of clinical Dengue virus (DENV) isolates (D2/BR/RP/RMB/09 and D3/BR/SL3/02) to infect immunocompetent C57BL/6 mice.MethodsTwo strategies of intraperitoneal infection, which were based on the concept of the antibody dependent enhancement phenomenon, were used. In one strategy, the animals were inoculated with macrophages infected in vitro with dengue viruses, which were incubated with enhancing antibodies, and in the other strategy, the animals were inoculated with a complex of enhancing antibodies and dengue viruses.ResultsThe D3/BR/SL3/08 isolate showed a higher ability of infection (virus RNA was more frequently detected in the serum and in several organs) in the experimental model compared to both the D2/BR/RP/RMB/2009 isolate and a laboratory adapted DENV-1 strain (Mochizuki strain), regardless of the infection strategy used. The main features of the D3/BR/SL3/08 isolate were its neuroinvasiveness and the induction of an extended period of viremia. Enhancing antibodies did not influence on the infection of animals when macrophages were used, but the level of viremia was increased when they were used as a complex with a D3/BR/SL3/02 isolate.DiscussionWe showed that DENV isolates could infect immunocompetent C57BL/6 mice, which have has been previously used to study some aspect of dengue disease when infected with laboratory adapted strains. DENV genome was detected in the same organs found in humans when autopsy and biopsy samples were analyzed, showing that C57BL/6 mice reproduce some aspects of the DENV tropism observed in humans. The main difference observed between the D3/BR/SL3/02 and D2/BR/RP/RMB/2009 clinical isolates was the neuroinvasive ability of the first one. Neuroinvasiveness has been described in some DENV infected cases and is common for other members of the Flavivirus genus.ConclusionsThese results suggest that C57BL/6 mice can be used as an experimental model to evaluate virulence differences among DENV clinical isolates.

Association of combinations of interleukin-10 and pro-inflammatory cytokine gene polymorphisms with dengue hemorrhagic fever

Pro-inflammatory and anti-inflammatory cytokines have been shown to play an important role in dengue disease pathogenesis. In the present study, to find out whether single nucleotide polymorphisms (SNPs) in the pro-inflammatory and anti-inflammatory cytokine genes are associated with dengue disease severity, SNPs in TNF, IFNG, IL1B, IL8, IL0, IL17A and IL17F genes were investigated using polymerase chain reaction based methods in 132 dengue (DEN) cases [87 dengue fever (DF), 45 dengue hemorrhagic fever (DHF) cases] and 108 apparently healthy controls (HC) from Pune, Maharashtra, western India. Under recessive genetic model (C/C vs. T/T+T/C), the TNF rs1799964 C/C genotype was significantly associated with DEN [P=0.014, OR with 95% CI 3.07 (1.18–7.98)]. Frequency of T/C genotype of IL17F rs763780 was significantly lower in DEN group as compared to HC [P=0.033, OR with 95% CI 0.43 (0.19–0.95)]. Under overdominant genetic model (A/T vs. A/A+T/T), IL8 rs4973 A/T genotype was negatively associated with DHF compared to HCs [p=0.029, OR with 95% CI 0.43 (0.20–0.93)]. Under overdominant genetic model, A/G genotype of IL10 rs1800871 was significantly negatively associated with DHF compared to DF cases [p=0.014, OR with 95% CI 0.35 (0.15–0.84)]. Significantly higher frequency of the combined genotype IL10 A/A-IFNG A/T and lower frequency of the combined genotypes IL10 A/G-IL1B A/A, IL10 A/G-IL8 A/T and IL10 A/G-IL17F T/T were observed in DHF cases compared to DF. The results suggest that heterozygous genotypes of IL8 rs4973 and IL10 rs1800871 are associated with reduced risk of DHF. Combinations of IL10 rs1800871 and pro-inflammatory cytokine genotypes influence the risk of DHF.

Polymorphisms of Transporter Associated with Antigen Presentation, Tumor Necrosis Factor-α and Interleukin-10 and their Implications for Protection and Susceptibility to Severe Forms of Dengue Fever in Patients in Sri Lanka.

Context:To date, a clear understanding of dengue disease pathogenesis remains elusive. Some infected individuals display no symptoms while others develop severe life-threatening forms of the disease. It is widely believed that host genetic factors influence dengue severity.Aims:This study evaluates the relationship between certain polymorphisms and dengue severity in Sri Lankan patients.Settings and Design:Polymorphism studies are carried out on genes for; transporter associated with antigen presentation (TAP), promoter of tumor necrosis factor-α (TNF-α), and promoter of interleukin-10 (IL-10). In other populations, TAP1 (333), TAP2 (379), TNF-α (−308), and IL-10 (−1082, −819, −592) have been associated with dengue and a number of different diseases. Data have not been collected previously for these polymorphisms for dengue patients in Sri Lanka.Materials and Methods:The polymorphisms were typed by amplification refractory mutation system polymerase chain reaction in 107 dengue hemorrhagic fever (DHF) patients together with 62 healthy controls.Statistical Analysis Used:Pearson's Chi-square contingency table analysis with Yates′ correction.Results:Neither the TAP nor the IL-10 polymorphisms considered individually can define dengue disease outcome with regard to severity. However, the genotype combination, IL-10 (−592/−819/−1082) CCA/ATA was significantly associated with development of severe dengue in these patients, suggesting a risk factor to developing DHF. Also, identified is the genotype combination IL-10 (−592/−819/−1082) ATA/ATG which suggested a possibility for protection from DHF. The TNF-α (−308) GG genotype was also significantly associated with severe dengue, suggesting a significant risk factor.Conclusions:The results reported here are specific to the Sri Lankan population. Comparisons with previous reports imply that data may vary from population to population.

Induksi Ekspresi Gen Sitokin/Kemokin pada Sel Makrofag Manusia yang Dipapar Virus Dengue Isolat Indonesia

Dengue is one of the world's most important arbovirus disease. Dengue pathogenesis has not been yet fully understood. It has been reported that there is involvement of the host immune factors and viral factors. Several studies have shown that concentrations of cytokines/chemokines on blood are significantly increased during infection and viral factors are also involved in disease severity. Therefore, characterization of host gene expression profiles in response to dengue virus infection of different serotypes could provide input for understanding the pathogenesis of dengue. The purpose of this research was to determine expression profiles of the genes (mRNA) of cytokines/chemokines as immune response that are released by monocyte derived macrophages (MDM) cells (host gene) exposed dengue viruses. Four dengue serotypes of Indonesia isolate were used in this study. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from blood cells of healthy donors by Ficoll gradient centrifugation techniques and then differentiated into MDM cells. Quantitative real time RT-PCR was used to quantify expression levels of cytokine/chemokine-encoding genes from MDM cells infected dengue. Four cytokine/chemokine-encoding genes i.e IP-10, MCP-1, IL-10, and MIP-1β known involved in dengue pathogenesis. Measurement of the expression levels of cytokines/ chemokines showed that the dengue virus of serotypes DENV-1 and DENV-3 caused an increase in the expression of genes encoding cytokine IL-10 and chemokine IP-10 is higher than other serotypes. Further research is needed to better determine the pathogenesis of dengue disease.

Cloning and expression of the NS1 protein of dengue virus in a prokaryotic system

Background Dengue virus nonstructural protein 1 (NS1) is a highly conserved glycoprotein involved in the production of infectious virus and the pathogenesis of dengue disease [1]. Serum or plasma DENV NS1 level has been found to correlate with viremia titer and disease severity. It can be found in the peripheral blood circulation for up to 9 days from illness onset, but can persist for up to 18 days from illness onset in some cases. Thus NS1 detection offers a larger window of opportunity for diagnosis of dengue compared with virus isolation, RT-PCR or NASBA [2]. In this context, the aim of this work has been the establishment of conditions for expression and purification of the recombinant protein NS1 of dengue virus serotype 2 produced in E. coli for further development of a serological diagnostic method at low cost.

Differential Oxidative Stress Induced by Dengue Virus in Monocytes from Human Neonates, Adult and Elderly Individuals

Changes in immune response during lifespan of man are well known. These changes involve decreased neonatal and elderly immune response. In addition, it has been shown a relationship between immune and oxidative mechanisms, suggesting that altered immune response could be associated to altered oxidative response. Increased expression of nitric oxide (NO) has been documented in dengue and in monocyte cultures infected with different types of dengue virus. However, there is no information about the age-dependent NO oxidative response in humans infected by dengue virus. In this study, monocyte cultures from neonatal, elderly and adult individuals (n = 10 each group) were infected with different dengue virus types (DENV- 1 to 4) and oxidative/antioxidative responses and apoptosis were measured at days 1 and 3 of culture. Increased production of NO, lipid peroxidation and enzymatic and nonenzymatic anti-oxidative responses in dengue infected monocyte cultures were observed. However, neonatal and elderly monocytes had lower values of studied parameters when compared to those in adult-derived cultures. Apoptosis was present in infected monocytes with higher values at day 3 of culture. This reduced oxidant/antioxidant response of neonatal and elderly monocytes could be relevant in the pathogenesis of dengue disease.

The Battle between Infection and Host Immune Responses of Dengue Virus and Its Implication in Dengue Disease Pathogenesis

Dengue virus (DENV) is a mosquito-transmitted single stranded RNA virus belonging to genus Flavivirus. The virus is endemic in the tropical and subtropical countries of the world, causing diseases classified according to symptoms and severity (from mild to severe) as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Among a variety of human cell types targeted by DENV, monocytes, macrophages, and dendritic cells are members of innate immunity, capable of mounting rapid inflammatory responses. These cells are also major antigen presenting cells, responsible for activating the adaptive immunity for long-term memory. This paper is an overview of the current understanding of the following mutually affected aspects: DENV structure, viral infectivity, cellular receptors, innate immune response, and adaptive immunity.

Can non-human primates serve as models for investigating dengue disease pathogenesis?

Dengue Virus (DV) infects between 50 and 100 million people globally, with public health costs totaling in the billions. It is the causative agent of dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), vector-borne diseases that initially predominated in the tropics. Due to the expansion of its mosquito vector, Aedes spp., DV is increasingly becoming a global problem. Infected individuals may present with a wide spectrum of symptoms, spanning from a mild febrile to a life-threatening illness, which may include thrombocytopenia, leucopenia, hepatomegaly, hemorrhaging, plasma leakage and shock. Deciphering the underlining mechanisms responsible for these symptoms has been hindered by the limited availability of animal models that can induce classic human pathology. Currently, several permissive non-human primate (NHP) species and mouse breeds susceptible to adapted DV strains are available. Though virus replication occurs in these animals, none of them recapitulate the cardinal features of human symptomatology, with disease only occasionally observed in NHPs. Recently our group established a DV serotype 2 intravenous infection model with the Indian rhesus macaque, which reliably produced cutaneous hemorrhages after primary virus exposure. Further manipulation of experimental parameters (virus strain, immune cell expansion, depletion, etc.) can refine this model and expand its relevance to human DF. Future goals include applying this model to elucidate the role of pre-existing immunity upon secondary infection and immunopathogenesis. Of note, virus titers in primates in vivo and in vitro, even with our model, have been consistently 1000-fold lower than those found in humans. We submit that an improved model, capable of demonstrating severe pathogenesis may only be achieved with higher virus loads. Nonetheless, our DV coagulopathy disease model is valuable for the study of select pathomechanisms and testing DV drug and vaccine candidates.

Molecular mimicry between virus and host and its implications for dengue disease pathogenesis

Numerous infectious agents may trigger autoimmunity or even result in autoimmune diseases. Several mechanisms have been proposed for pathogen-triggered autoimmunity including molecular mimicry, cryptic antigens, epitope spreading, bystander activation and polyclonal activation. In the case of dengue virus infection which causes serious public health problems, the mechanisms regarding the pathogenesis of dengue hemorrhagic syndrome are not fully resolved. Our previous studies suggest a mechanism of molecular mimicry in which antibodies directed against dengue virus non-structural protein 1 (NS1) cross-react with human platelets and endothelial cells and cause their damage and dysfunction, which may be related to the clinical features of dengue disease. Several cell surface proteins recognized by patient serum samples and anti-NS1 antibodies have been identified. Based on proteomic studies and sequence analysis, the C-terminal region of dengue virus NS1 shows sequence homology with target proteins. In addition, different regions of dengue virus proteins including core, prM, E and NS1 proteins show sequence homology with different coagulatory molecules. As an example, the amino acid sequence 101-106 of E protein (WGNGCG) shows sequence homology with factors XI, X, IX, VII, II (thrombin), plasminogen and tissue plasminogen activator. Furthermore, single chain variable region against NS1 can interfere with fibrin formation, which leads to prolonged thrombin time. We hypothesize that molecular mimicry between dengue virus proteins and coagulatory molecules may induce cross-reactive autoantibodies that can interfere with coagulation activation. A molecular mimicry pathogenesis for dengue disease which involves cross-reactivity of dengue virus with human endothelial cells, platelets and coagulatory molecules is proposed.

Frequency Alterations in Key Innate Immune Cell Components in the Peripheral Blood of Dengue Patients Detected by FACS Analysis

Dengue is a timing disease, and frequently dengue patients do not seek help until 2–3 days after prodrome. Thus, the innate immune parameters in dengue patients remain largely unexplored. A comprehensive FACS profiling of key innate immune cells in the peripheral blood of dengue patients was performed. Twenty-seven dengue patients varying in days of fever onset were enrolled and the majority of them had primary infection based on serological results. FACS panels were used to evaluate NK cells, platelet-leukocyte aggregates (PLA), inflammatory monocytes, and plasmacytoid (pDC) and myeloid dendritic cells (mDC). FACS results showed the following findings: (i) NK cells significantly dropped on day 5 after the onset of fever and gradually returned to normal within 2 weeks of illness; (ii) biphasic PLA were observed, maximum levels were reached on days 6–8 and 11–16, and platelet-monocyte aggregates (PMA) were the most frequent event; (iii) inflammatory monocytes were consistently lower on days 5–8 and gradually returned to the normal level in the second week of illness, and (iv) pDC reached the maximum level on day 5 and gradually declined to the baseline level after 1 week of illness. In contrast, mDC fluctuated somewhat during the first week of illness and returned to the baseline level in the second week of illness. The most interesting finding was the biphasic PLA, in particular the PMA. These innate immunological parameters could be a crucial factor dictating the complicated pathogenesis of dengue disease.

Dengue virus–induced hemorrhage in a nonhuman primate model

AbstractLack of a dengue hemorrhagic animal model recapitulating human dengue virus infection has been a significant impediment in advancing our understanding of the early events involved in the pathogenesis of dengue disease. In efforts to address this issue, a group of rhesus macaques were intravenously infected with dengue virus serotype 2 (strain 16 681) at 1 × 107 PFU/animal. A classic dengue hemorrhage developed 3 to 5 days after infection in 6 of 6 animals. Blood chemistry appeared to be normal with exception of creatine phosphokinase, which peaked at 7 days after infection. A modest thrombocytopenia and noticeable neutropenia concomitant with slight decrease of hemoglobin and hematocrit were registered. In addition, the concentration of D-dimer was elevated significantly. Viremia peaked at 3 to 5 days after infection followed by an inverse relationship between T and B lymphocytes and a bimodal pattern for platelet-monocytes and platelet-neutrophil aggregates. Dengue virus containing platelets engulfed by monocytes was noted at 8 or 9 days after infection. Thus, rhesus macaques inoculated intravenously with a high dose of dengue virus produced dengue hemorrhage, which may provide a unique platform to define the early events in dengue virus infection and help identify which blood components contribute to the pathogenesis of dengue disease.