Phenotypic and genotypic characterization of dengue virus isolates differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome

Dengue viruses (DENV) cause 50-100 million cases of acute febrile disease every year, including 500,000 reported cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Viral factors have been proposed to influence the severity of the disease, but markers of virulence have never been identified on DENV. Three DENV serotype-1 isolates from the 2007 epidemic in Cambodia that are derived from patients experiencing the various clinical forms of dengue were characterized both phenotypically and genetically. Phenotypic characteristics in vitro, based on replication kinetics in different cell lines and apoptosis response, grouped isolates from DF and DHF patients together, whereas the virus isolate from a DSS patient showed unique features: a lower level of replication in mammalian cells and extensive apoptosis in mosquito cells. Genomic comparison of viruses revealed six unique amino acid residues in the membrane, envelope, and in non-structural genes in the virus isolated from the DSS patient.     

Race: a risk factor for dengue hemorrhagic fever

Summary. Cuban DHF/DSS outbreaks have provided evidence of a reduced risk of people of Negroid race for DHF/DSS compared to those of Caucasoid race. These observations from Cuban dengue outbreaks have significant epidemiological interest, as the differences in susceptibility to DHF/DSS among racial groups in Cuba coincide with that reported in African and Black Caribbean populations. In this article, we review the literature on race as a risk factor for DHF/DSS and discuss recent results from onging studies. Taking into consideration the origins of contemporary Cuban inhabitants, we propose that the Cuban, Caribbean Black and African populations share a common gene pool that could explain, at least partially, the low incidence of dengue hemorrhagic fever in Cuba and Caribbean and African countries. The central role played by immunological mechanisms in the pathogenesis of DHF/DSS has led us to consider that the polymorphic genes associated with the immune response must be carefully considered among those human genes regulating dengue disease severity that might be distributed unequally in Blacks and Whites.     

Dengue Virus Pathogenesis: an Integrated View

Summary: Much remains to be learned about the pathogenesis of the different manifestations of dengue virus (DENV) infections in humans. They may range from subclinical infection to dengue fever, dengue hemorrhagic fever (DHF), and eventually dengue shock syndrome (DSS). As both cell tropism and tissue tropism of DENV are considered major determinants in the pathogenesis of dengue, there is a critical need for adequate tropism assays, animal models, and human autopsy data. More than 50 years of research on dengue has resulted in a host of literature, which strongly suggests that the pathogenesis of DHF and DSS involves viral virulence factors and detrimental host responses, collectively resulting in abnormal hemostasis and increased vascular permeability. Differential targeting of specific vascular beds is likely to trigger the localized vascular hyperpermeability underlying DSS. A personalized approach to the study of pathogenesis will elucidate the basis of individual risk for development of DHF and DSS as well as identify the genetic and environmental bases for differences in risk for development of severe disease.     

Antibodies play a greater role than immune cells in heterologous protection against secondary dengue virus infection in a mouse model

The four serotypes of dengue virus (DENV1–4) are causative agents of dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Previous DENV infection is a risk factor for DHF/DSS during subsequent infection by a different serotype. Nonetheless, most primary and secondary DENV infections are asymptomatic. To investigate the possible mechanisms of immune protection in vivo, 129/Pas mice lacking IFN-α/β and -γ receptors (AG129) were used to model secondary infection using both DENV1–DENV2 and DENV2–DENV4 sequences. At intervals between sequential infections of 4 to 52 weeks, protection against secondary heterologous DENV infection was observed. Passive transfer of DENV-immune serum was protective against replication of heterologous challenge virus in all tissues tested, whereas adoptive transfer of DENV-immune cells significantly protected mice from replication of the challenge virus only when a lower inoculum was administered. These findings are relevant for understanding both natural and vaccine-induced immunity to DENV.     

Antibodies from patients with dengue viral infection mediate cellular cytotoxicity.

Acute and late convalescent sera (collected at day 5 of disease onset and 1 year later) from dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) laboratory confirmed cases, were tested for antibody-dependent cell-mediated cytotoxicity (ADCC) activity using dengue 1 (DENV-1) or dengue 2 (DENV-2) infected cells as target. All patients experienced their first dengue virus (DENV) infection 20 years before. ADCC activity was detected in acute sera from DHF/DSS but not in sera from DF patients. However, 1 year after illness, ADCC activity was observed in all cases. This preliminary report represents one of the few studies of ADCC in dengue patients and suggests that ADCC could be implicated in dengue pathogenesis.     

Subversion of early innate antiviral responses during antibody-dependent enhancement of Dengue virus infection induces severe disease in immunocompetent mice

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1–4). Epidemiologic and observational studies demonstrate that the majority of severe dengue cases, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), occurs predominantly in either individuals with cross-reactive immunity following a secondary heterologous infection or in infants with primary DENV infections born from dengue-immune mothers, suggesting that B-cell-mediated and antibody responses impact on disease evolution. We demonstrate here that B cells play a pivotal role in host responses against primary DENV infection in mice. After infection, μMT^?/? mice showed increased viral loads followed by severe disease manifestation characterized by intense thrombocytopenia, hemoconcentration, cytokine production and massive liver damage that culminated in death. In addition, we show that poly and monoclonal anti-DENV-specific antibodies can sufficiently increase viral replication through a suppression of early innate antiviral responses and enhance disease manifestation, so that a mostly non-lethal illness becomes a fatal disease resembling human DHF/DSS. Finally, treatment with intravenous immunoglobulin containing anti-DENV antibodies confirmed the potential enhancing capacity of subneutralizing antibodies to mediate virus infection and replication and induce severe disease manifestation of DENV-infected mice. Thus, our results show that humoral responses unleashed during DENV infections can exert protective or pathological outcomes and provide insight into the pathogenesis of this important human pathogen.     

Senescence Affects Endothelial Cells Susceptibility to Dengue Virus Infection

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.     

2型登革病毒诱导RAW264.7细胞凋亡的机制及凋亡对病毒复制的影响

 目的：探讨2型登革病毒（DENV2）感染能否诱导RAW264.7细胞凋亡,并初步探讨凋亡对病毒复制的影响。方法：用DENV2感染RAW264.7细胞,MTT检测细胞活性,Hoechst 33342染色检测细胞核变化,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡,Western blotting检测caspase-3和caspase-8活化片段的变化,比色法检测caspase-9活性变化,JC-1染色检测线粒体膜电位变化,Z-VAD-FMK抑制细胞凋亡后以TCID50检测感染细胞上清病毒滴度。结果：DENV2感染RAW264.7细胞24 h、36 h及48 h后细胞活性受到抑制,免疫荧光检测有核固缩现象,流式细胞术检测发现病毒感染诱导了细胞凋亡,Western blotting检测发现活化caspase-3和caspase-8的表达增加,caspase-9活性也增加,JC-1染色发现病毒感染诱导RAW264.7细胞线粒体膜电位降低,用Z-VAD-FMK抑制凋亡后感染细胞上清病毒滴度增加。结论：登革病毒感染可以通过内、外源性途径诱导RAW264.7细胞发生凋亡;凋亡发生抑制了病毒的产生。     

Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection

Today, dengue viruses are the most prevalent arthropod-borne viruses in the world. Since the 1960s, numerous reports have identified a second heterologous dengue virus (DENV) infection as a principal risk factor for severe dengue disease (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS). Modifiers of dengue disease response include the specific sequence of two DENV infections, the interval between infections, and contributions from the human host, such as age, ethnicity, chronic illnesses and genetic background. Antibody-dependent enhancement (ADE) of dengue virus infection has been proposed as the early mechanism underlying DHF/DSS. Dengue cross-reactive antibodies raised following a first dengue infection combine with a second infecting virus to form infectious immune complexes that enter Fc-receptor-bearing cells. This results in an increased number of infected cells and increased viral output per cell. At the late illness stage, high levels of cytokines, possibly the result of T cell elimination of infected cells, result in vascular permeability, leading to shock and death. This review is focused on the etiological role of secondary infections (SI) and mechanisms of ADE.     

High Producing Tumor Necrosis Factor Alpha Gene Alleles in Protection against Severe Manifestations of Dengue

Dengue virus (DENV) infection usually presents with mild self-limiting dengue fever (DF). Few however, would present with the more severe form of the disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the present study, the association between IL-12B, IL-10 and TNF-α gene polymorphisms and dengue severity was investigated. Methods: A case-control study was performed on a total of 120 unrelated controls, 86 DF patients and 196 DHF/DSS patients. The polymorphisms in IL-12B, IL-10 and TNF-α genes were genotyped using PCR-RFLP and PCR-sequencing methods. Results: A protective association of TNF-α -308A allele and -308GA genotype against DHF/DSS was observed, while TNF-α -238A allele and -238GA genotype were associated with DHF/DSS. A combination of TNF-α -308GA+AA genotype and IL-10 non-GCC haplotypes, IL-12B pro homozygotes (pro1/pro1, pro2/pro2) and IL-12B 3''UTR AC were significantly correlated with protective effects against DHF/DSS. An association between the cytokine gene polymorphisms and protection against the clinical features of severe dengue including thrombocytopenia and increased liver enzymes was observed in this study. Conclusion: The overall findings of the study support the correlation of high-producer TNF-α genotypes combined with low-producer IL-10 haplotypes and IL-12B genotypes in reduced risk of DHF/DSS.     

Tissue plasminogen activator induced by dengue virus infection of human endothelial cells

Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are severe complications of dengue virus (DV) infection. However, the pathogenesis of hemorrhage induced by dengue virus infection is poorly understood. Since endothelial cells play a pivotal role in the regulation of hemostasis, we studied the effect of DV infection on the production of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) in vitro using both primary isolated endothelial cells, human umbilical cord veins cells, and a human microvascular endothelial cell line. DV infection significantly induced the secretion of tPA but not PAI-1 of human endothelial cells. In addition, tPA mRNA of endothelial cells was induced by DV as demonstrated by RT-PCR. Antibody against IL-6 but not control antibody inhibited DV-induced tPA production of endothelial cells. Furthermore, a good correlation between sera levels of IL-6 and tPA was found in DHF but not DF patients. These results suggest that IL-6 can regulate DV-induced tPA production of endothelial cells, which may play important roles in the pathogenic development of DHF/DSS.     

Autoimmune pathogenesis in dengue virus infection

The pathogenic mechanisms of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) caused by dengue virus (DV) infection remain unresolved. Patients with DHF/DSS are characterized by several manifestations, including severe thrombocytopenia, vascular leakage, and hepatomegaly. In addition to the effect of virus load and virus variation, abnormal immune responses of the host after DV infection may also account for the progression of DHF/DSS. Actually, viral autoimmunity is involved in the pathogenesis of numerous viral infections, such as human immunodeficiency virus, human hepatitis C virus, human cytomegalovirus, herpes simplex virus, Epstein- Barr virus, and DV. In this review, we discuss the implications of autoimmunity in dengue pathogenesis. Antibodies directed against DV nonstructural protein 1 (NS1) showed cross-reactivity with human platelets and endothelial cells, which lead to platelet and endothelial cell damage and inflammatory activation. Based on these findings, we hypothesize that anti-DV NS1 is involved in the pathogenesis of DF and DHF/DSS, and this may provide important information in dengue vaccine development.     

Antibodies from dengue patient sera cross-react with endothelial cells and induce damage

Dengue virus infection causes a wide range of diseases from the mild febrile illness dengue fever to the life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage and hemorrhagic syndrome are the clinical features associated with dengue infection, yet the mechanisms remain unclear. In this study, the cross-reactivity of dengue patient sera with endothelial cells was demonstrated. There were higher percentages of endothelial cells reactive with dengue hemorrhagic fever/dengue shock syndrome patient sera than those with dengue fever patient sera. The percentages of endothelial cells reactive with patient serum IgM were higher than those with IgG. Further studies showed that the endothelial cell binding activity was inhibited by pretreatment with dengue virus nonstructural protein 1 (NS1). The antibodies against NS1 produced after dengue virus infection may, at least in part, account for the cross-reactivity of patient sera with endothelial cells. Furthermore, dengue patient sera induced endothelial cell apoptosis via a caspase-dependent pathway that was also inhibited by NS1 pretreatment. In addition to apoptosis, patient sera caused cell lysis in the presence of complement, and DHF/DSS patient sera showed higher percentages of cytotoxicity than dengue fever patient sera. Thus, the generation of cross-reactive autoantibodies against endothelial cells would lead to their dysfunction, which may play a role in the pathogenesis of dengue virus infection.     

A simple one-step real-time RT-PCR for diagnosis of dengue virus infection

Dengue is the most important arbovirus disease in tropical and sub-tropical countries, and can be caused by infection with any of the four-dengue virus (DENV) serotypes. Infection with DENV can lead to a broad clinical spectrum, ranging from sub-clinical infection or an influenza-like disease known as dengue fever (DF) to a severe, sometimes fatal, disease characterized by hemorrhage and plasma leakage that can lead to shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The diagnosis of dengue is routinely accomplished by serologic assays, such as IgM and IgG ELISAs, as well as HI tests, analyzing serum samples obtained from patients with at least 7 days of symptoms onset. These tests cannot be used for diagnosis during the early symptomatic phase. In addition, antibodies against dengue are broad reactive with other flaviviruses. Therefore, a specific diagnostic method for acute DENV infection is of great interest. In that sense, the real-time RT-PCR has become an important tool that can be used for early and specific detection of dengue virus genome in human serum samples. This study describes a simple, specific, and sensitive real-time RT-PCR for early diagnosis of dengue virus infection.     

Generation of IgM anti-platelet autoantibody in dengue patients

Dengue virus infection causes a wide range of diseases from dengue fever to life-threatening dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). The mechanisms involved in DHF/DSS pathogenesis remain unclear. Patient sera collected from an outbreak in southern Taiwan from November 1998 to January 1999 were studied. The presence of antibodies which cross-reacted with platelets could be detected in patient sera, and the isotype of these autoantibodies was IgM. The anti-platelet IgM levels were higher in DHF/DSS than in dengue fever patient sera in disease acute phase. These autoantibodies were still detectable in convalescent stage (1-3 weeks after acute phase) and even eight to nine months after illness. The platelet binding activity was not observed in other virus-infected patient sera tested. Further investigation showed that dengue patient sera caused platelet lysis in the presence of complement. The platelet cytotoxicity induced by DHF/DSS patient sera was higher than that by dengue fever sera. Dengue patient sera also inhibited platelet aggregation which, however, appeared to be not related to DHF/DSS development.     

The pathology of dengue hemorrhagic fever

An estimated 2.5 billion people are at risk of dengue infection, and of the 100 million cases of dengue fever per year, up to 500,000 develop dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), the life-threatening forms of the infection. The large majority of DHF/DSS occurs as the result of a secondary infection with a different serotype of the virus. While not completely understood, there is evidence that the target cells include dendritic reticulum cells, monocytes, lymphocytes, hepatocytes, and vascular endothelial cells. Viral replication appears to occur in dendritic cells, monocytes, and possibly circulating lymphoid cells, and damage to these and other target cells occurs through immune-mediated mechanisms related to cross-reacting antibodies and cytokines released by dendritic cells, monocytes, and vascular endothelium. There is evidence of a concomitant cellular activation as well as immune suppression during the infection. The activation of memory T cells results in cascades of inflammatory cytokines, including tumor necrosis factor-alpha, interleukins (IL-2, IL-6, and IL-8), and other chemical mediators that increase vascular endothelial permeability or trigger death of target cells through apoptosis. Pathological studies in humans are uncommon, and a suitable animal model of DHF/DSS does not exist. The current treatment of DHF/DSS is symptomatic, and prevention is through vector control. As such, there is a great impetus for the development of vaccines and novel therapeutic molecules to impede viral replication in infected cells or counteract the effects of specific inflammatory mediators on target cells. The role of genetics in relation to resistance to DHF/DSS also requires clarification.     

Cytokine IP-10 and GM-CSF are prognostic biomarkers for severity in secondary dengue infection

Dengue virus (DENV) infection is mostly prevalent in tropical and sub-tropical regions of the world. Though most DENV infections are self-limiting febrile like-illness, a small proportion of secondary infection is fatal, if untreated symptomatically. Among various factors involved in severe dengue, immune enhancement by cytokine is the major one.The objective of the study is to elucidate serum cytokine expression among primary and secondary infection and determine if any signature cytokine is correlated with disease severity. Seventy-six serum samples at acute time points were collected during the 2017 DENV outbreak in Madurai, Tamil Nadu. Among the 76 serum samples, 49 belong to primary and 27 to secondary DENV infection. Interestingly, a large number of primary infection presented with DHF/DSS symptoms and, children were found prone to DHF and DSS in secondary infection. The serum samples were analysed for inflammatory cytokines, namely IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17A, IFN-γ, TNF-α, IP-10 and GM-CSF using ELISA assay as well as mRNA analysis using qPCR.Among the 12 inflammatory cytokines analysed IP-10 and GMCSF mRNA and protein shows significant upregulation in secondary infection. Similarly, a strong correlation was observed between GM-CSF and IP-10 with thrombocytopenia, ascites, serous effusion and spontaneous bleeding. Based on the observations, GM-CSF and IP-10 could be a potential prognostic biomarkers for secondary DENV infection.     

Immunity and immunopathology in dengue virus infections.

Dengue virus infections are a serious public health problem in tropical and subtropical areas of the world. Based on epidemiological data, it has been postulated that immune responses to dengue virus contribute to the pathogenesis of severe dengue illness, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Host immune responses are also important for controlling dengue virus infection. Therefore, dengue virus infections are an interesting model to explore the interactions between viruses and the immune system which result in immunopathology or recovery from infection. In this paper, we review immune responses to dengue viruses with an emphasis on the human T cell responses, and discuss possible roles of these immune responses in the control of dengue virus infection and in the pathogenesis of DHF/DSS.     

Galectin-9 plasma levels reflect adverse hematological and immunological features in acute dengue virus infection

BackgroundDengue virus (DENV) infection remains a major public health burden worldwide. Soluble mediators may play a critical role in the pathogenesis of acute DENV infection. Galectin-9 (Gal-9) is a soluble β-galactoside-binding lectin, with multiple immunoregulatory and inflammatory properties.ObjectiveTo investigate plasma Gal-9 levels as a biomarker for DENV infection.Study designWe enrolled 65 DENV infected patients during the 2010 epidemic in the Philippines and measured their plasma Gal-9 and cytokine/chemokine levels, DENV genotypes, and copy number during the critical and recovery phases of illness.ResultsDuring the critical phase, Gal-9 levels were significantly higher in DENV infected patients compared to healthy or those with non-dengue febrile illness. The highest Gal-9 levels were observed in dengue hemorrhagic fever (DHF) patients (DHF: 2464 pg/ml; dengue fever patients (DF): 1407 pg/ml; non-dengue febrile illness: 616 pg/ml; healthy: 196 pg/ml). In the recovery phase, Gal-9 levels significantly declined from peak levels in DF and DHF patients. Gal-9 levels tracked viral load, and were associated with multiple cytokines and chemokines (IL-1α, IL-8, IP-10, and VEGF), including monocyte frequencies and hematologic variables of coagulation. Further discriminant analyses showed that eotaxin, Gal-9, IFN-α2, and MCP-1 could detect 92% of DHF and 79.3% of DF, specifically (P < 0.01).ConclusionGal-9 appears to track DENV inflammatory responses, and therefore, it could serve as an important novel biomarker of acute DENV infection and disease severity.