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Dengue viruses cause two severe diseases that alter vascular fluid barrier functions, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Preexisting antibodies to dengue virus disposes patients to immune-enhanced edema (DSS) or hemorrhagic (DHF) disease following infection by a discrete dengue virus serotype. Although the endothelium is the primary vascular fluid barrier, direct effects of dengue virus on endothelial cells (ECs) have not been considered primary factors in pathogenesis. Here, we show that dengue virus infection of human ECs elicits immune-enhancing EC responses. Our results suggest that rapid early dengue virus proliferation within ECs is permitted by dengue virus regulation of early, but not late, beta interferon (IFN-β) responses. The analysis of EC responses following synchronous dengue virus infection revealed the high-level induction and secretion of immune cells (T cells, B cells, and mast cells) as well as activating and recruiting cytokines BAFF (119-fold), IL-6/8 (4- to 7-fold), CXCL9/10/11 (45- to 338-fold), RANTES (724-fold), and interleukin-7 (IL-7; 128-fold). Moreover, we found that properdin factor B, an alternative pathway complement activator that directs chemotactic anaphylatoxin C3a and C5a production, was induced 34-fold. Thus, dengue virus-infected ECs evoke key inflammatory responses observed in dengue virus patients which are linked to DHF and DSS. Our findings suggest that dengue virus-infected ECs directly contribute to immune enhancement, capillary permeability, viremia, and immune targeting of the endothelium. These data implicate EC responses in dengue virus pathogenesis and further rationalize therapeutic targeting of the endothelium as a means of reducing the severity of dengue virus disease.

Dengue virus causes leakage of the vascular endothelium, resulting in dengue hemorrhagic fever and dengue shock syndrome. The endothelial cell lining of the vasculature regulates capillary permeability and is altered by immune and chemokine responses which affect fluid barrier functions of the endothelium. Our findings indicate that human endothelial cells are highly susceptible to infection by dengue virus (type 4). We found that dengue virus productively infects ∼80% of primary human endothelial cells, resulting in the rapid release of ∼105 virions 1 day postinfection. Analysis of potential inhibitors of dengue virus entry demonstrated that antibodies and ligands to integrins and cellular receptors were unable to inhibit dengue virus infection of endothelial cells. In contrast, pretreating cells with heparin or heparan sulfate resulted in a 60 to 80% reduction in dengue virus-infected cells, and pretreatment of endothelial cells with heparinase III or protease reduced dengue infectivity by >80%. Dengue virus bound specifically to resin immobilized heparin, and binding was competitively inhibited by excess heparin but not other ligands. Collectively, these findings suggest that dengue virus specifically attaches to heparan sulfate-containing proteoglycan receptors on endothelial cells. Following attachment to human endothelial cell receptors, dengue virus causes a highly productive infection that has the potential to increase viral dissemination and viremia. This provides the potential for dengue virus-infected endothelial cells to directly alter barrier functions of the endothelium, contribute to enhancement of immune cell activation, and serve as potential targets of immune responses which play a central role in dengue pathogenesis.

Dengue has been recognized as one of the most important vector-borne emerging infectious diseases globally. Though dengue normally causes a self-limiting infection, some patients may develop a life-threatening illness, dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). The reason why DHF/DSS occurs in certain individuals is unclear. Studies in the endemic regions suggest that the preexisting antibodies are a risk factor for DHF/DSS. Viremia and thrombocytopenia are the key clinical features of dengue virus infection in patients. The amounts of virus circulating in patients are highly correlated with severe dengue disease, DHF/DSS. Also, the disturbance, mainly a transient depression, of hematological cells is a critical clinical finding in acute dengue patients. However, the cells responsible for the dengue viremia are unresolved in spite of the intensive efforts been made. Dengue virus appears to replicate and proliferate in many adapted cell lines, but these in vitro properties are extremely difficult to be reproduced in primary cells or in vivo. This paper summarizes reports on the permissive cells in vitro and in vivo and suggests a hematological cell lineage for dengue virus infection in vivo, with the hope that a new focus will shed light on further understanding of the complexities of dengue disease.

Although endothelial cells have been speculated to be a target in the pathogenesis of dengue hemorrhagic fever (DHF), there has been little evidence linking dengue virus infection to any alteration in endothelial cell function. In this study, we show that human umbilical vein endothelial cells become activated when exposed to culture fluids from dengue virus-infected peripheral blood monocytes. Maximum activation was achieved with culture fluids from monocytes in which virus infection was enhanced by the addition of dengue virus-immune serum, thus correlating with epidemiological evidence that prior immunity to dengue virus is a major risk factor for DHF. Activation was strongest for endothelial cell expression of VCAM-1 and ICAM-1. In contrast, activation of endothelial cell E-selectin expression appeared to be more transient, as indicated by its detection at 3 h, but not at 16 h, of treatment. Treatment of monocyte culture fluids with anti-tumor necrosis factor alpha (TNF-alpha) antibody largely abolished the activation effect (as measured by endothelial cell expression of ICAM-1), whereas treatment with IL-1beta receptor antagonist had a much smaller inhibitory effect on activation. Endothelial cells inoculated directly with dengue virus or with virus-antibody combinations were poorly infectable (compared to Vero cells or peripheral blood monocytes), and virus-inoculated endothelial cells showed no increased expression of VCAM-1, ICAM-1, or E-selectin. Taken together, the results strongly indicate that dengue virus can modulate endothelial cell function by an indirect route, in which a key intermediary is TNF-alpha released from virus-infected monocytes.

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2Kd-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8+ CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.

The four serotypes of dengue virus (DENV) cause dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Severe disease has been associated with heterotypic secondary DENV infection, mediated by cross-reactive antibodies (Abs) and/or cross-reactive T cells. The role of cross-reactive immunity in mediating enhanced disease versus cross-protection against secondary heterotypic DENV infection is not well defined. A better understanding of the cross-reactive immune response in natural infections is critical for development of safe and effective tetravalent vaccines. We studied the B cell phenotype of circulating B cells in the blood of pediatric patients suspected of dengue during the 2010–2011 dengue season in Managua, Nicaragua (n = 216), which was dominated by the DENV-3 serotype. We found a markedly larger percentage of plasmablast/plasma cells (PB/PCs) circulating in DENV-positive patients as compared to patients with Other Febrile Illnesses (OFIs). The percentage of DENV-specific PB/PCs against DENV-3 represented 10% of the circulating antibody-producing cells (ASCs) in secondary DENV-3 infections. Importantly, the cross-reactive DENV-specific B cell response was higher against a heterotypic serotype, with 46% of circulating PB/PCs specific to DENV-2 and 10% specific to DENV-3 during acute infection. We also observed a higher cross-reactive DENV-specific IgG serum avidity directed against DENV-2 as compared to DENV-3 during acute infection. The neutralization capacity of the serum was broadly cross-reactive against the four DENV serotypes both during the acute phase and at 3 months post-onset of symptoms. Overall, the cross-reactive B cell immune response dominates during secondary DENV infections in humans. These results reflect our recent findings in a mouse model of DENV cross-protection. In addition, this study enabled the development of increased technical and research capacity of Nicaraguan scientists and the implementation of several new immunological assays in the field.

Author Summary

Dengue is the most common mosquito-borne viral infection of humans, with half the world's population at risk for infection. Four different dengue virus serotypes (DENV-1 to -4) can cause the disease, which can be either inapparent or present with flu-like symptoms (Dengue Fever), also known as “breakbone fever”. In a number of cases, the disease can be more severe and sometimes fatal, with signs of bleeding and vascular leakage leading to shock (Dengue Hemorrhagic Fever/Dengue Shock Syndrome). Severe disease has been associated with secondary sequential DENV infections, i.e., infection with a second DENV serotype different from the serotype causing the first infection. No specific treatment or vaccine is available. Understanding how the human immune response develops during a natural infection can be beneficial for future vaccine studies and trials. B cells are a subset of cells that produce antibodies and are thus essential in the response to natural infections and vaccines. We show here that during secondary DENV infections in humans, the B cell immune response to a previous infecting DENV serotype is stronger than the response against the current infecting serotype. In addition, this study allowed the development of research capacity and implementation of new immunological assays in Nicaragua.

Dengue is a major public health problem worldwide and continues to increase in incidence. Dengue virus (DENV) infection leads to a range of outcomes, including subclinical infection, undifferentiated febrile illness, Dengue Fever (DF), life-threatening syndromes with fluid loss and hypotensive shock, or other severe manifestations such as bleeding and organ failure. The long-standing World Health Organization (WHO) dengue classification and management scheme was recently revised, replacing DF, Dengue Hemorrhagic Fever (DHF), and Dengue Shock Syndrome (DSS) with Dengue without Warning Signs, Dengue with Warning Signs (abdominal pain, persistent vomiting, fluid accumulation, mucosal bleeding, lethargy, liver enlargement, increasing hematocrit with decreasing platelets) and Severe Dengue (SD; dengue with severe plasma leakage, severe bleeding, or organ failure). We evaluated the traditional and revised classification schemes against clinical intervention levels to determine how each captures disease severity using data from five years (2005–2010) of a hospital-based study of pediatric dengue in Managua, Nicaragua. Laboratory-confirmed dengue cases (n = 544) were categorized using both classification schemes and by level of care (I–III). Category I was out-patient care, Category II was in-patient care that did not meet criteria for Category III, which included ICU admission, ventilation, administration of inotropic drugs, or organ failure. Sensitivity and specificity to capture Category III care for DHF/DSS were 39.0% and 75.5%, respectively; sensitivity and specificity for SD were 92.1% and 78.5%, respectively. In this data set, DENV-2 was found to be significantly associated with DHF/DSS; however, this association was not observed with the revised classification. Among dengue-confirmed cases, the revised WHO classification for severe dengue appears to have higher sensitivity and specificity to identify cases in need of heightened care, although it is no longer as specific for a particular pathogenic entity as was the traditional schema.

Author Summary

Dengue is a mosquito-transmitted viral disease that is a major public health problem worldwide. Dengue virus (DENV) infection leads to Dengue Fever (DF) and a spectrum of life-threatening syndromes with fluid loss and hypotensive shock or other severe manifestations. Recently, the traditional World Health Organization (WHO) dengue classification scheme (classic DF, Dengue Hemorrhagic Fever (DHF), and Dengue Shock Syndrome (DSS)) was replaced with Dengue without Warning Signs, Dengue with Warning Signs and Severe Dengue (SD). Using data from 544 laboratory-confirmed dengue cases recruited over five years of a hospital-based study of pediatric dengue in Managua, Nicaragua, we evaluated the traditional and revised classification schemes against clinical intervention levels (I–III) to determine how each captures disease severity. The sensitivity and specificity to capture Category III care for DHF/DSS were 39.0% and 75.5%, respectively, and for SD were 92.1% and 78.5%, respectively. Interestingly, DENV-2 was significantly associated with DHF/DSS; however, this association was not observed with the revised classification. This study indicates that among dengue-confirmed cases, the revised WHO classification appears to have higher sensitivity and specificity for identifying cases in need of heightened care, although it is no longer as specific for a particular pathogenic entity as was the traditional schema.

Background

Increased vascular permeability is a hallmark feature in severe dengue virus (DV) infection, and dysfunction of endothelial cells has been speculated to contribute in the pathogenesis of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Rho-family GTPase Rac1 is a significant element of endothelial barrier function regulation and has been implicated in the regulation of actin remodeling and intercellular junction formation. Yet there is little evidence linking Rac1 GTPase to alteration in endothelial cell function induced by DV infection.

Methods and Findings

Here, we showed that actin is essential for DV serotype 2 (DV2) entry into and release from ECV304 cells, and Rac1 signaling is involved these processes. At early infection, actin cytoskeleton rearranged significantly during 1 hour post infection, and disrupting actin filament dynamics with jasplakinolide or cytochalasin D reduced DV2 entry. DV2 entry induced reduction of Rac1 activity within 1 hour post infection. The expression of dominant-negative forms of Rac1 established that DV2 entry is negatively regulated by Rac1. At late infection, actin drugs also inhibited the DV2 release and induced accumulation of viral proteins in the cytoplasm. Meanwhile, the activity of Rac1 increased significantly with the progression of DV2 infection and was up-regulated in transfected cells expressing E protein. Confocal microscopy showed that DV2 E protein was closely associated with either actin or Rac1 in DV2-infected cells. The interaction between E protein and actin was further confirmed by co-immunoprecipitation assay.

Conclusions

These results defined roles for actin integrity in DV2 entry and release, and indicated evidence for the participation of Rac1 signaling pathways in DV2-induced actin reorganizations and E-actin interaction. Our results may provide further insight into the pathogenesis of DHF/DSS.

Author Summary

An important clinical characteristic of dengue hemorrhagic fever/dengue shock syndrome is increased vascular permeability. Actin cytoskeleton is a significant element of endothelial barrier function regulation. In vitro study showed that dengue virus infection could induce redistributions of actin cytoskeleton. It is not precisely clear the roles of actin and the mechanisms of its reorganization during the infection. Using immunochemical assays, drug inhibition assays and protein interaction profiling methods, we aimed to identify the ways in which dengue virus serotype 2 interacts with actin cytoskeleton. The study showed that dynamic treadmilling of actin is necessary for dengue virus entry, production and release, while small GTPase Rac1 also plays multiple roles during these processes. In addition, we demonstrated the association of viral E protein with actin, indicating a direct effect of viral protein on the structural modifications of actin cytoskeleton. Our results provide evidence for the participation of Rac1 signaling pathways in viral protein-induced actin reorganizations, which may be a mechanism involved in the etiology of dengue hemorrhagic fever.

The severe complications of dengue virus infections, hemorrhagic manifestation and shock, are much more commonly observed during secondary infections caused by a different serotype of dengue virus than that which caused the primary infections. It has been speculated, therefore, that dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are caused by serotype crossreactive immunopathological mechanisms. We analyzed clones of dengue serotype crossreactive T lymphocytes derived from the PBMC of a donor who had been infected with dengue 3 virus. These PBMC responded best to dengue 3 antigen, but also responded to dengue 1, 2, and 4 antigens, in bulk culture proliferation assays. 12 dengue antigen-specific clones were established using a limiting dilution technique. All of the clones had CD3+ CD4+ CD8 phenotypes. Eight clones responded to dengue 1, 2, 3, and 4 antigens and are crossreactive, while four other clones responded predominantly to dengue 3 antigen. These results indicate that the serotype crossreactive dengue-specific T lymphocyte proliferation observed in bulk cultures reflects the crossreactive responses detected at the clonal level. Serotype crossreactive clones produced high titers of IFN- gamma after stimulation with dengue 3 antigens, and also produced IFN- gamma to lower levels after stimulation with dengue 1, 2, and 4 antigens. The crossreactive clones lysed autologous lymphoblastoid cell line (LCL) pulsed with dengue antigens, and the crossreactivity of CTL lysis by T cell clones was consistent with the crossreactivity observed in proliferation assays. Epidemiological studies have shown that secondary infections with dengue 2 virus cause DHF/DSS at a higher rate than the other serotypes. We hypothesized that the lysis of dengue virus-infected cells by CTL may lead to DHF/DSS; therefore, the clones were examined for cytotoxic activity against dengue 2 virus-infected LCL. All but one of the serotype crossreactive clones lysed dengue 2 virus-infected autologous LCL, and they did not lyse uninfected autologous LCL. The lysis of dengue antigen-pulsed or virus-infected LCL by the crossreactive CTL clones that we have examined is restricted by HLA DP or DQ antigens. These results indicate that primary dengue virus infections induce predominantly crossreactive memory CD4+ T lymphocytes. These crossreactive T lymphocytes proliferate and produce IFN-gamma after stimulation with a virus strain of another serotype, and demonstrate crossreactive cyotoxic activity against autologous cells infected with heterologous dengue viruses.(ABSTRACT TRUNCATED AT 400 WORDS)

The World Health Organization (WHO) estimates that there are over 50 million cases of dengue fever reported annually and approximately 2.5 billion people are at risk. Mild dengue fever presents with headache, fever, rash, myalgia, osteogenic pain, and lethargy. Severe disease can manifest as dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF). Symptoms of DSS/DHF are leukopenia, low blood volume and pressure encephalitis, cold and sweaty skin, gastrointestinal bleeding, and spontaneous bleeding from gums and nose. Currently, there are no therapeutics available beyond supportive care and untreated complicated dengue fever can have a 50% mortality rate. According to WHO DSS/DHF is the leading cause of childhood mortality in some Asian countries. Dendritic cells are professional antigen presenting cells that are primary targets in a dengue infection. Dengue binds to Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN). DC-SIGN has a high affinity for ICAM3 which is expressed in activating T-cells. Previous studies have demonstrated an altered T-cell phenotype expressed in dengue infected patients that could be potentially mediated by dengue-infected DCs.

Dengue is enhanced by three interacting components of the immune system. Dengue begins by infecting dendritic cells which in immature dendritic cells is mediated by DC-SIGN. In mature dendritic cells, antibodies can enhance dengue infection via Fc receptors. Downstream of dendritic cells T-cells become activated and generate the very cytokines implicated in vascular leak and shock in addition to activating effector cells. Both the virus and the antibodies are involved in release of complement and anaphylatoxins which can cause or exacerbate DHF/DSS. These systems are inextricable and strongly associated with dengue pathogenesis.

Background

Dengue virus (DV) infection is one of the most important mosquito-borne diseases in the tropics. Recently, the severe forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), have become the leading cause of death among children in Southern Vietnam. Protective and/or pathogenic T cell immunity is supposed to be important in the pathogenesis of DHF and DSS.

Methodology/Principal Findings

To identify HLA alleles controlling T cell immunity against dengue virus (DV), we performed a hospital-based case control study at Children's Hospital No.2, Ho Chi Minh City (HCMC), and Vinh Long Province Hospital (VL) in Southern Vietnam from 2002 to 2005. A total of 211 and 418 patients with DHF and DSS, respectively, diagnosed according to the World Health Organization (WHO) criteria, were analyzed for their characteristic HLA-A, -B and -DRB1 alleles. Four hundred fifty healthy children (250 from HCMC and 200 from VL) of the same Kinh ethnicity were also analyzed as population background. In HLA class I, frequency of the HLA-A*24 showed increased tendency in both DHF and DSS patients, which reproduced a previous study. The frequency of A*24 with histidine at codon 70 (A*2402/03/10), based on main anchor binding site specificity analysis in DSS and DHF patients, was significantly higher than that in the population background groups (HCMC 02-03 DSS: OR = 1.89, P = 0.008, DHF: OR = 1.75, P = 0.033; VL 02-03 DSS: OR = 1.70, P = 0.03, DHF: OR = 1.46, P = 0.38; VL 04-05 DSS: OR = 2.09, P = 0.0075, DHF: OR = 2.02, P = 0.038). In HLA class II, the HLA-DRB1*0901 frequency was significantly decreased in secondary infection of DSS in VL 04-05 (OR = 0.35, P = 0.0025, Pc = 0.03). Moreover, the frequency of HLA-DRB1*0901 in particular was significantly decreased in DSS when compared with DHF in DEN-2 infection (P = 0.02).

Conclusion

This study improves our understanding of the risk of HLA-class I for severe outcome of DV infection in the light of peptide anchor binding site and provides novel evidence that HLA-class II may control disease severity (DHF to DSS) in DV infection.

Author Summary

Dengue has become one of the most common viral diseases transmitted by infected mosquitoes (with any of the four dengue virus serotypes: DEN-1, -2, -3, or -4). It may present as asymptomatic or illness, ranging from mild to severe disease. Recently, the severe forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), have become the leading cause of death among children in Southern Vietnam. The pathogenesis of DHF/DSS, however, is not yet completely understood. The immune response, virus virulence, and host genetic background are considered to be risk factors contributing to disease severity. Human leucocyte antigens (HLA) expressed on the cell surface function as antigen presenting molecules and those polymorphism can change individuals' immune response. We investigated the HLA-A, -B (class I), and -DRB1 (class II) polymorphism in Vietnamese children with different severity (DHF/DSS) by a hospital-based case-control study. The study showed persons carrying HLA-A*2402/03/10 are about 2 times more likely to have severe dengue infection than others. On the other hand, HLA-DRB1*0901 persons are less likely to develop DSS with DEN-2 virus infection. These results clearly demonstrated that HLA controlled the susceptibility to severe forms of DV infection.

The severe complications of dengue virus infections, hemorrhagic manifestations and shock, are more commonly observed during secondary dengue virus infections than during primary infections. It has been speculated that these complications are mediated by cross-reactive host-immune responses. We have begun to analyze human T cell responses to dengue antigens in vitro to explain the possible role of T lymphocytes in the pathogenesis of these complications. Dengue antigens induce proliferative responses of PBMC from dengue antibody-positive donors, but do not induce specific proliferative responses of PBMC from dengue antibody-negative donors. IFN gamma is detected in the culture fluids of dengue-immune PBMC stimulated with dengue antigens. The cells that proliferate in the dengue antigen-stimulated bulk cultures have CD3+, CD4+, CD8-, CD16-, and CD20- phenotypes. Dengue-specific T cell lines were established using limiting dilution techniques. They have CD3+, CD4+, and CD8- phenotypes, and produce IFN gamma in response to dengue antigens. Culture fluids from dengue-immune PBMC stimulated with dengue antigens, which contain IFN gamma, augment dengue virus infection of human monocytes by dengue virus-antibody complexes. These results indicate that PBMC from dengue-immune donors contain CD4+ T cells that proliferate and produce IFN gamma after stimulation with dengue antigens, and suggest that the IFN gamma that is produced by these stimulated dengue-specific T cells may contribute to the pathogenesis of dengue hemorrhagic fever and dengue shock syndrome by increasing the number of dengue virus-infected monocytes in the presence of cross-reactive anti-dengue antibodies.

Images

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.

Lack of an appropriate animal model for dengue virus (DEN), which causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), has impeded characterization of the mechanisms underlying the disease pathogenesis. The cardinal feature of DHF/DSS, the severe form of DEN infection, is increased vascular permeability. To develop a murine model that is more relevant to DHF/DSS, a novel DEN strain, D2S10, was generated by alternately passaging a non-mouse-adapted DEN strain between mosquito cells and mice, thereby mimicking the natural transmission cycle of the virus between mosquitoes and humans. After infection with D2S10, mice lacking interferon receptors died early without manifesting signs of paralysis, carried infectious virus in both non-neuronal and neuronal tissues, and exhibited signs of increased vascular permeability. In contrast, mice infected with the parental DEN strain developed paralysis at late times after infection, contained detectable levels of virus only in the central nervous system, and displayed normal vascular permeability. In the mice infected with D2S10, but not the parental DEN strain, significant levels of serum tumor necrosis factor alpha (TNF-α) were produced, and the neutralization of TNF-α activity prevented early death of D2S10-infected mice. Sequence analysis comparing D2S10 to its parental strain implicated a conserved region of amino acid residues in the envelope protein as a possible source for the D2S10 phenotype. These results demonstrate that D2S10 causes a more relevant disease in mice and that TNF-α may be one of several key mediators of severe DEN-induced disease in mice. This report represents a significant advance in animal models for severe DEN disease, and it begins to provide mechanistic insights into DEN-induced disease in vivo.

Background

All four dengue virus (DV) serotypes (D1V, D2V, D3V and D4V) can cause a series of disorders, ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). Previous studies have revealed that DV serotype-specific CD8+ T cells are involved in controlling DV infection. Serotype cross-reactive CD8+ T-cells may contribute to the immunopathogenesis of DHF/DSS. The aim of the study was to identify HLA-A*0201-binding peptides from four DV serotypes. We then examined their immunogenicity in vivo and cross-reactivity within heterologous peptides.

Methods

D1V-derived candidate CD8+ T-cell epitopes were synthesized and evaluated for their affinity to the HLA-A*0201 molecule. Variant peptides representing heterologous D2V, D3V, D4V serotypes were synthesized. The immunogenicity of the high-affinity peptides were evaluated in HLA-A*0201 transgenic mice.

Results

Of the seven D1V-derived candidate epitopes [D1V-NS4a56–64(MLLALIAVL), D1V-C46–54(LVMAFMAFL), D1V-NS4b562–570(LLATSIFKL), D1V-NS2a169–177(AMVLSIVSL), D1V-NS4a140–148(GLLFMILTV), D1V-NS2a144–152(QLWAALLSL) and D1V-NS4b183–191(LLMRTTWAL)], three peptides [D1V-NS4a140–148, D1V-NS2a144–152 and D1V-NS4b183–191] had a high affinity for HLA-A*0201 molecules. Moreover, their variant peptides for D2V, D3V and D4V [D2V-NS4a140–148(AILTVVAAT), D3V-NS4a140-148(GILTLAAIV), D4V-NS4a140-148(TILTIIGLI), D2V-NS2a144–152(QLAVTIMAI), D3V-NS2a144–152(QLWTALVSL), D4V-NS2a143–151(QVGTLALSL), D2V-NS4b182–190(LMMRTTWAL), D3V-NS4b182–190 (LLMRTSWAL) and D4V-NS4b179–187(LLMRTTWAF)] also had a high affinity for HLA-A*0201 molecules. Furthermore, CD8+ T cells directed to these twelve peptides were induced in HLA-A*0201 transgenic mice following immunization with these peptides. Additionally, cross-reactivity within four peptides (D1V-NS4b183–191, D2V-NS4b182–190, D3V-NS4b182–190 and D4V-NS4b179–187) was observed.

Conclusions

Two novel serotype-specific HLA-A*0201-restricted CD8+ T-cell epitopes (NS4a140-148 and NS2a144–152) and one cross-reactive HLA-A*0201-restricted CD8+ T-cell epitopes which is similar to a previously identified epitope were identified in D1V-D4V. Combining prediction algorithms and HLA transgenic mice is an effective strategy to identify HLA-restricted epitopes. Serotype-specific epitopes would be used to determine the protective role of serotype-specific CD8+ T cells, while cross-reactive epitopes may provide assistance in exploring the role of serotype cross-reactive CD8+ T cells in the immunopathogenesis of DHF/DSS.

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.

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.

Dengue hemorrhagic fever is characterized by a unique vascular leakage syndrome. The mechanisms of endothelial barrier dysfunction in dengue hemorrhagic fever are not well understood. We examined the modulation of endothelial barrier function in dengue virus type 2 (DENV2) infections using primary human umbilical vein endothelial cells. We demonstrated that the increase in endothelial barrier function within 72 hours after DENV2 infection is mediated by type I interferon–dependent CD73 up-regulation. After 72 hours, DENV2 slowed the recovery of endothelial barrier function in response to tumor necrosis factor-α or vascular endothelial growth factor. This phenomenon was likely caused by type I interferon receptor signaling inhibition and lower CD73 levels in DENV2-infected endothelial cells. Our findings suggest that during DENV2 infection, endothelial barrier homeostasis is maintained by a balance between pro-inflammatory and pro-angiogenic cytokines, and type I interferon–dependent CD73 expression and activity.

The age distribution of cases of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) in infants under the age of 1 year are reported from Bangkok, Thailand, and for the first time for Ho Chi Minh City, Vietnam; Yangon, Myanmar; and Surabaya, Indonesia. The four dengue viruses were isolated from Thai infants, all of whom were having a primary dengue infection. Progress studying the immunologically distinct infant DHF/DSS has been limited; most contemporary research has centered on DHF/DSS accompanying secondary dengue infections. In designing research results obtained in studies on a congruent animal model, feline infectious peritonitis virus (FIPV) infections of kittens born to FIPV-immune queens should be considered. Research challenges presented by infant DHF/DSS are discussed.

Dengue is the most prevalent mosquito-borne human illness worldwide. The ability to predict disease severity during the earliest days of the illness is a long-sought, but unachieved goal.

We examined human genome-wide transcript abundance patterns in daily peripheral blood mononuclear cell (PBMC) samples from 41 children hospitalized with dengue virus (DENV) infection in Nicaragua, as well as 8 healthy control subjects. Nine patients had primary dengue fever (DF1), 11 had dengue fever with serologic evidence of prior DENV infection, i.e., secondary dengue fever (DF2), 12 had dengue hemorrhagic fever (DHF), and 9 had dengue shock syndrome (DSS). We identified 2,092 genes for which transcript abundance differed significantly between patients on days 3–6 of fever and healthy subjects (FDR<1%). Prior DENV infection explained the greatest amount of variation in gene expression among patients. The number of differentially expressed genes was greatest on fever day 3 in patients with DF1, while the number in patients with DF2 or DHF/DSS was greatest on day 5. Genes associated with the mitotic cell cycle and B cell differentiation were expressed at higher levels, and genes associated with signal transduction and cell adhesion were expressed at lower levels, in patients versus healthy controls. On fever day 3, a set of interferon-stimulated gene transcripts was less abundant in patients who subsequently developed DSS than in other patient groups (p<0.05, ranksum). Patients who later developed DSS also had higher levels of transcripts on day 3 associated with mitochondrial function (p<0.01, ranksum). These day 3 transcript abundance findings were not evident on subsequent fever days.

In conclusion, we identified differences in the timing and magnitude of human gene transcript abundance changes in DENV patients that were associated with serologic evidence of prior infection and with disease severity. Some of these differential features may predict the outcome of DENV infection.

Author Summary

Infection with dengue virus (DENV) causes dengue fever, the most prevalent mosquito-borne illness of humans worldwide. Tens of millions of cases occur annually; up to 500,000 patients develop additional life-threatening complications, including hemorrhage and shock. The clinical course of the disease evolves rapidly, making it difficult to identify patients at risk for severe disease and suggesting that biological events associated with the development of severe disease may be short-lived. We examined gene expression patterns in the blood of children hospitalized with DENV infection, and found that patients with differences in disease severity and history of previous DENV infection shared a common set of gene expression features, but the timing and magnitude of these features differed. In our study, prior DENV infection explained the greatest amount of variation in gene expression among patients. We discovered features of gene expression on day 3 that were associated with subsequent disease severity—features that were not apparent on subsequent days, emphasizing the importance of looking at temporal patterns of gene expression in acute infection.

Summary

Dengue virus (DENV) causes disease ranging from dengue fever (DF), a self-limited febrile illness, to the potentially lethal dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). Epidemiological studies have suggested that DHF/DSS usually occurs in patients who, prior to infection, have acquired DENV-reactive antibodies, either from a previous infection with a heterologous DENV serotype or, in the case of infants, passively from an immune mother. Therefore, it has been hypothesized that subneutralizing levels of DENV-specific antibodies exacerbate disease, a phenomenon termed antibody-dependent enhancement (ADE). To date, the mechanism of ADE and its contribution to pathology remain elusive. Here, we demonstrate that administration of DENV-specific antibodies to DENV-infected mice can sufficiently increase severity of disease so that a mostly non-lethal illness becomes a fatal disease resembling human DHF/DSS. Antibodies promote massive infection of liver sinusoidal endothelial cells (LSECs), which results in increased systemic levels of virus. Our findings demonstrate that a subprotective humoral response may, under some circumstances, have pathological consequences.

Background

Despite the seriousness of dengue-related disease, with an estimated 50–100 million cases of dengue fever and 250,000–500,000 cases of dengue hemorrhagic fever/dengue shock syndrome each year, a clear understanding of dengue pathogenesis remains elusive. Because of the lack of a disease model in animals and the complex immune interaction in dengue infection, the study of host response and immunopathogenesis is difficult. The development of genomics technology, microarray and high throughput quantitative PCR have allowed researchers to study gene expression changes on a much broader scale. We therefore used this approach to investigate the host response in dengue virus-infected cell lines and in patients developing dengue fever.

Methodology/Principal Findings

Using microarray and high throughput quantitative PCR method to monitor the host response to dengue viral replication in cell line infection models and in dengue patient blood samples, we identified differentially expressed genes along three major pathways; NF-κB initiated immune responses, type I interferon (IFN) and the ubiquitin proteasome pathway. Among the most highly upregulated genes were the chemokines IP-10 and I-TAC, both ligands of the CXCR3 receptor. Increased expression of IP-10 and I-TAC in the peripheral blood of ten patients at the early onset of fever was confirmed by ELISA. A highly upregulated gene in the IFN pathway, viperin, was overexpressed in A549 cells resulting in a significant reduction in viral replication. The upregulation of genes in the ubiquitin-proteasome pathway prompted the testing of proteasome inhibitors MG-132 and ALLN, both of which reduced viral replication.

Conclusion/Significance

Unbiased gene expression analysis has identified new host genes associated with dengue infection, which we have validated in functional studies. We showed that some parts of the host response can be used as potential biomarkers for the disease while others can be used to control dengue viral replication, thus representing viable targets for drug therapy.

Author Summary

Dengue is the most prevalent mosquito-born viral disease affecting humans, yet there is, at present, no drug treatment for the disease nor are there any validated host targets for therapeutic intervention. Using microarray technology to monitor the response of virtually every human gene, we aimed to identify the ways in which humans interact with dengue virus during infection in order to discover new therapeutic targets that could be exploited to control viral replication. From the activated genes, we identified three pathways common to in vitro and in vivo infection; the NF-κB initiated immune pathway, the type I interferon pathway, and the ubiquitin proteasome pathway. We next found that inhibiting the ubiquitin proteasome pathway, or activating the type I interferon pathway, resulted in significant inhibition of viral replication. However, inhibiting the NF-κB initiated immune pathway had no effect on viral replication. We suggest that drugs that target the ubiquitin proteasome pathway may prove effective at killing the dengue virus, and, if used therapeutically, improve clinical outcome in dengue disease.

Background

Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase) and -related cytokines (IL-4, -9, and -17) between patients with differing severity of Dengue fever and healthy controls.

Methodology/Principal Findings

The study was performed at Children's Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF), Dengue hemorrhagic fever (DHF), and Dengue shock syndrome (DSS), as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9.

Conclusions

As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.

Author Summary

To clarify the involvement of mast cells in the development of severe Dengue diseases, plasma levels of mast cell-derived mediators, namely vascular endothelial cell growth factor (VEGF), tryptase, and chymase, were estimated in Dengue patients and control subjects in Vietnam. The levels of the mediators were significantly increased in Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS) patients compared with those of Dengue fever (DF) and control (febrile illness and healthy subjects) patients, and the soluble form of VEGF receptors (sVEGFR)-1 and -2 levels were significantly changed in the patients with severe disease. After 2–4 days of admission, the mediator levels had returned to similar levels as those of DF and control subjects. Furthermore, the levels of the Th17 cell-derived mast-cell activators IL-9 and -17 were increased in DHF and DSS. In-vitro production of VEGF in human mast cells was significantly enhanced in the presence of IL-9 when these cells were inoculated with Dengue virus in the presence of human Dengue virus-immune serum. As mast cells are an important source of VEGF, and tryptase and chymase are considered to be specific markers for mast cell activation, mast cells and mast cell-derived mediators might participate in the development of DHF/DSS.

Analyses of a prospective case-control study of infant dengue by Daniel Libraty and colleagues casts doubt on the antibody-dependent enhancement model for dengue hemorrhagic fever.

Background

Dengue hemorrhagic fever (DHF) is the severe and life-threatening syndrome that can develop after infection with any one of the four dengue virus (DENV) serotypes. DHF occurs almost exclusively in individuals with secondary heterologous DENV infections and infants with primary DENV infections born to dengue immune mothers. The widely accepted explanation for the pathogenesis of DHF in these settings, particularly during infancy, is antibody-dependent enhancement (ADE) of DENV infection.

Methods and Findings

We conducted a prospective nested case-control study of DENV infections during infancy. Clinical data and blood samples were collected from 4,441 mothers and infants in up to two pre-illness study visits, and surveillance was performed for symptomatic and inapparent DENV infections. Pre-illness plasma samples were used to measure the associations between maternally derived anti-DENV3 antibody-neutralizing and -enhancing capacities at the time of DENV3 infection and development of infant DHF.

The study captured 60 infants with DENV infections across a wide spectrum of disease severity. DENV3 was the predominant serotype among the infants with symptomatic (35/40) and inapparent (15/20) DENV infections, and 59/60 infants had a primary DENV infection. The estimated in vitro anti-DENV3 neutralizing capacity at birth positively correlated with the age of symptomatic primary DENV3 illness in infants. At the time of symptomatic DENV3 infection, essentially all infants had low anti-DENV3 neutralizing activity (50% plaque reduction neutralizing titers [PRNT50] ≤50) and measurable DENV3 ADE activity. The infants who developed DHF did not have significantly higher frequencies or levels of DENV3 ADE activity compared to symptomatic infants without DHF. A higher weight-for-age in the first 3 mo of life and at illness presentation was associated with a greater risk for DHF from a primary DENV infection during infancy.

Conclusions

This prospective nested case-control study of primarily DENV3 infections during infancy has shown that infants exhibit a full range of disease severity after primary DENV infections. The results support an initial in vivo protective role for maternally derived antibody, and suggest that a DENV3 PRNT50 >50 is associated with protection from symptomatic DENV3 illness. We did not find a significant association between DENV3 ADE activity at illness onset and the development of DHF compared with less severe symptomatic illness. The results of this study should encourage rethinking or refinement of the current ADE pathogenesis model for infant DHF and stimulate new directions of research into mechanisms responsible for the development of DHF during infancy.

Trial registration

ClinicalTrials.gov NCT00377754

Please see later in the article for the Editors' Summary

Editors' Summary

Background

Every year, dengue infects 50–100 million people living in tropical and subtropical areas. The four closely related viruses that cause dengue (DENV1–4) are transmitted to people through the bites of female Aedes aegypti mosquitoes, which acquire the viruses by feeding on the blood of an infected person. Many people who become infected with DENV have no symptoms but some develop dengue fever, a severe, flu-like illness that lasts a few days. Other people—about half a million a year—develop a potentially fatal condition called dengue hemorrhagic fever (DHF). In DHF, which can be caused by any of the DENVs, small blood vessels become leaky and friable. This leakiness causes nose and gum bleeds, bruising and, in the worst cases, failure of the circulatory system and death. There is no vaccine to prevent dengue and no specific treatment for dengue fever or DHF. However, with standard medical care—in particular, replacement of lost fluids—most people can survive DHF.

Why Was This Study Done?

DHF is increasingly common, but why do only some people develop DHF after infection with DENV? The widely accepted explanation for the development of DHF is “antibody-dependent enhancement” (ADE) of DENV infection. DHF occurs almost exclusively in two settings; (i) children and adults who become infected with a second DENV serotype after an initial “primary” DENV infection with a different serotype, and (ii) infants with primary DENV infections whose mothers have some DENV immunity. The ADE model suggests that in individuals who develop DHF, although there are some antibodies (proteins made by the immune system to fight infections) against DENV in their blood (in secondary heterologous infections, antibodies left over from the primary infection; in infants with primary infections, antibodies acquired from their mothers before birth), these antibodies cannot “neutralize” the virus. Instead, they bind to it and enhance its uptake by certain immune system cells, thus increasing viral infectivity and triggering an immunological cascade that results in DHF. In this prospective, nested case-control study, the researchers directly test the ADE model for infant DHF. In a prospective study, a group of people is selected and followed to see if they develop a disease; in a nested case-control study, each case is compared with people in the group who do not develop the disease.

What Did the Researchers Do and Find?

The researchers collected clinical data and blood samples from 4,441 mothers and their babies at up to two pre-illness study visits. They then followed the infants for a year to see which of them developed symptomatic and symptom-free DENV infections. Finally, they used the pre-illness blood samples to estimate the maternally derived anti-DENV antibody-neutralizing and -enhancing capacities in the infants at the time of DENV infection. 60 infants were infected with DENV—mainly DENV3—during the study. All but one infection was a primary infection. The infected infants showed a wide range of disease severity. Infants who had a high DENV3 neutralizing capacity at birth tended to develop symptomatic DENV3 infections later than infants who had a low DENV3 neutralizing capacity at birth. All the infants who developed a symptomatic DENV3 infection had a low estimated DENV3 neutralizing activity at the time of infection, and nearly all had measurable levels of DENV3 ADE activity. Infants who developed DHF did not have significantly higher frequencies or levels of DENV3 ADE activity than DENV3-infected infants with less severe symptoms.

What Do These Findings Mean?

These findings indicate that maternally derived anti-DENV3 antibody initially provides protection against dengue infections. That is, babies born to DENV immune mothers are protected against dengue infections by maternally derived antibodies. Over time, the level of these antibodies declines until eventually the infant becomes susceptible to DENV infections. However, the lack of a significant association between the estimated level of DENV3 ADE activity at illness onset and the development of DHF rather than a less severe illness throws doubt onto (but does not completely rule out) the current ADE pathogenesis model for infant DHF, at least for DENV3 infections. The results of this study, the researchers conclude, should encourage rethinking or refinement of the ADE model for infant DHF and should promote further prospective studies into the development of DHF during infancy.

Additional Information

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000171.

TropIKA.net provides review articles, news, opinions, research articles, and reports on dengue (in English)

The US Centers for Disease Control and Prevention provide detailed information about dengue fever and dengue hemorrhagic fever (in English and Spanish)

The World Health Organization provides information on dengue fever and dengue hemorrhagic fever around the world (in several languages)

Links to additional resources about dengue are provided by MedlinePlus (in English and Spanish)

Wikipedia has a page on antibody-dependent enhancement of viral infections (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)

Dengue hemorrhagic fever and/or dengue shock syndrome represent the most serious pathophysiological manifestations of human dengue virus infection. Despite intensive research, the mechanisms and important cellular players that contribute to dengue disease are unclear. Mast cells are tissue-resident innate immune cells that play a sentinel cell role in host protection against infectious agents via pathogen-recognition receptors by producing potent mediators that modulate inflammation, cell recruitment and normal vascular homeostasis. Most importantly, mast cells are susceptible to antibody-enhanced dengue virus infection and respond with selective cytokine and chemokine responses. In order to obtain a global view of dengue virus-induced gene regulation in mast cells, primary human cord blood-derived mast cells (CBMCs) and the KU812 and HMC-1 mast cell lines were infected with dengue virus in the presence of dengue-immune sera and their responses were evaluated at the mRNA and protein levels. Mast cells responded to antibody-enhanced dengue virus infection or polyinosiniċpolycytidylic acid treatment with the production of type I interferons and the rapid and potent production of chemokines including CCL4, CCL5 and CXCL10. Multiple interferon-stimulated genes were also upregulated as well as mRNA and protein for the RNA sensors PKR, RIG-I and MDA5. Dengue virus-induced chemokine production by KU812 cells was significantly modulated by siRNA knockdown of RIG-I and PKR, in a negative and positive manner, respectively. Pretreatment of fresh KU812 cells with supernatants from dengue virus-infected mast cells provided protection from subsequent infection with dengue virus in a type I interferon-dependent manner. These findings support a role for tissue-resident mast cells in the early detection of antibody-enhanced dengue virus infection via RNA sensors, the protection of neighbouring cells through interferon production and the potential recruitment of leukocytes via chemokine production.