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1.
Clin Infect Dis ; 2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33462580

RESUMO

BACKGROUND: Dengue is the most significant mosquito-borne viral disease; there are no specific therapeutics. The antiparasitic drug ivermectin efficiently inhibits the replication of all 4 dengue virus serotypes in vitro. METHODS: We conducted 2 consecutive randomized, double-blind, placebo-controlled trials in adult dengue patients to evaluate safety and virological and clinical efficacies of ivermectin. After a phase 2 trial with 2 or 3 days of 1 daily dose of 400 µg/kg ivermectin, we continued with a phase 3, placebo-controlled trial with 3 days of 400 µg/kg ivermectin. RESULTS: The phase 2 trial showed a trend in reduction of plasma nonstructural protein 1 (NS1) clearance time in the 3-day ivermectin group compared with placebo. Combining phase 2 and 3 trials, 203 patients were included in the intention to treat analysis (100 and 103 patients receiving ivermectin and placebo, respectively). Dengue hemorrhagic fever occurred in 24 (24.0%) of ivermectin-treated patients and 32 (31.1%) patients receiving placebo (P = .260). The median (95% confidence interval [CI]) clearance time of NS1 antigenemia was shorter in the ivermectin group (71.5 [95% CI 59.9-84.0] hours vs 95.8 [95% CI 83.9-120.0] hours, P = .014). At discharge, 72.0% and 47.6% of patients in the ivermectin and placebo groups, respectively had undetectable plasma NS1 (P = .001). There were no differences in the viremia clearance time and incidence of adverse events between the 2 groups. CONCLUSIONS: A 3-day 1 daily dose of 400 µg/kg oral ivermectin was safe and accelerated NS1 antigenemia clearance in dengue patients. However, clinical efficacy of ivermectin was not observed at this dosage regimen.

2.
PLoS Negl Trop Dis ; 14(11): e0008835, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33216752

RESUMO

Suitable cell models are essential to advance our understanding of the pathogenesis of liver diseases and the development of therapeutic strategies. Primary human hepatocytes (PHHs), the most ideal hepatic model, are commercially available, but they are expensive and vary from lot-to-lot which confounds their utility. We have recently developed an immortalized hepatocyte-like cell line (imHC) from human mesenchymal stem cells, and tested it for use as a substitute model for hepatotropic infectious diseases. With a special interest in liver pathogenesis of viral infection, herein we determined the suitability of imHC as a host cell target for dengue virus (DENV) and as a model for anti-viral drug testing. We characterized the kinetics of DENV production, cellular responses to DENV infection (apoptosis, cytokine production and lipid droplet metabolism), and examined anti-viral drug effects in imHC cells with comparisons to the commonly used hepatoma cell lines (HepG2 and Huh-7) and PHHs. Our results showed that imHC cells had higher efficiencies in DENV replication and NS1 secretion as compared to HepG2 and Huh-7 cells. The kinetics of DENV infection in imHC cells showed a slower rate of apoptosis than the hepatoma cell lines and a certain similarity of cytokine profiles to PHHs. In imHC, DENV-induced alterations in levels of lipid droplets and triacylglycerols, a major component of lipid droplets, were more apparent than in hepatoma cell lines, suggesting active lipid metabolism in imHC. Significantly, responses to drugs with DENV inhibitory effects were greater in imHC cells than in HepG2 and Huh-7 cells. In conclusion, our findings suggest superior suitability of imHC as a new hepatocyte model for studying mechanisms underlying viral pathogenesis, liver diseases and drug effects.

3.
J Gen Virol ; 101(1): 59-72, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31682220

RESUMO

Dengue virus assembly involves the encapsidation of genomic RNA by the capsid protein (C) and the acquisition of an envelope comprising the premembrane (prM) and envelope (E) glycoproteins. This rapid process, lacking in detectable nucleocapsid intermediates, may impose authentic C-prM-E arrangement as a prerequisite for efficient particle assembly. A mosquito cell-based complementation system was employed in this study to investigate the possibility that expression of the three structural proteins in trans allows the efficient production of a partially C-deleted dengue virus as compared to the presence of C alone. Following the transfection of ΔC56-capped RNA transcripts into C6/36 cells transiently expressing C or CprME, the production of the single-cycle virus was comparable. Subsequent propagation in the stable CprME-expressing clone, however, supported virus adaptation leading to acquisition of the L29P and S101F (PF) dual mutations in the C protein. The triple mutant, ΔC56(PF), exhibited enhanced levels of virus replication, specific infectivity and frequent increases of intracellular C dimer, as compared with ΔC56 in the CprME-clone. The PF mutations were associated with the accumulation of truncated CprM in ΔC56(PF)-infected cells, and uncleaved CprM as well as reduced intracellular C-dimer when the dual mutations were introduced into the wild-type dengue virus genetic background. These results indicate that the PF mutations may exert a replication-enhancing effect for the triple mutant virus by relieving the interference of trans-complementing structural proteins during viral assembly and suggest that the C-prM-E arrangement may be advantageous for pseudoinfectious virus production.


Assuntos
Vírus da Dengue/genética , Nucleocapsídeo/genética , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Montagem de Vírus/genética , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Chlorocebus aethiops , Culicidae/virologia , Dengue/virologia , RNA Viral/genética , Células Vero , Replicação Viral/genética
4.
Trop Med Infect Dis ; 4(2)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013708

RESUMO

Recent advances in electron microscopy and tomography have revealed distinct virus-induced endoplasmic reticulum (ER) structures unique for dengue virus (DV) and other flaviviruses in cell culture models, including hepatocytes. These altered ultrastructures serve as sites for viral replication. In this study, we used transmission electron microscopy to investigate whether such structures were present in the liver of fatal dengue hemorrhagic fever (DHF) autopsy cases. In parallel, electron microscopic examination of suckling mouse brains experimentally infected with DV was performed as an in vivo model of acute DV infection. Typical features of ER changes containing abundance of replicative virions were observed in neurons and microglia of DV-infected suckling mouse brains (SMB). This indicated that the in vivo DV infection could induce similar viral replication structures as previously described in the in vitro DV-infected cell model. Nevertheless, liver tissues from autopsy of patients who died of DHF showed scant changes of ER membrane structures and rare particles of virions in hepatocytes, despite overwhelming evidence for the presence of viral antigens and RNA-indicating active virus replication. Instead hepatocytes contained an abundance of steatotic vesicles and structural damages. This lack of structural changes indicative of virus replication in human hepatocytes is discussed.

5.
J Immunol ; 197(10): 4053-4065, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27798151

RESUMO

Flavivirus nonstructural protein 1 (NS1) is a unique secreted nonstructural glycoprotein. Although it is absent from the flavivirus virion, intracellular and extracellular forms of NS1 have essential roles in viral replication and the pathogenesis of infection. The fate of NS1 in insect cells has been more controversial, with some reports suggesting it is exclusively cell associated. In this study, we confirm NS1 secretion from cells of insect origin and characterize its physical, biochemical, and functional properties in the context of dengue virus (DENV) infection. Unlike mammalian cell-derived NS1, which displays both high mannose and complex type N-linked glycans, soluble NS1 secreted from DENV-infected insect cells contains only high mannose glycans. Insect cell-derived secreted NS1 also has different physical properties, including smaller and more heterogeneous sizes and the formation of less stable NS1 hexamers. Both mammalian and insect cell-derived NS1 bind to complement proteins C1s, C4, and C4-binding protein, as well as to a novel partner, mannose-binding lectin. Binding of NS1 to MBL protects DENV against mannose-binding lectin-mediated neutralization by the lectin pathway of complement activation. As we detected secreted NS1 and DENV together in the saliva of infected Aedes aegypti mosquitoes, these findings suggest a mechanism of viral immune evasion at the very earliest phase of infection.


Assuntos
Lectina de Ligação a Manose da Via do Complemento , Vírus da Dengue/imunologia , Evasão da Resposta Imune , Lectina de Ligação a Manose/imunologia , Lectina de Ligação a Manose/metabolismo , Proteínas não Estruturais Virais/metabolismo , Aedes/virologia , Animais , Linhagem Celular , Ativação do Complemento , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Vírus da Dengue/patogenicidade , Humanos , Ligação Proteica , Saliva/virologia , Suínos , Proteínas não Estruturais Virais/química
6.
J Virol ; 89(3): 1587-607, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25410854

RESUMO

UNLABELLED: Shedding of microparticles (MPs) is a consequence of apoptotic cell death and cellular activation. Low levels of circulating MPs in blood help maintain homeostasis, whereas increased MP generation is linked to many pathological conditions. Herein, we investigated the role of MPs in dengue virus (DENV) infection. Infection of various susceptible cells by DENV led to apoptotic death and MP release. These MPs harbored a viral envelope protein and a nonstructural protein 1 (NS1) on their surfaces. Ex vivo analysis of clinical specimens from patients with infections of different degrees of severity at multiple time points revealed that MPs generated from erythrocytes and platelets are two major MP populations in the circulation of DENV-infected patients. Elevated levels of red blood cell-derived MPs (RMPs) directly correlated with DENV disease severity, whereas a significant decrease in platelet-derived MPs was associated with a bleeding tendency. Removal by mononuclear cells of complement-opsonized NS1-anti-NS1 immune complexes bound to erythrocytes via complement receptor type 1 triggered MP shedding in vitro, a process that could explain the increased levels of RMPs in severe dengue. These findings point to the multiple roles of MPs in dengue pathogenesis. They offer a potential novel biomarker candidate capable of differentiating dengue fever from the more serious dengue hemorrhagic fever. IMPORTANCE: Dengue is the most important mosquito-transmitted viral disease in the world. No vaccines or specific treatments are available. Rapid diagnosis and immediate treatment are the keys to achieve a positive outcome. Dengue virus (DENV) infection, like some other medical conditions, changes the level and composition of microparticles (MPs), tiny bag-like structures which are normally present at low levels in the blood of healthy individuals. This study investigated how MPs in culture and patients' blood are changed in response to DENV infection. Infection of cells led to programmed cell death and MP release. In patients' blood, the majority of MPs originated from red blood cells and platelets. Decreased platelet-derived MPs were associated with a bleeding tendency, while increased levels of red blood cell-derived MPs (RMPs) correlated with more severe disease. Importantly, the level of RMPs during the early acute phase could serve as a biomarker to identify patients with potentially severe disease who require immediate care.


Assuntos
Biomarcadores/sangue , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/metabolismo , Dengue/patologia , Adulto , Animais , Apoptose , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Prognóstico , Proteínas do Envelope Viral/análise , Proteínas não Estruturais Virais/análise
7.
Elife ; 2: e00767, 2013 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-23638302

RESUMO

Immune cells called mast cells can hinder rather than help the body's response to dengue virus, which suggests that mast cell products could be used as biomarkers to identify severe forms of the disease.


Assuntos
Dengue/imunologia , Mastócitos/imunologia , Humanos
8.
mBio ; 2(6)2011.
Artigo em Inglês | MEDLINE | ID: mdl-22167226

RESUMO

Mannose-binding lectin (MBL) is a key soluble pathogen recognition protein of the innate immune system that binds specific mannose-containing glycans on the surfaces of microbial agents and initiates complement activation via the lectin pathway. Prior studies showed that MBL-dependent activation of the complement cascade neutralized insect cell-derived West Nile virus (WNV) in cell culture and restricted pathogenesis in mice. Here, we investigated the antiviral activity of MBL in infection by dengue virus (DENV), a related flavivirus. Using a panel of naïve sera from mouse strains deficient in different complement components, we showed that inhibition of infection by insect cell- and mammalian cell-derived DENV was primarily dependent on the lectin pathway. Human MBL also bound to DENV and neutralized infection of all four DENV serotypes through complement activation-dependent and -independent pathways. Experiments with human serum from naïve individuals with inherent variation in the levels of MBL in blood showed a direct correlation between the concentration of MBL and neutralization of DENV; samples with high levels of MBL in blood neutralized DENV more efficiently than those with lower levels. Our studies suggest that allelic variation of MBL in humans may impact complement-dependent control of DENV pathogenesis. IMPORTANCE Dengue virus (DENV) is a mosquito-transmitted virus that causes a spectrum of clinical disease in humans ranging from subclinical infection to dengue hemorrhagic fever and dengue shock syndrome. Four serotypes of DENV exist, and severe illness is usually associated with secondary infection by a different serotype. Here, we show that mannose-binding lectin (MBL), a pattern recognition molecule that initiates the lectin pathway of complement activation, neutralized infection of all four DENV serotypes through complement activation-dependent and -independent pathways. Moreover, we observed a direct correlation with the concentration of MBL in human serum and neutralization of DENV infection. Our studies suggest that common genetic polymorphisms that result in disparate levels and function of MBL in humans may impact DENV infection, pathogenesis, and disease severity.


Assuntos
Proteínas do Sistema Complemento/imunologia , Vírus da Dengue/imunologia , Lectina de Ligação a Manose/imunologia , Inativação de Vírus , Animais , Ativação do Complemento , Humanos , Camundongos , Testes de Neutralização
9.
J Immunol ; 187(1): 424-33, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21642539

RESUMO

The complement system plays a pivotal protective role in the innate immune response to many pathogens including flaviviruses. Flavivirus nonstructural protein 1 (NS1) is a secreted nonstructural glycoprotein that accumulates in plasma to high levels and is displayed on the surface of infected cells but absent from viral particles. Previous work has defined an immune evasion role of flavivirus NS1 in limiting complement activation by forming a complex with C1s and C4 to promote cleavage of C4 to C4b. In this study, we demonstrate a second mechanism, also involving C4 and its active fragment C4b, by which NS1 antagonizes complement activation. Dengue, West Nile, or yellow fever virus NS1 directly associated with C4b binding protein (C4BP), a complement regulatory plasma protein that attenuates the classical and lectin pathways. Soluble NS1 recruited C4BP to inactivate C4b in solution and on the plasma membrane. Mapping studies revealed that the interaction sites of NS1 on C4BP partially overlap with the C4b binding sites. Together, these studies further define the immune evasion potential of NS1 in reducing the functional capacity of C4 in complement activation and control of flavivirus infection.


Assuntos
Ativação do Complemento/imunologia , Flavivirus/imunologia , Antígenos de Histocompatibilidade/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Linhagem Celular , Complemento C4b/antagonistas & inibidores , Complemento C4b/metabolismo , Proteína de Ligação ao Complemento C4b , Cricetinae , Vírus da Dengue/imunologia , Vírus da Dengue/patogenicidade , Flavivirus/patogenicidade , Antígenos de Histocompatibilidade/metabolismo , Humanos , Ligação Proteica/imunologia , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/patogenicidade , Vírus da Febre Amarela/imunologia , Vírus da Febre Amarela/patogenicidade
10.
Virology ; 413(2): 253-64, 2011 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-21429549

RESUMO

Dengue virus (DENV) NS1 is a versatile non-structural glycoprotein that is secreted as a hexamer, binds to the cell surface of infected and uninfected cells, and has immune evasive functions. DENV NS1 displays two conserved N-linked glycans at N130 and N207. In this study, we examined the role of these two N-linked glycans on NS1 secretion, stability, and function. Because some groups have reported reduced yields of infectious DENV when N130 and N207 are changed, we analyzed glycosylation-deficient NS1 phenotypes using a transgenic expression system. We show that the N-linked glycan at position 130 is required for stabilization of the secreted hexamer whereas the N-linked glycan at residue 207 facilitates secretion and extracellular protein stability. Moreover, NS1 mutants lacking an N-linked glycan at N130 did not interact efficiently with complement components C1s and C4. In summary, our results elucidate the contribution of N-linked glycosylation to the function of DENV NS1.


Assuntos
Proteínas do Sistema Complemento/metabolismo , Vírus da Dengue/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Proteínas não Estruturais Virais/metabolismo , Vírus da Dengue/classificação , Glicosilação , Humanos , Mutação , Polissacarídeos , Vírus Sindbis , Proteínas não Estruturais Virais/genética , Eliminação de Partículas Virais
11.
J Exp Med ; 207(4): 793-806, 2010 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-20308361

RESUMO

The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection.


Assuntos
Complemento C4/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Animais , Biocatálise/efeitos dos fármacos , Células CHO , Complemento C1/metabolismo , Proteína Inibidora do Complemento C1/metabolismo , Complemento C1s/agonistas , Complemento C1s/antagonistas & inibidores , Complemento C1s/metabolismo , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Complemento C4/metabolismo , Complemento C4b/metabolismo , Fator I do Complemento/metabolismo , Ensaio de Atividade Hemolítica de Complemento , Via Clássica do Complemento/efeitos dos fármacos , Via Clássica do Complemento/imunologia , Lectina de Ligação a Manose da Via do Complemento/efeitos dos fármacos , Lectina de Ligação a Manose da Via do Complemento/imunologia , Cricetinae , Cricetulus , Vírus da Dengue/imunologia , Precursores Enzimáticos/metabolismo , Cobaias , Humanos , Cinética , Testes de Neutralização , Ligação Proteica/imunologia , Proteínas não Estruturais Virais/farmacologia
12.
Biochem Biophys Res Commun ; 379(2): 196-200, 2009 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-19105951

RESUMO

Dengue virus infection is an important mosquito-borne disease and a public health problem worldwide. A better understanding of interactions between human cellular host and dengue virus proteins will provide insight into dengue virus replication and cellular pathogenesis. The glycosylated envelope protein of dengue virus, DENV E, is processed in the endoplasmic reticulum of host cells and therefore reliant on host processing functions. The complement of host ER functions involved and nature of the interactions with DENV E has not been thoroughly investigated. By employing a yeast two-hybrid assay, we found that domain III of DENV E interacts with human immunoglobulin heavy chain binding protein (BiP). The relevance of this interaction was demonstrated by co-immunoprecipitation and co-localization of BiP and DENV E in dengue virus-infected cells. Using the same approach, association of DENV E with two other chaperones, calnexin and calreticulin was also observed. Knocking-down expression of BiP, calnexin, or calreticulin by siRNA significantly decreased the production of infectious dengue virions. These results indicate that the interaction of these three chaperones with DENV E plays an important role in virion production, likely facilitating proper folding and assembly of dengue proteins.


Assuntos
Vírus da Dengue/fisiologia , Dengue/virologia , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Calnexina/genética , Calnexina/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Chlorocebus aethiops , Dengue/genética , Dengue/metabolismo , Vírus da Dengue/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , Técnicas do Sistema de Duplo-Híbrido , Células Vero
13.
J Gen Virol ; 89(Pt 10): 2492-2500, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18796718

RESUMO

During the replication of dengue virus, a viral non-structural glycoprotein, NS1, associates with the membrane on the cell surface and in the RNA replication complex. NS1 lacks a transmembrane domain, and the mechanism by which it associates with the membrane remains unclear. This study aimed to investigate whether membrane-bound NS1 is present in lipid rafts in dengue virus-infected cells. Double immunofluorescence staining of infected HEK-293T cells revealed that NS1 localized with raft-associated molecules, ganglioside GM1 and CD55, on the cell surface. In a flotation gradient centrifugation assay, a small proportion of NS1 in Triton X-100 cell lysate consistently co-fractionated with raft markers. Association of NS1 with lipid rafts was detected for all four dengue serotypes, as well as for Japanese encephalitis virus. Analysis of recombinant NS1 forms showed that glycosylated NS1 dimers stably expressed in HEK-293T cells without an additional C-terminal sequence, or with a heterologous transmembrane domain, failed to associate with lipid rafts. In contrast, glycosylphosphatidylinositol-linked recombinant NS1 exhibited a predilection for lipid rafts. These results indicate an association of a minor subpopulation of NS1 with lipid rafts during dengue virus infection and suggest that modification of NS1, possibly lipidation, is required for raft association.


Assuntos
Antígenos CD55/metabolismo , Vírus da Dengue/patogenicidade , Células Epiteliais/virologia , Gangliosídeo G(M1)/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Centrifugação com Gradiente de Concentração , Vírus da Dengue/classificação , Vírus da Dengue/metabolismo , Células Epiteliais/metabolismo , Imunofluorescência , Humanos
14.
Vaccine ; 26 Suppl 8: I100-7, 2008 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-19388173

RESUMO

The complement system is a family of serum and cell surface proteins that recognize pathogen-associated molecular patterns, altered-self ligands, and immune complexes. Activation of the complement cascade triggers several antiviral functions including pathogen opsonization and/or lysis, and priming of adaptive immune responses. In this review, we will examine the role of complement activation in protection and/or pathogenesis against infection by Flaviviruses, with an emphasis on experiments with West Nile and Dengue viruses.


Assuntos
Proteínas do Sistema Complemento/fisiologia , Infecções por Flavivirus/imunologia , Ativação do Complemento , Infecções por Flavivirus/etiologia , Humanos , Imunidade Inata
15.
PLoS Pathog ; 3(11): e183, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18052531

RESUMO

Dengue virus (DENV) nonstructural protein-1 (NS1) is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection.


Assuntos
Sulfatos de Condroitina/metabolismo , Vírus da Dengue/metabolismo , Heparitina Sulfato/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Camundongos , Microscopia Confocal , Proteínas não Estruturais Virais/imunologia
16.
Biochem Biophys Res Commun ; 362(2): 334-9, 2007 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-17707345

RESUMO

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are important public health problems in tropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF. However, hepatomegaly, hepatocellular necrosis and fulminant hepatic failure are occasionally observed in patients with DHF. Dengue virus-infected liver cells undergo apoptosis but the underlying molecular mechanism remains unclear. Using a yeast two-hybrid screen, we found that dengue virus capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx, a Fas-associated protein. The interaction between DENV C and Daxx in dengue virus-infected liver cells was also demonstrated by co-immunoprecipitation and double immunofluorescence staining. The two proteins were predominantly co-localized in the cellular nuclei. Fas-mediated apoptotic activity in liver cells constitutively expressing DENV C was induced by anti-Fas antibody, indicating that the interaction of DENV C and Daxx involves in apoptosis of dengue virus-infected liver cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proteínas do Capsídeo/metabolismo , Proteínas Nucleares/metabolismo , Receptor fas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Western Blotting , Proteínas do Capsídeo/genética , Linhagem Celular Tumoral , Proteínas Correpressoras , Fragmentação do DNA , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Citometria de Fluxo , Humanos , Imunoprecipitação , Microscopia Confocal , Chaperonas Moleculares , Proteínas Nucleares/genética , Plasmídeos/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Receptor fas/genética
17.
J Immunol ; 176(6): 3821-9, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16517753

RESUMO

Dengue virus infection poses a growing public health and economic burden in a number of tropical and subtropical countries. Dengue circulates as a number of quasispecies, which can be divided by serology into four groups or serotypes. An interesting feature of Dengue, recognized over five decades ago, is that most severe cases that show hemorrhagic fever are not suffering from a primary infection. Instead, they are reinfected with a virus of different serotype. This observation poses considerable problems in vaccine design, and it is therefore imperative to gain a full understanding of the mechanisms underlying this immunological enhancement of disease. In this study, we examined a T cell epitope restricted by HLA-A*24, a major MHC class I allele, in Southeast Asia in a cohort of children admitted to a hospital with acute Dengue infection. The cytokine profiles and the degranulation capacity of T cells generated to this epitope are defined and compared across different viral serotypes. Cross-reactive Dengue-specific T cells seem to show suboptimal degranulation but high cytokine production, which may contribute to the development of the vascular leak characteristic of Dengue hemorrhagic fever.


Assuntos
Vírus da Dengue/imunologia , Dengue Grave/imunologia , Dengue Grave/virologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Células Cultivadas , Reações Cruzadas/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Antígeno HLA-A1/química , Antígeno HLA-A1/imunologia , Antígeno HLA-A2/imunologia , Humanos , Modelos Moleculares , Fenótipo , Estrutura Quaternária de Proteína , Linfócitos T/química
18.
J Infect Dis ; 193(8): 1078-88, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16544248

RESUMO

BACKGROUND: Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown. METHODS: The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patients with DSS were also analyzed. RESULTS: Soluble NS1 activated complement to completion, and activation was enhanced by polyclonal and monoclonal antibodies against NS1. Complement was also activated by cell-associated NS1 in the presence of specific antibodies. Plasma levels of NS1 and terminal SC5b-9 complexes correlated with disease severity. Large amounts of NS1, complement anaphylatoxin C5a, and the terminal complement complex SC5b-9 were present in pleural fluids from patients with DSS. CONCLUSIONS: Complement activation mediated by NS1 leads to local and systemic generation of anaphylatoxins and SC5b-9, which may contribute to the pathogenesis of the vascular leakage that occurs in patients with DHF/DSS.


Assuntos
Proteínas do Sistema Complemento/fisiologia , Vírus da Dengue/fisiologia , Dengue/fisiopatologia , Doenças Vasculares/virologia , Proteínas não Estruturais Virais/fisiologia , Adolescente , Anticorpos Antivirais/imunologia , Estudos de Casos e Controles , Linhagem Celular , Criança , Pré-Escolar , Complemento C5a/análise , Complexo de Ataque à Membrana do Sistema Complemento , Proteínas do Sistema Complemento/análise , Feminino , Glicoproteínas/análise , Glicoproteínas/fisiologia , Humanos , Masculino , Cavidade Pleural/química , RNA Viral/análise , Carga Viral , Proteínas não Estruturais Virais/análise
19.
Nat Genet ; 37(5): 507-13, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15838506

RESUMO

Dengue fever and dengue hemorrhagic fever are mosquito-borne viral diseases. Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN1, encoded by CD209), an attachment receptor of dengue virus, is essential for productive infection of dendritic cells. Here, we report strong association between a promoter variant of CD209, DCSIGN1-336, and risk of dengue fever compared with dengue hemorrhagic fever or population controls. The G allele of the variant DCSIGN1-336 was associated with strong protection against dengue fever in three independent cohorts from Thailand, with a carrier frequency of 4.7% in individuals with dengue fever compared with 22.4% in individuals with dengue hemorrhagic fever (odds ratio for risk of dengue hemorrhagic fever versus dengue fever: 5.84, P = 1.4 x 10(-7)) and 19.5% in controls (odds ratio for protection: 4.90, P = 2 x 10(-6)). This variant affects an Sp1-like binding site and transcriptional activity in vitro. These results indicate that CD209 has a crucial role in dengue pathogenesis, which discriminates between severe dengue fever and dengue hemorrhagic fever. This may have consequences for therapeutic and preventive strategies.


Assuntos
Moléculas de Adesão Celular/genética , Dengue/genética , Lectinas Tipo C/genética , Regiões Promotoras Genéticas , Receptores de Superfície Celular/genética , Índice de Gravidade de Doença , Dengue/fisiopatologia , Humanos , Polimorfismo Genético
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