Differential proinflammatory and angiogenesis-specific cytokine production in human pulmonary endothelial cells, HPMEC-ST1.6R infected with dengue-2 and dengue-3 virus

In this study, the ability of dengue virus serotypes 2 (DENV-2) and 3 (DENV-3) to infect and induce increased production of proinflammatory cytokines in a pulmonary endothelial cell line (HPMEC-ST1.6R) was investigated. This cell line exhibits the major constitutive and inducible endothelial cell characteristics, as well as angiogenic response. DENV-2 and DENV-3 infection was confirmed by an observed cytopathic effect (CPE), as well as RT-PCR and immunofluorescence assays. Increases in Th-1 and Th-2 cytokines IL-4, IL-8, IL-6, IL-10, GM-CSF, INF-gamma, and tumor necrosis factor (TNF-alpha) within DENV-2- and DENV-3-infected cells were demonstrated using a microbead-based Bio-plex assay. Proinflammatory cytokine increases and the expression of a potent angiogenic inducer protein, VEGF were confirmed by dot-blot analysis using the TranSignal Human Angiogenesis Antibody Array. Dengue virus-infected HPMEC-ST1.6R cells exhibited an elongated cytoplasmic morphology, possibly representing a response to VEGF and activation of angiogenesis. The increased levels of Th-1 cytokines and VEGF in DENV-2 virus infected-HPMEC-ST1.6R could be distinguished from those infected by DENV-3. This suggests that cytokine patterns associated with DENV infections may be serotype and strain-specific. The experimental approaches described here could be developed further into a useful diagnostic tool for the characterization of dengue hemorrhagic fever cases, leading to enhancement of treatment therapy.     

Phylogenetic and evolutionary analyses of dengue viruses isolated in Jakarta,Indonesia

Dengue has affected Indonesia for the last five decades and become a major health problem in many cities in the country. Jakarta, the capital of Indonesia, reports dengue cases annually, with several outbreaks documented. To gain information on the dynamic and evolutionary history of dengue virus (DENV) in Jakarta, we conducted phylogenetic and evolutionary analyses of DENV isolated in 2009. Three hundred thirty-three dengue-suspected patients were recruited. Our data revealed that dengue predominantly affected young adults, and the majority of cases were due to secondary infection. A total of 171 virus isolates were successfully serotyped. All four DENV serotypes were circulating in the city, and DENV-1 was the predominant serotype. The DENV genotyping of 17 isolates revealed the presence of Genotypes I and IV in DENV-1, while DENV-2 isolates were grouped into the Cosmopolitan genotype. The grouping of isolates into Genotype I and II was seen for DENV-3 and DENV-4, respectively. Evolutionary analysis revealed the relatedness of Jakarta isolates with other isolates from other cities in Indonesia and isolates from imported cases in other countries. We revealed the endemicity of DENV and the role of Jakarta as the potential source of imported dengue cases in other countries. Our study provides genetic information regarding DENV from Jakarta, which will be useful for upstream applications, such as the study of DENV epidemiology and evolution and transmission dynamics.     

Endothelial dysfunction in dengue virus pathology

Dengue virus (DENV) is a leading cause of illness and death, mainly in the (sub)tropics, where it causes dengue fever and/or the more serious diseases dengue hemorrhagic fever and dengue shock syndrome that are associated with changes in vascular permeability. Despite extensive research, the pathogenesis of DENV is still poorly understood and, although endothelial cells represent the primary fluid barrier of the blood vessels, the extent to which these cells contribute to DENV pathology is still under debate. The primary target cells for DENV are dendritic cells and monocytes/macrophages that release various chemokines and cytokines upon infection, which can activate the endothelium and are thought to play a major role in DENV‐induced vascular permeability. However, recent studies indicate that DENV also replicates in endothelial cells and that DENV‐infected endothelial cells may directly contribute to viremia, immune activation, vascular permeability and immune targeting of the endothelium. Also, the viral non‐structural protein‐1 and antibodies directed against this secreted protein have been reported to be involved in endothelial cell dysfunction. This review provides an extensive overview of the effects of DENV infection on endothelial cell physiology and barrier function. Copyright © 2014 John Wiley & Sons, Ltd.     

In vitro assessment of human endothelial cell permeability: effects of inflammatory cytokines and dengue virus infection

Electrical resistance across human umbilical vein endothelial cells (HUVECs) was measured using an electrical cell sensor system. The transendothelial electrical resistance (TEER) value was used to estimate the permeability through endothelial cells in vitro. Decrease in the TEER value was associated with increase in the passage of albumin through endothelial cells in the albumin permeability assay. The effects of cytokines and dengue virus infection on the permeability of HUVECs were examined by measuring the TEER value. Tumor necrosis factor alpha (TNF-alpha) at 1 and 0.1 microg/ml decreased the TEER value, but TNF-alpha at lower dose did not. Interferon-gamma (IFN-gamma) at 1 microg/ml also decreased the TEER value. In contrast, interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) or interferon-beta (IFN-beta) did not decrease the TEER value. The decrease in the TEER value was associated with the morphological changes of HUVECs. Dengue virus infection at a multiplicities of infection (m.o.i.) of 5 pfu/cell decreased the TEER value. Infection at an m.o.i. of 0.5 pfu/cell did not decrease the TEER value; however, addition of 0.01 microg/ml of TNF-alpha to these infected endothelial cells decreased the TEER value. The results suggest that TNF-alpha and dengue virus infection decrease synergistically the TEER value of endothelial cells. The TEER method is easy, reliable and can be applicable to further analysis of the increase in the permeability of endothelial cells in vitro induced by inflammatory cytokines and dengue virus infection.     

Apoptosis in tissues from fatal dengue shock syndrome.

BACKGROUND: Apoptosis, or programmed cell death, has been implicated in dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) pathogenesis. OBJECTIVES: To determine the in vivo apoptosis contribution to the pathogenesis of fatal DHF/DSS during a Cuban dengue epidemic. STUDY DESIGN: We detected apoptosis by the TdT-mediated dUTP Nick-End Labeling (TUNEL) technique and dengue virus (DENV) antigens by an immunohistochemical assay in different tissues from six individuals who died of DHF/DSS during the Santiago de Cuba DENV-2 epidemic in 1997. RESULTS: DENV antigens were immunolocalized mainly in hepatocytes. Apoptotic cells were found in five of the six cases studied. Apoptosis was demonstrated in liver, brain, intestinal and lung tissues. Severe brain hypoxia and ischemia in the studied subjects during DHF/DSS probably might induce apoptosis in cerebral cells. Apoptotic microvascular endothelial cells (ECs) in pulmonary and intestinal tissues, a finding only previously reported in vitro, are likely related to vascular plasma leakage manifested by the individuals. CONCLUSIONS: Apoptosis was demonstrated in cerebral cells, white blood cells, intestinal and pulmonary microvascular ECs from Cuban fatal cases of DHF/DSS. As far as we know, these findings have not been previously reported in DHF/DSS. Our results indicate there is very likely an in vivo contribution of apoptosis to the pathophysiological mechanisms of DHF/DSS.     

登革病毒对人血管内皮细胞分泌ET-1及PGI2的影响

目的　研究登革病毒感染对人血管内皮细胞分泌重要的血管活性物质ＥＴ１ 及ＰＧＩ２ 的影响,以了解登革出血热及登革休克综合征（ＤＨＦＤＳＳ）的发病机制。方法　用登革病毒Ⅱ型,感染人脐静脉内皮细胞（ＨＵＶＥＣ） ,于感染后４ 、２４ 、４８ 、７２ 及９６ 小时,分别收集病毒感染上清液,用放射免疫检测法测定ＥＴ１ 及ＰＧＩ２ 的含量。结果　登革病毒感染可使ＨＵＶＥＣ分泌ＥＴ１ 及ＰＧＩ２ 的能力受到明显抑制。在病毒感染早期（４ 小时）,ＨＵＶＥＣ分泌ＥＴ１ 及ＰＧＩ２ 的能力即受到明显抑制。登革病毒对ＨＵＶＥＣ分泌ＥＴ１ 抑制作用强烈而持久,至感染后９６ 小时,ＨＵＶＥＣ分泌ＥＴ１ 的能力与未受感染的阴性对照组比较,差异仍有显著性。然而,登革病毒对ＨＵＶＥＣ 分泌ＰＧＩ２ 的抑制作用,可随时间的推移而减弱,至感染后９６ 小时,ＨＵＶＥＣ分泌ＰＧＩ２ 的能力已达正常水平。结论　登革病毒感染可影响血管内皮细胞分泌血管活性物质ＥＴ１ 及ＰＧＩ２ 的功能,导致血管通透性增加和凝血、止血功能障碍。因此,登革病毒所致的血管内皮细胞功能障碍,可能是ＤＨＦＤＳＳ重要的发病机制     

Primary human endothelial cells support direct but not antibody‐dependent enhancement of dengue viral infection

Microvascular plasma leakage is the hallmark of dengue hemorrhagic fever and dengue shock syndrome. The precise molecular mechanisms leading to microvascular leakage are yet to be determined, but dengue virus (DENV) infection and consequent endothelial cell death has been suggested as its major cause. However, the extent of endothelial cell permissiveness to DENV infection and the magnitude of cell death following DENV infection are controversial. To clarify this issue, we analyzed the kinetics and consequences of DENV infection of human umbilical vein endothelial cells (HUVEC) using a novel molecularly cloned DENV2‐16681 virus. Viral replication was detected as early as 24 hr post‐infection by RT‐PCR and plaque assays. However, merely 2% of HUVEC were DENV antigen‐positive even after 96 hr of infection as measured by the FACS indirect immunofluorescence assays. Unlike monocytes/macrophages, HUVEC did not support antibody dependent enhancement of dengue viral infection due to a lack of FcγRI and FcγRII. Furthermore, DENV infection did not increase HUVEC apoptosis as compared to mock‐infected cells. Because in vitro only a small percentage of endothelial cells were productively infected in vitro with no significant apoptosis occurring in either infected or bystander cells, it would be important to re‐examine whether direct dengue viral infection of endothelium is the major cause of the extensive vascular leakage observed in patients with dengue hemorrhagic fever and dengue shock syndrome. J. Med. Virol. 81:519–528, 2009. © 2009 Wiley‐Liss, Inc.     

Antibodies from patients with dengue viral infection mediate cellular cytotoxicity.

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

Inhibition of Dengue Virus Entry into Target Cells Using Synthetic Antiviral Peptides

Despite the importance of DENV as a human pathogen, there is no specific treatment or protective vaccine. Successful entry into the host cells is necessary for establishing the infection. Recently, the virus entry step has become an attractive therapeutic strategy because it represents a barrier to suppress the onset of the infection. Four putative antiviral peptides were designed to target domain III of DENV-2 E protein using BioMoDroid algorithm. Two peptides showed significant inhibition of DENV when simultaneously incubated as shown by plaque formation assay, RT-qPCR, and Western blot analysis. Both DET4 and DET2 showed significant inhibition of virus entry (84.6% and 40.6% respectively) using micromolar concentrations. Furthermore, the TEM images showed that the inhibitory peptides caused structural abnormalities and alteration of the arrangement of the viral E protein, which interferes with virus binding and entry. Inhibition of DENV entry during the initial stages of infection can potentially reduce the viremia in infected humans resulting in prevention of the progression of dengue fever to the severe life-threatening infection, reduce the infected vector numbers, and thus break the transmission cycle. Moreover these peptides though designed against the conserved region in DENV-2 would have the potential to be active against all the serotypes of dengue and might be considered as Hits to begin designing and developing of more potent analogous peptides that could constitute as promising therapeutic agents for attenuating dengue infection.     

A clinical isolate of dengue virus and its proteins induce apoptosis in HMEC-1 cells: a possible implication in pathogenesis

Cumulative studies have demonstrated that dengue virus infection results in the induction of apoptosis of certain cells in vitro. Moreover, apoptosis of microvascular endothelial cells in the brain and in the intestinal serosa has been demonstrated postmortem in dengue virus (DENV)-infected patients. In this work, human microvascular endothelial cells (HMEC-1) infected with a DENV-2 clinical isolate, or HMEC-1 cells transfected with its protease sequence (NS3pro) or its complex (NS2BNS3pro) were able to trigger apoptosis after 24 h of infection or transfection. The infected or transfected HMEC-1 cells displayed the distinctive apoptotic hallmarks, which include cytoplasmic shrinkage and plasma membrane blebbing. In addition, the transfected HMEC-1 cells showed biochemical changes such as exposure of phosphatidylserine on the outer leaflet of the plasma membrane, TUNEL positivity, caspase 3 activation and cleaved PARP, a central regulator of apoptosis. These findings suggest the role of such proteins from the clinical isolate in the induction of apoptosis. Melina Vásquez Ochoa and Julio García Cordero contributed equally to this work.     

Induced autophagy reduces virus output in dengue infected monocytic cells

While several studies have shown a role for autophagy in the replication of dengue virus (DENV), these studies have been performed in directly infected cells. However, in severe cases of DENV infection the critical cell in the disease is believed to be monocytes which are poorly infected directly, but are highly susceptible to antibody enhanced infection. This study sought to determine the involvement of autophagy in the DENV infection of monocytic cells, using U937 cells as a model system. While the induction of autophagy was seen in response to DENV-2 infection, biochemical induction of autophagy resulted in a significant decrease in virus output. Down regulation of autophagy resulted in only a very slight increase in intracellular virus levels. In monocytic cells autophagy is not a significant part of the DENV replication mechanism, and there are distinct cell type specific differences in the DENV-autophagy interaction.     

Human keratinocyte cultures (HaCaT) can be infected by DENV,triggering innate immune responses that include IFNλ and LL37

The skin is the first anatomical region that dengue virus (DENV) encounters during the natural infection. Although the role of some skin resident cells like dendritic cells and fibroblasts has been demonstrated to be crucial to elucidate the role of resident cells and molecules participating during the early events of the innate immune response, the participation of keratinocytes during DENV infection has not been fully elucidated. In this paper we aimed to evaluate the use of the HaCaT cell line as a model to study the immune responses of skin keratinocytes to DENV infection. We demonstrated productive DENV-2 infection of HaCaT cells and their capability to establish an antiviral response through production of type I and type III interferons (IFN-β and IFN-λ). The production of these cytokines by HaCaT cells correlated with upregulation of IFN-inducible transmembrane protein-3 (IFITM3) and viperin in bystander, uninfected cells. We also observed an increase in secretion of IL-6 and IL-8. Skin keratinocytes are known to secrete antimicrobial peptides (AMPs) during viral infections. In our model, DENV-2 infected HaCaT cells upregulate the production of cytoplasmic LL-37. We evaluated the dual role of LL-37, HBD2, and HBD3 antiviral activity and immunoregulation during DENV-2 infection of HaCaT cells and found that LL-37 significantly reduced DENV-2 replication. This indicates that the HaCaT cell line can be used as a model for studying the innate response of keratinocytes to DENV infection. Our results also suggest that skin keratinocytes play an important role in the skin microenvironment after DENV infection by secreting molecules like type I and type III IFNs, pro-inflammatory molecules, and LL-37, which may contribute to the protection against arboviral infections.     

CD16(+) natural killer cells play a limited role against primary dengue virus infection in tamarins

CD16 is a major molecule expressed on NK cells. To directly assess the role of natural killer (NK) cells in dengue virus (DENV) infection in vivo, CD16 antibody-treated tamarins were inoculated with a DENV-2 strain. This resulted in the transient depletion of CD16(+) NK cells, whereas no significant effects on the overall levels or kinetics of plasma viral loads and antiviral antibodies were observed in the treated monkeys when compared to control monkeys. It remains elusive whether the CD16(-) NK subpopulation could play an important role in the control of primary DENV infection.     

Dengue hemorrhagic fever-associated immunomediators induced via maturation of dengue virus nonstructural 4B protein in monocytes modulate endothelial cell adhesion molecules and human microvascular endothelial cells permeability

We previously demonstrated that dengue virus (DENV) nonstructural 4B protein (NS4B) induced dengue hemorrhagic fever (DHF)-associated immunomediators in THP-1 monocytes. Moreover, cleavage of NS4AB polyprotein by the NS2B3 protease, significantly increased immunomediator production to levels found after DENV infection. In this report using primary human microvascular endothelial cells (HMVEC) transwell permeability model and HMVEC monolayer, we demonstrate that the immunomediators secreted in the supernatants of DENV-infected monocytes increase HMVEC permeability and expression of ICAM-1, VCAM-1 and E-selectin. Moreover, maturation of NS4B via cleavage of 2KNS4B is sufficient to induce immunomediators that cause HMVEC phenotypic changes, which appear to be synergistically induced by TNFα and IL-8. These data suggest that therapies targeting the maturation steps of NS4B, particularly 2KNS4B processing, may reduce overall DHF-associated immunomediator levels, thereby reducing DHF-associated morbidity and mortality. Alternatively, TNFα inhibitors may be a valid intervention strategy during the later stages of infection to prevent DHF progression.     

Antibody-dependent enhancement of dengue virus infection in vitro by undiluted sera from monkeys infected with heterotypic dengue virus

Two monkeys (Macaca fascicularis) each were infected with dengue virus type 1 (DENV-1) and type 2 (DENV-2). High levels of neutralizing antibody to homotypic serotype were detected from day 10 to week 58 after infection. Levels of cross-reactive neutralizing antibody to other serotypes were at lower levels or undetectable. Serum samples collected from day 10 to week 58 enhanced infection by homotypic and heterotypic serotypes of DENV when diluted, demonstrating antibody-dependent enhancement (ADE). The ADE activities to heterotypic and homotypic dengue virus infections peaked at dilutions of 1:10–1:100 and 1:100–1:1,000, respectively. Serum samples collected enhanced heterotypic dengue virus infection without any dilution. The results indicate that sera from infected monkeys have an ability to enhance heterotypic dengue virus infection in vitro without dilution, although some of these sera also possess neutralizing activity.     

Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.     

A retrospective survey of dengue virus infection in fatal cases from an epidemic in Brazil

Dengue virus can infect many cell types from the vascular, muscular and hematological systems causing diverse clinical and pathological signs. The purpose of the present study was to investigate by different diagnostic methods dengue virus in human tissue specimens obtained from fatal cases (n=29) during a large-scale dengue fever epidemic in 2002 in the State of Rio de Janeiro, Brazil. The combination of four procedures provided diagnostic confirmation of DENV-3 infection in 26 (89.6%) out of the 29 suspected fatal cases. Dengue virus (DENV) was isolated from 2/74 (2.7%) tissue samples, inoculated into C6/36 cells and identified as DENV-3, nested RT-PCR accusing 22/72 (30.5%) samples as DENV-3. Real-time RT-PCR yielded the highest positivity rate, detecting viral RNA in 45/77 (58.4%) clinical specimens, including the liver (n=18), lung (n=8), spleen (n=8), brain (n=6), kidney (n=3), bone marrow (n=1) and heart (n=1). Immunohistochemical tests recognized the DENV antigen in 26/59 (44%) specimens. Given the accuracy and effectiveness of real-time RT-PCR in this investigation, this approach may play an important role for rapid diagnosis of dengue infections.     

Dengue virus serotypes and genotypic characterization from northeast India

Dengue is a rapidly spreading acute arboviral infection transmitted through a human and Aedes mosquito cycle. Though northeast region of India has been experiencing dengue outbreaks regularly for over a decade, reports on genetic characterization of the virus from this region are limited. The present study was undertaken to detect the genotype and genetic composition of circulating dengue virus (DENV) in this region. Blood samples were collected from 918 suspected dengue patients of five northeast Indian states. Serological investigations, viz, nonstructural 1 (NS1) enzyme-linked immunosorbent assay (ELISA), immunoglobulin M (IgM) ELISA, and immunoglobulin G (IgG) ELISA were performed followed by molecular detection. Sequence analysis and phylogenetic tree construction based on capsid-premembrane (C-prM) gene junction was done by BioEdit and MEGA6 software, respectively. Serological detection showed 35.34% NS1 and 18.12% IgM positivity. Secondary infection was observed in 24.53%. All four serotypes were detected. Phylogenetic analysis demonstrated circulation of genotype III of DENV-1, genotype IV of DENV-2, and genotype III of DENV-3. Sequences from this region form distinct clades in the phylogenetic tree. Characterization of the C-prM gene junction reveals divergence among the DENV strains. As genetic variation within the DENV is known to be associated with diverse clinical outcomes, information regarding the genetic composition of circulating virus could be beneficial in designing an effective intervention strategy.