scholarly journals Altered Moesin and Actin Cytoskeleton Protein Rearrangements Affect Transendothelial Permeability in Human Endothelial Cells upon Dengue Virus Infection and TNF-α Treatment

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2042
Author(s):  
Aroonroong Suttitheptumrong ◽  
Thanaporn Mahutchariyakul ◽  
Nantapon Rawarak ◽  
Onrapak Reamtong ◽  
Kobporn Boonnak ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Vascular Permeability ◽  
Synergistic Effects ◽  
Denv Infection ◽  
Severe Dengue ◽  
Actin Stress Fiber ◽  
Tnf Α ◽  
Ea.Hy926 Cells ◽  
Transendothelial Permeability

It has been hypothesized that the host, viral factors, and secreted cytokines (especially TNF-α) play roles in the pathogenesis of secondary dengue infections. Mass spectrometry-based proteomic screening of cytoskeleton fractions isolated from human endothelial (EA.hy926) cells upon dengue virus (DENV) infection and TNF-α treatment identified 450 differentially altered proteins. Among them, decreased levels of moesin, actin stress fiber rearrangements, and dot-like formations of vinculin were observed with western blot analyses and/or immunofluorescence staining (IFA). In vitro vascular permeability assays using EA.hy926 cells, seeded on collagen-coated transwell inserts, showed low levels of transendothelial electrical resistance in treated cells. The synergistic effects of DENV infection and TNF-α treatment caused cellular permeability changes in EA.hy926 cells, which coincided with decreasing moesin levels and the production of abnormal organizations of actin stress fibers and vinculin. Functional studies demonstrated moesin overexpression restored transendothelial permeability in DENV/TNF-α-treated EA.hy926 cells. The present study improves the understanding of the disruption mechanisms of cytoskeleton proteins in enhancing vascular permeability during DENV infection and TNF-α treatment. The study also suggests that these disruption mechanisms are major factors contributing to vascular leakage in severe dengue patients.

scholarly journals Viperin is anti-viral in vitro but is dispensable for restricting dengue virus replication or induction of innate and inflammatory responses in vivo

2021 ◽  
Vol 102 (10) ◽  
Author(s):  
Wisam-Hamzah Al Shujairi ◽  
Luke P. Kris ◽  
Kylie van der Hoek ◽  
Evangeline Cowell ◽  
Gustavo Bracho-Granado ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Immune Cell ◽  
Inflammatory Responses ◽  
Denv Infection ◽  
Brain Morphology ◽  
Moderate Increase ◽  
Tnf Α ◽  
Significant Difference

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip−/−) showed enhanced DENV infection, accompanied by increased IFN-β and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip−/− mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip−/− compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-β, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip−/− mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip−/− DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-β, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip−/− mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.

scholarly journals Dengue Virus Induces Increased Activity of the Complement Alternative Pathway in Infected Cells

2018 ◽  
Vol 92 (14) ◽  
Author(s):  
Sheila Cabezas ◽  
Gustavo Bracho ◽  
Amanda L. Aloia ◽  
Penelope J. Adamson ◽  
Claudine S. Bonder ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Dengue Virus ◽  
Vascular Permeability ◽  
Alternative Pathway ◽  
Denv Infection ◽  
Factor H ◽  
Severe Dengue ◽  
Complement Alternative Pathway ◽  
Viral Pathogen ◽  
Infected Cells

ABSTRACTSevere dengue virus (DENV) infection is associated with overactivity of the complement alternative pathway (AP) in patient studies. Here, the molecular changes in components of the AP during DENV infectionin vitrowere investigated. mRNA for factor H (FH), a major negative regulator of the AP, was significantly increased in DENV-infected endothelial cells (EC) and macrophages, but, in contrast, production of extracellular FH protein was not. This discord was not seen for the AP activator factor B (FB), with DENV induction of both FB mRNA and protein, nor was it seen with Toll-like receptor 3 or 4 stimulation of EC and macrophages, which induces both FH and FB mRNA and protein. Surface-bound and intracellular FH protein was, however, induced by DENV, but only in DENV antigen-positive cells, while in two other DENV-susceptible immortalized cell lines (ARPE-19 and human retinal endothelial cells), FH protein was induced both intracellularly and extracellularly by DENV infection. Regardless of the cell type, there was an imbalance in AP components and an increase in markers of complement AP activity associated with DENV-infected cells, with lower FH relative to FB protein, an increased ability to promote AP-mediated lytic activity, and increased deposition of complement component C3b on the surface of DENV-infected cells. For EC in particular, these changes are predicted to result in higher complement activity in the local cellular microenvironment, with the potential to induce functional changes that may result in increased vascular permeability, a hallmark of dengue disease.IMPORTANCEDengue virus (DENV) is a significant human viral pathogen with a global medical and economic impact. DENV may cause serious and life-threatening disease, with increased vascular permeability and plasma leakage. The pathogenic mechanisms underlying these features remain unclear; however, overactivity of the complement alternative pathway has been suggested to play a role. In this study, we investigate the molecular events that may be responsible for this observed alternative pathway overactivity and provide novel findings of changes in the complement system in response to DENV infection in primary cell types that are a major target for DENV infection (macrophages) and pathogenesis (endothelial cells)in vivo. Our results suggest a new dimension of cellular events that may influence endothelial cell barrier function during DENV infection that could expand strategies for developing therapeutics to prevent or control DENV-mediated vascular disease.

scholarly journals Antiviral Efficacy of the Anesthetic Propofol against Dengue Virus Infection and Cellular Inflammation

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Ting-Jing Shen ◽  
Chia-Ling Chen ◽  
Ming-Kai Jhan ◽  
Po-Chun Tseng ◽  
Rahmat Dani Satria ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Cell Model ◽  
Antiviral Agents ◽  
Denv Infection ◽  
Severe Dengue ◽  
Cellular Inflammation ◽  
Antiviral Effects ◽  
Endosomal Acidification ◽  
Adults And Children

Propofol, 2,6-diisopropylphenol, is a short-acting intravenous sedative agent used in adults and children. Current studies show its various antimicrobial as well as anti-inflammatory effects. Dengue virus (DENV) is an emerging infectious pathogen transmitted by mosquitoes that causes mild dengue fever and progressive severe dengue diseases. In the absence of safe vaccines and antiviral agents, adjuvant treatments and supportive care are generally administered. This study investigated the antiviral effects of propofol against DENV infection and cellular inflammation by using an in vitro cell model. Treatment with propofol significantly inhibited DENV release 24 h postinfection in BHK-21 cells. Furthermore, it also blocked viral protein expression independent of the translational blockade. Propofol neither caused inhibitory effects on endosomal acidification nor prevented dsRNA replication. Either the proinflammatory TNF-α or the antiviral STAT1 signaling was reduced by propofol treatment. These results provide evidence to show the potential antiviral effects of the sedative propofol against DENV infection and cellular inflammation.

scholarly journals Soluble ST2 Does Not Regulate TNF-α and IL-6 Production in Dengue Virus-Infected Human Monocytes

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Marisol Pérez-Acosta ◽  
Félix Giovanni Delgado ◽  
Jaime E. Castellanos
Keyword(s):  
Dengue Virus ◽  
Viral Antigen ◽  
Mononuclear Cells ◽  
Acute Infection ◽  
Denv Infection ◽  
Human Monocytes ◽  
Peripheral Blood Mononuclear ◽  
Soluble St2 ◽  
Tnf Α

Dengue virus (DENV) produces an acute infection that results in the overproduction of proinflammatory cytokines. Although increased levels of the immunoregulator soluble ST2 (sST2) protein have been reported in the serum of patients with dengue, its importance during DENV infection remains unclear. The purpose of this study was to evaluate the effect of a recombinant human sST2 protein on the production of TNF-α and IL-6 in an in vitro model of DENV infection. Peripheral blood mononuclear cells (PBMCs) were permissive to in vitro DENV infection since viral antigen was detected in CD14+ monocytes by flow cytometry (median, 1%; range, 0–2.2), and in their supernatants TNF-α and IL-6 were detected. However, sST2 protein was not detected. Using multiple staining on infected PBMC we found that only CD14+ cells produced TNF-α and IL-6. Treatment with human recombinant sST2 protein decreased lipopolysaccharide-induced monocyte TNF-α and IL-6 production. However, this effect was not observed when the monocytes were pretreated with sST2 and later infected with DENV-2. These results suggest that sST2 has different roles in the regulation of TNF-α and IL-6 expression in human monocytes stimulated with LPS and DENV-2.

scholarly journals Recent advances in understanding dengue

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1279 ◽  
Author(s):  
Scott Halstead
Keyword(s):  
Vascular Permeability ◽  
Vaccine Development ◽  
Severe Disease ◽  
Denv Infection ◽  
Severe Dengue ◽  
Structural Protein ◽  
Control Programs ◽  
Past Half Century

This is a selective review of recent publications on dengue clinical features, epidemiology, pathogenesis, and vaccine development placed in a context of observations made over the past half century. Four dengue viruses (DENVs) are transmitted by urban cycle mosquitoes causing diseases whose nature and severity are influenced by interacting factors such as virus, age, immune status of the host, and human genetic variability. A phenomenon that controls the kinetics of DENV infection, antibody-dependent enhancement, best explains the correlation of the vascular permeability syndrome with second heterotypic DENV infections and infection in the presence of passively acquired antibodies. Based on growing evidence in vivo and in vitro, the tissue-damaging DENV non-structural protein 1 (NS1) is responsible for most of the pathophysiological features of severe dengue. This review considers the contribution of hemophagocytic histiocytosis syndrome to cases of severe dengue, the role of movement of humans in dengue epidemiology, and modeling and planning control programs and describes a country-wide survey for dengue infections in Bangladesh and efforts to learn what controls the clinical outcome of dengue infections. Progress and problems with three tetravalent live-attenuated vaccines are reviewed. Several research mysteries remain: why is the risk of severe disease during second heterotypic DENV infection so low, why is the onset of vascular permeability correlated with defervescence, and what are the crucial components of protective immunity?

scholarly journals Dengue Virus Infection with Highly Neutralizing Levels of Cross-Reactive Antibodies Causes Acute Lethal Small Intestinal Pathology without a High Level of Viremia in Mice

2015 ◽  
Vol 89 (11) ◽  
pp. 5847-5861 ◽  
Author(s):  
Satoru Watanabe ◽  
Kitti Wing Ki Chan ◽  
Jiaqi Wang ◽  
Laura Rivino ◽  
Shee-Mei Lok ◽  
...  
Keyword(s):  
Small Intestine ◽  
Viral Load ◽  
Virus Infection ◽  
Dengue Virus ◽  
Vascular Permeability ◽  
Severe Dengue ◽  
Intestinal Pathology ◽  
Tnf Α ◽  
Viremia Level ◽  
Small Intestinal

ABSTRACTSevere dengue virus (DENV)-associated diseases can occur in patients who have preexisting DENV antibodies (Abs) through antibody-dependent enhancement (ADE) of infection. It is well established that during ADE, DENV-antibody immune complexes (ICs) infect Fcγ receptor-bearing cells and increase the systemic viral burden that can be measured in the blood. For protection against infection with DENV serotypes 1 to 4, strongly neutralizing Abs must be elicited to overcome the effect of ADE. Clinical observations in infants who have maternal DENV Abs or recent phase II/III clinical trials with a leading tetravalent dengue vaccine suggested a lack of correlation between Ab neutralization andin vivodisease prevention. In addressing this gap in knowledge, we found that inoculation of ICs formed with serotype cross-reactive Abs that are more than 98% neutralizedin vitropromotes high mortality in AG129 mice even though peak viremia was lower than that in direct virus infection. This suggests that the serum viremia level is not always correlated with disease severity. We further demonstrated that infection with the ICs resulted in increased vascular permeability, specifically in the small intestine, accompanied with increased tissue viral load and cytokine production, which can be suppressed by anti-tumor necrosis factor alpha (anti-TNF-α) Abs. Flow cytometric analysis identified increased infection in CD11bintCD11cint/hiCD103−antigen-presenting cells by IC inoculation, suggesting that these infected cells may be responsible for the increase in TNF-α production and vascular permeability in the small intestine that lead to mortality in mice. Our findings may have important implications for the development of dengue therapeutics.IMPORTANCEWe examined the relationship between the neutralizing level of Abs at the time of infection and subsequent disease progression in a mouse model in order to understand why patients who are shown to have a neutralizing quantity of Abs still allow sufficient DENV replication to induce severe dengue manifestations, which sometimes do not correlate with viremia level. Strikingly, we found that high mortality was induced in AG129 mice by the increase in TNF-α-induced vascular permeability accompanied by an increased viral load, specifically in the small intestine, even when the initial infection level is suppressed to less than 5% and the peak viremia level is not enhanced. This suggests that ADE overcomes the protective efficacy of Abs in a tissue-dependent manner that leads to severe small intestinal pathology. Our findings may serve to address the pathogenic role of Abs on severe dengue disease and also help to develop safe Ab-based therapeutic strategies.

scholarly journals Dengue Virus Targets Nrf2 for NS2B3-Mediated Degradation Leading to Enhanced Oxidative Stress and Viral Replication

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Matteo Ferrari ◽  
Alessandra Zevini ◽  
Enrico Palermo ◽  
Michela Muscolini ◽  
Magdalini Alexandridi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Dengue Virus ◽  
Redox Homeostasis ◽  
Reactive Oxygen ◽  
Hemorrhagic Fever ◽  
Denv Infection ◽  
Progressive Increase ◽  
Severe Dengue ◽  
Oxygen Species

ABSTRACT Dengue virus (DENV) is a mosquito-borne virus that infects upward of 300 million people annually and has the potential to cause fatal hemorrhagic fever and shock. While the parameters contributing to dengue immunopathogenesis remain unclear, the collapse of redox homeostasis and the damage induced by oxidative stress have been correlated with the development of inflammation and progression toward the more severe forms of disease. In the present study, we demonstrate that the accumulation of reactive oxygen species (ROS) late after DENV infection (>24 hpi) resulted from a disruption in the balance between oxidative stress and the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant response. The DENV NS2B3 protease complex strategically targeted Nrf2 for degradation in a proteolysis-independent manner; NS2B3 licensed Nrf2 for lysosomal degradation. Impairment of the Nrf2 regulator by the NS2B3 complex inhibited the antioxidant gene network and contributed to the progressive increase in ROS levels, along with increased virus replication and inflammatory or apoptotic gene expression. By 24 hpi, when increased levels of ROS and antiviral proteins were observed, it appeared that the proviral effect of ROS overcame the antiviral effects of the interferon (IFN) response. Overall, these studies demonstrate that DENV infection disrupts the regulatory interplay between DENV-induced stress responses, Nrf2 antioxidant signaling, and the host antiviral immune response, thus exacerbating oxidative stress and inflammation in DENV infection. IMPORTANCE Dengue virus (DENV) is a mosquito-borne pathogen that threatens 2.5 billion people in more than 100 countries annually. Dengue infection induces a spectrum of clinical symptoms, ranging from classical dengue fever to severe dengue hemorrhagic fever or dengue shock syndrome; however, the complexities of DENV immunopathogenesis remain controversial. Previous studies have reported the importance of the transcription factor Nrf2 in the control of redox homeostasis and antiviral/inflammatory or death responses to DENV. Importantly, the production of reactive oxygen species and the subsequent stress response have been linked to the development of inflammation and progression toward the more severe forms of the disease. Here, we demonstrate that DENV uses the NS2B3 protease complex to strategically target Nrf2 for degradation, leading to a progressive increase in oxidative stress, inflammation, and cell death in infected cells. This study underlines the pivotal role of the Nrf2 regulatory network in the context of DENV infection.

scholarly journals Epitope Determinants of a Chimpanzee Dengue Virus Type 4 (DENV-4)-Neutralizing Antibody and Protection against DENV-4 Challenge in Mice and Rhesus Monkeys by Passively Transferred Humanized Antibody

2007 ◽  
Vol 81 (23) ◽  
pp. 12766-12774 ◽  
Author(s):  
Ching-Juh Lai ◽  
Ana P. Goncalvez ◽  
Ruhe Men ◽  
Claire Wernly ◽  
Olivia Donau ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Virus Type ◽  
High Titer ◽  
Neutralizing Antibody ◽  
Denv Infection ◽  
Passive Transfer ◽  
Escape Mutant ◽  
Protective Capacity

ABSTRACT The chimpanzee monoclonal antibody (MAb) 5H2 is specific for dengue virus type 4 (DENV-4) and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. One variant contained a Lys174-Glu substitution and another contained a Pro176-Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity for the antibody 18- to >100-fold. Humanized immunoglobulin G (IgG) 5H2, originally produced from an expression vector, has been shown to be a variant containing a nine-amino-acid deletion in the Fc region which completely ablates antibody-dependent enhancement of DENV replication in vitro. The variant MAb, termed IgG 5H2 ΔD, is particularly attractive for exploring its protective capacity in vivo. Passive transfer of IgG 5H2 ΔD at 20 μg/mouse afforded 50% protection of suckling mice against challenge with 25 50% lethal doses of mouse neurovirulent DENV-4 strain H241. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge. Monkeys that received 2 mg/kg of body weight of IgG 5H2 ΔD were completely protected against 100 50% monkey infectious doses (MID50) of DENV-4, as indicated by the absence of viremia and seroconversion. A DENV-4 escape mutant that contained a Lys174-Glu substitution identical to that found in vitro was isolated from monkeys challenged with 106 MID50 of DENV-4. This substitution was also present in all naturally occurring isolates belonging to DENV-4 genotype III. These studies have important implications for possible antibody-mediated prevention of DENV infection.

scholarly journals Discovery of Dengue Virus NS4B Inhibitors

2015 ◽  
Vol 89 (16) ◽  
pp. 8233-8244 ◽  
Author(s):  
Qing-Yin Wang ◽  
Hongping Dong ◽  
Bin Zou ◽  
Ratna Karuna ◽  
Kah Fei Wan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dengue Virus ◽  
Transmembrane Protein ◽  
Selective Inhibition ◽  
Denv Infection ◽  
Viral Pathogens ◽  
Replication Complex ◽  
First Time

ABSTRACTThe four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV bothin vitroandin vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has goodin vivopharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients.IMPORTANCEDengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) bothin vitroandin vivo. Our results validate, for the first time, that NS4B inhibitors could potentially be developed for antiviral therapy for treatment of DENV infection in humans.

scholarly journals Differential replication of dengue virus serotypes 2 and 3 in coinfections of C6/36 cells and Aedes aegypti mosquitoes

2014 ◽  
Vol 8 (07) ◽  
pp. 876-884 ◽  
Author(s):  
Diana Carolina Quintero-Gil ◽  
Marta Ospina ◽  
Jorge Emilio Osorio-Benitez ◽  
Marlen Martinez-Gutierrez
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Cell Viability ◽  
Denv Infection ◽  
Replication Capacity ◽  
Denv Serotypes ◽  
Viral Genomes ◽  
Diphenyltetrazolium Bromide

Introduction: Different dengue virus (DENV) serotypes have been associated with greater epidemic potential. In turn, the increased frequency in cases of severe forms of dengue has been associated with the cocirculation of several serotypes. Because Colombia is a country with an endemic presence of all four DENV serotypes, the aim of this study was to evaluate the in vivo and in vitro replication of the DENV-2 and DENV-3 strains under individual infection and coinfection conditions. Methodology: C6/36HT cells were infected with the two strains individually or simultaneously (coinfection). Replication capacity was evaluated by RT-qPCR, and the effects on cell viability were assessed with an MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Additionally, Aedes aegypti mosquitoes were artificially fed the two strains of each serotype individually or simultaneously. The viral genomes were quantified by RT-qPCR and the survival of the infected mosquitoes was compared to that of uninfected controls. Results: In single infections, three strains significantly affected C6/36HT cell viability, but no significant differences were found in the replication capacities of the strains of the same serotype. In the in vivo infections, mosquito survival was not affected, and no significant differences in replication between strains of the same serotype were found. Finally, in coinfections, serotype 2 replicated with a thousandfold greater efficiency than serotype 3 did both in vitro and in vivo. Conclusions: Due to the cocirculation of serotypes in endemic regions, further studies of coinfections in a natural environment would further an understanding of the transmission dynamics that affect DENV infection epidemiology.

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