scholarly journals Dengue virus type 2 replication is limited by activation of NOD2 and its interactions with RIP2 and MAVS adaptors in THP-1 macrophage-like cells

2021 ◽  
Author(s):  
Diana Domínguez-Martínez ◽  
Daniel Nuñez Avellaneda ◽  
Juan Castillo Cruz ◽  
Gloria León-Avila ◽  
BLANCA GARCIA-PEREZ ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Infections ◽  
Denv Infection ◽  
Human Macrophage ◽  
Infection Period ◽  
Viral Loads ◽  
Protein Levels ◽  
Mock Control

The nucleotide-binding domain (NBD) and leucine-rich repeat receptors, such as NOD-like receptors (NLRs), have pivotal functions in the innate immune response to various viral infections participating during the recognition of pathogens and activation of signaling pathways. One NLR, NOD2, is a dynamic protein that is activated in the presence of viral genomes and metabolites. However, its participation in combating a dengue virus (DENV) infection remains unclear. The aim of this study was to determine the role of NOD2 in macrophage-like THP-1 cells during an in vitro infection with DENV type 2 (DENV2). The interactions of NOD2 with RIP2 and MAVS was examined in DENV2-infected and agonist-stimulated cells. The effects of downregulating NOD2 expression or signaling on virus loads was also evaluated. The cellular mRNA expression and protein levels of NOD2 on cells under the stimuli were quantified with RT-PCR, Western blot and indirect immunofluorescence. Both the mRNA and protein expression of NOD2 was enhanced in response to DENV-2 infection. Interactions of NOD2 with RIP2 and MAVS, analyzed with confocal microscopy and co-immunoprecipitation assays, were time-dependent and increased in the post-infection period, between 6 and 24 h. After silencing NOD2 expression, DENV2-infected cells displayed greater viral loads and decreased expression of IL-8 and IFN-α (measured in supernatants obtained from the cells), compared to the uninfected (mock control) cells or those transfected with irrelevant-siRNA. Thus, in response to a DENV2 infection, NOD2 was activated in THP-1 human macrophage-like cells, the production of IL-8 and IFN-α was enhanced, and viral replication was limited.

In-vitro effect of human cathelicidin antimicrobial peptide LL-37 on dengue virus type 2

Peptides ◽  
2017 ◽  
Vol 92 ◽  
pp. 23-30 ◽  
Author(s):  
K. Alagarasu ◽  
P.S. Patil ◽  
P. Shil ◽  
M. Seervi ◽  
M.B. Kakade ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Antimicrobial Peptide ◽  
Virus Type ◽  
Vitro Effect ◽  
Dengue Virus Type 2

scholarly journals ILC2 Cells Promote Th2 Cell Differentiation in AECOPD Through Activated Notch-GATA3 Signaling Pathway

2021 ◽  
Vol 12 ◽  
Author(s):  
Min Jiang ◽  
Ren Cai ◽  
Jing Wang ◽  
Zheng Li ◽  
Dan Xu ◽  
...  
Keyword(s):  
Chronic Obstructive Pulmonary Disease ◽  
Peripheral Blood ◽  
Culture Supernatant ◽  
Th2 Cells ◽  
Chronic Obstructive ◽  
Obstructive Pulmonary Disease ◽  
Protein Levels ◽  
Th2 Cell

This study is to investigate the capacity of type 2 innate lymphoid cells (ILC2s) in regulating the Th2 type adaptive immune response of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The study enrolled healthy people, stable chronic obstructive pulmonary disease (COPD) patients, and AECOPD patients. Flow cytometry was used to detect Th2 and ILC2 cells in the peripheral blood. In addition, ILC2s from the peripheral blood of AECOPD patients were stimulated with PBS, IL-33, Jagged1, DAPT, IL-33+Jagged1, IL-33+DAPT, and IL-33+Jagged-1+DAP in vitro. The levels of cytokines in the culture supernatant were detected by ELISA and the culture supernatant was used to culture CD4 + T cells. The mRNA and protein levels of Notch1, hes1, GATA3, RORα, and NF-κB of ILC2s were detected by real-time PCR and Western blot. The proportion of Th2 and ILC2s was significantly increased in the peripheral blood of AECOPD patients, alone with the increased Notch1, hes1, and GATA3 mRNA levels. In vitro results showed that the mRNA and protein levels of Notch1, hes1, GATA3 and NF-κB were significantly increased after stimulation with Notch agonist, meanwhile, the level of type 2 cytokines were increased in the supernatant of cells stimulated with Notch agonist, and significantly promoted differentiation of Th2 cells in vitro. Disruption of Notch pathway weakened GATA3 expression and cytokine production, and ultimately affected the differentiation of Th2 cells. In conclusion, our results suggest that ILC2s can promote Th2 cell differentiation in AECOPD via activated Notch-GATA3 signal pathway.

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.

Transcriptional response of Wolbachia-transinfected Aedes aegypti mosquito cells to dengue virus at early stages of infection

2022 ◽  
Vol 103 (1) ◽  
Author(s):  
Michael Leitner ◽  
Kayvan Etebari ◽  
Sassan Asgari
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Viral Infections ◽  
Transcriptional Response ◽  
Denv Infection ◽  
Content Type ◽  
Public Health Burden ◽  
Immune Genes ◽  
Link Type ◽  
Early Stages

Mosquito-borne flaviviruses are responsible for viral infections and represent a considerable public health burden. Aedes aegypti is the principal vector of dengue virus (DENV), therefore understanding the intrinsic virus–host interactions is vital, particularly in the presence of the endosymbiont Wolbachia, which blocks virus replication in mosquitoes. Here, we examined the transcriptional response of Wolbachia -transinfected Ae. aegypti Aag2 cells to DENV infection. We identified differentially expressed immune genes that play a key role in the activation of anti-viral defence such as the Toll and immune deficiency pathways. Further, genes encoding cytosine and N6-adenosine methyltransferases and SUMOylation, involved in post-transcriptional modifications, an antioxidant enzyme, and heat-shock response were up-regulated at the early stages of DENV infection and are reported here for the first time. Additionally, several long non-coding RNAs were among the differentially regulated genes. Our results provide insight into Wolbachia -transinfected Ae. aegypti’s initial virus recognition and transcriptional response to DENV infection.

Cortisol-mediated potentiation of uterine artery contractility: effect of pregnancy

2002 ◽  
Vol 283 (1) ◽  
pp. H238-H246 ◽  
Author(s):  
Daliao Xiao ◽  
Xiaohui Huang ◽  
Soochan Bae ◽  
Charles A. Ducsay ◽  
Lubo Zhang
Keyword(s):  
Uterine Artery ◽  
Vascular Reactivity ◽  
Maternal Plasma ◽  
Apparent Dissociation Constant ◽  
Protein Levels ◽  
No Release ◽  
Endothelial Nitric Oxide

During pregnancy, maternal plasma cortisol concentrations approximately double. Given that cortisol plays an important role in the regulation of vascular reactivity, the present study investigated the potential role of cortisol in potentiation of uterine artery (UA) contractility and tested the hypothesis that pregnancy downregulated the cortisol-mediated potentiation. In vitro cortisol treatment (3, 10, or 30 ng/ml for 24 h) produced a dose-dependent increase in norepinephrine (NE)-induced contractions in both nonpregnant and pregnant (138–143 days gestation) sheep UA. However, this cortisol-mediated response was significantly attenuated by ∼50% in pregnant UA. The 11β-hydroxysteroid dehydrogenase (11-βHSD) inhibitor carbenoxolone did not change the effect of cortisol in nonpregnant UA but abolished its effect in pregnant UA by increasing the NE pD2 in control tissues from 6.20 ± 0.05 to 6.59 ± 0.11. The apparent dissociation constant value of NE α1-adrenoceptors was not changed by cortisol in pregnant UA but was decreased in nonpregnant UA. There was no difference in glucocorticoid receptor density between nonpregnant and pregnant UA. Cortisol significantly decreased endothelial nitric oxide (NO) synthase protein levels and NO release in both nonpregnant and pregnant UA, but the effect of cortisol was attenuated in pregnant UA by ∼50%. Carbenoxolone alone had no effects on NO release in nonpregnant UA but was decreased in pregnant UA. These results suggest that cortisol potentiates NE-mediated contractions by decreasing NO release and increasing NE-binding affinity to α1-adrenoceptors in nonpregnant UA. Pregnancy attenuates UA sensitivity to cortisol, which may be mediated by increasing type-2 11-βHSD activity in UA.

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.

scholarly journals 169. AT-752, an Oral Guanosine Nucleotide Prodrug, Exhibits Potent in Vitro Activity Against Flaviviruses and Prevents Disease Progression in a Dengue Mouse Model

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S213-S214
Author(s):  
Steven S Good ◽  
Adel Moussa ◽  
Xiao-Jian Zhou ◽  
Jean-Pierre Sommadossi ◽  
Keith Pietropaolo
Keyword(s):  
Weight Loss ◽  
Viral Load ◽  
Mouse Model ◽  
Dengue Virus ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Health Score ◽  
Viral Loads ◽  
Eyes Closed

Abstract Background The increasing global prevalence of human Dengue virus infection and the potential for life-threatening sequelae highlight the significance of this unmet medical need. Here we report the potent in vitro activity of AT-281, the free base form of AT-752, against Dengue virus and other flaviviruses and the in vivo efficacy of AT-752 in a mouse model of Dengue viral disease. Methods Antiviral activities of serial dilutions of AT-281 were evaluated in infected Huh-7 cells. Effective concentrations of AT-281 required to inhibit virus yield reduction by 90% (EC90) and to prevent cytopathic effect by 50% (EC50) were determined, respectively, by visual examination and by neutral red staining, as was cytotoxicity. AG129 (α-, β- and γ-interferon knock-out) mice received an oral dose of AT-752 (1000 mg/kg) 4 h before s.c. inoculation with Dengue virus type 2 (strain D2Y98P, 1x105 virus particles) followed by b.i.d. doses (500 mg/kg) for 7 days starting 1 h post-inoculation (p.i.). Six groups each (n=5) of treated and control mice were scheduled to be sacrificed on days 4, 6, 7, 8, 10 and 21 p.i. with serum and spleen viral RNA levels determined by plaque assay. AT-281 efficacy was evaluated based on overall health score, survival, weight loss and viral load in serum and spleen. Results In vitro EC90 values for AT-281 against Dengue, West Nile and Yellow Fever viruses ranged from 0.26 to 0.64 µM and EC50 values for Zika and Japanese encephalitis were 0.21 and 0.64 µM, respectively (Table 1). No toxicity was observed up to the highest concentrations tested (172 µM). Oral administration of AT-752 to Dengue-infected AG129 mice substantially improved survival, prevented weight loss and lowered viral loads by day 6, with virus being undetectable on day 8 and thereafter (Figure 1). Serum and spleen viral loads in control mice declined between days 4 and 8 but no control mice survived beyond day 8. In contrast, AT-752 treated mice survived up to day 19, eventually succumbing to model-induced CNS sequelae. Table 1. Antiviral Activity of AT-281 Against Various Flaviviruses in Huh-7 Cell Cultures Figure 1. Efficacy of AT-752 in the AG129 mouse model of Dengue infection. Panel a: health score: 1, healthy; 2, coat slightly ruffled; 3, coat ruffled/wet; 4, coat very ruffled, eyes slightly closed/inset; 5, coat very ruffled, eyes closed/inset; 6, coat very ruffled, eyes closed/inset, moribund requiring humane euthanasia; 7, found dead. Panel b: Kaplan-Meier survival plot. Panel c: percent weight loss. Panel d: serum viremia. Panel e: spleen viral load. Conclusion The potent activity of AT-281 against Dengue virus in vitro and the efficacy of its salt form, AT-752, in the terminal AG129 mouse model warrant further clinical development of the drug. Preclinical safety studies are in progress and clinical trials will be initiated thereafter. Disclosures Steven S. Good, MS, Atea Pharmaceuticals, Inc. (Employee) Adel Moussa, PhD, Atea Pharmaceuticals, Inc. (Employee) Xiao-Jian Zhou, PhD, Atea Pharmaceuticals, Inc. (Employee) Jean-Pierre Sommadossi, PhD, Atea Pharmaceuticals, Inc. (Board Member) Keith Pietropaolo, BA, Atea Pharmaceuticals, Inc. (Employee)

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