DENV Replication Research Articles

Dengue virus is particularly sensitive to interference with long-chain fatty acid elongation and desaturation.

Orthoflaviviruses are emerging arthropod-borne pathogens whose replication cycle is tightly linked to host lipid metabolism. Previous lipidomic studies demonstrated that infection with the closely related hepatitis C virus (HCV) changes the fatty acid (FA) profile of several lipid classes. Lipids in HCV-infected cells had more very long-chain and desaturated FAs and viral replication relied on functional FA elongation and desaturation. Here, we systematically analyzed the role of FA elongases and desaturases in infection models of the most prevalent pathogenic orthoflaviviruses, dengue (DENV), Zika (ZIKV), West Nile (WNV), yellow fever (YFV), and tick-borne encephalitis virus (TBEV). Knockdown of desaturases and elongases in Huh7 cells only marginally affected ZIKV, WNV, YFV, and TBEV replication, while DENV titers were strongly reduced. This was most prominent for enzymes involved in very long-chain fatty acid synthesis. In detail, knockdown of the FA elongase ELOVL4, which catalyzes ultra long-chain FA synthesis, significantly reduced DENV titers, decreased the formation of replication intermediates, and lowered viral protein levels in DENV infected hepatoma cells, suggesting a function of ELOVL4 in DENV RNA replication. In contrast, the activity of FA desaturase FADS2, rate-limiting in poly-unsaturated FA biosynthesis, is not involved in viral RNA replication or translation, but is essentially required for formation of infectious DENV particles. Further, in immunocompetent immortalized microglial cells, FADS2 deletion additionally limits viral replication through increased expression of interferon-stimulated genes in response DENV infection. Taken together, enzymes involved in very long-chain FA synthesis are critical for different steps of DENV replication.

Functional Verification of Differentially Expressed Genes Following DENV2 Infection in Aedes aegypti.

The dengue virus (DENV) is primarily transmitted by Aedes aegypti. Investigating genes associated with mosquito susceptibility to DENV2 offers a theoretical foundation for targeted interventions to regulate or block viral replication and transmission within mosquitoes. Based on the transcriptomic analyses of the midgut and salivary glands from Aedes aegypti infected with DENV2, alongside analyses of Aag2 cell infections, 24 genes potentially related to the regulation of Aedes aegypti infection with DENV2 were selected. By establishing transient transfection and overexpression models of Aedes aegypti Aag2 cells, and mosquito target gene interference models, the difference in viral load before and after treatment was compared, and the effects of DEGs on viral replication were evaluated. After overexpressing 24 DEGs in Aag2 cells, 19 DEGs showed a significant difference in DENV2 RNA copies in the cell supernatant (p < 0.05). In adult mosquitoes, knocking down defensin-A, defensin-A-like, and SMCT1 respectively reduced the DENV2 RNA copies, while knocking down UGT2B1 and ND4 respectively increased the DENV2 RNA copies. In this study, to assess the role of genes related to DENV2 replication, and transient transfection and overexpression models in Aag2 cells and mosquito gene knockdown models were established, and five genes, defensin-A, defensin-A-like, SMCT1, UGT2B1, and ND4, were found to have an impact on the replication of DENV2, providing a reference basis for studying the complex mechanism of mosquito-virus interactions.

The Effectiveness of Cassava Leaf (Manihot esculenta C.) Flavonoid Quercetin as Dengue Virus-1 Antivirus in Vitro

Background: Dengue fever is a mosquito-borne disease that can cause severe symptoms and death, placing high burden on healthcare systems in tropical regions. Currently, there are no antiviral treatments. It has been proven that synthetic quercetin able to improve body performance and reduce the risk of infection as well as inhibit DENV replication. Cassava leaves (Manihot esculenta C.) contain anthocyanins (flavonoids) such as quercetin and it shows potential as antiviral agent as the synthetic as well. Aims: To describe the levels of self-compassion and procrastination among medical students and to examine how these factors relate to academic performance. Methods: This experimental study used post-test only with control group design consisted of 13 treatment groups and 1 control group. Treatment group were divided into 6 different concentrations for inhibition test (50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.125 µg/mL, 1.5 µg/mL) and 7 different concentrations for cytotoxicity test (80 µg/mL, 40 µg/mL, 20 µg/mL, 10 µg/mL, 5 µg/mL, 2.5 µg/mL, 1.25 µg/mL). The control group was a negative control treated with 0.2% DMSO. Inhibition was assessed by Focus Forming Unit Assay and cytotoxic was assessed by number of Microtiter Tetrazolium Assay (MTT Assay). The statistical analysis used in this study were the Shapiro-Wilk test for normality test, Kruskal-Wallis test for hypothesis test, and post-hoc test to determine whether the groups studied in this research had significant differences from each other. Results: The CC50 and IC50 values of quercetin of cassava leaf (Manihot esculenta C.) were found to be 3.44 μg/mL and 0.25 μg/mL, respectively, with an SI value of 123. Based on these results, it can be concluded that the flavonoid quercetin of cassava leaf (Manihot esculenta C.) exhibits selective properties in inhibiting the replication of DENV-1. Statistical analysis showed non-normal distribution (P&lt;0.05), hypothesis test was accepted (P&lt;0.05), and no significant differences between concentrations in the post-hoc test. Conclusion: Quercetin of cassava leaf (Manihot esculenta C.) is effective as an antiviral agent against dengue serotype 1 strain New Guinea C in vitro. Received: 25 September 2024 | Reviewed: 15 October 2024 | Revised: 30 November 2024 | Accepted: 15 December 2024.

E2 Ubiquitin-Conjugating Enzymes Regulates Dengue Virus-2 Replication in Aedes albopictus.

Aedes albopictus, a major vector of dengue virus (DENV), has a global distribution. Identifying the key components of the ubiquitin system of A. albopictus essential for the replication of viruses could help identify targets for developing broad-spectrum antiviral strategies. This study explores the interaction between E2 ubiquitin-conjugating enzymes (Ubc9) and DENV-2 proteins (NS1, NS5, and E) using cell culture and mosquito models. The replication of DENV-2 and the knockdown efficiency of the Ubc9 gene were assessed through reverse transcription-quantitative polymerase chain reaction. The DENV-2-related protein expression was evaluated via Western blot analysis. The interaction between Ubc9 and DENV E and NS5 proteins was investigated through confocal immunofluorescence and co-immunoprecipitation. RNA interference technology was employed to silence Ubc9 expression in C6/36 cells and in A. albopictus mosquitoes. The expression level of Ubc9 in the DENV-2-infected group was 3.5-fold higher than that in the control group. The Ubc9 gene expression in the midgut tissue of the mosquito was significantly upregulated. Transfection of C6/36 and BHK-21 cells with the pAc5.1b-EGFP-Ubc9-HA vector led to the overexpression of Ubc9, which decreased the transcription levels of DENV E and NS1, NS5 proteins. The difference was statistically significant (F = 24.27, p < 0.01). The expression levels of DENV NS5 and E proteins significantly decreased after infection with DENV-2, suggesting that the depletion of Ubc9 may limit the replication of DENV-2. Ubc9 regulates DENV-2 replication through SUMOylation in the cells and A. albopictus, potentially affecting vector competence and DENV transmission. This is the first study to demonstrate that the Ubc9 of A. albopictus plays a significant role in regulating the replication of DENV in both mosquito cells and the mosquito itself. The study results may prove useful in designing appropriate therapeutic approaches for dengue and associated complications.

Interaction of the Wolbachia surface protein with a novel pro-viral protein from Aedes aegypti.

Wolbachia is an endosymbiotic bacterium that blocks the replication of arboviruses in transinfected Aedes aegypti mosquitoes. In this study, we focused on identifying the potential interaction of Wolbachia proteins with the host pro-viral proteins. For this, we embarked on identifying the interacting proteins with a major Wolbachia protein, WSP, which is both structural and also secreted into the host cells. An Ae. aegypti STK was identified, which is induced in DENV and Wolbachia-infected cells. Silencing or induction of the gene led to reduced and increased DENV replication in vitro. Consistently, knocking down the gene in mosquitoes resulted in decreased virus replication. We hypothesize that WSP may sequester STK, which is pro-viral, contributing to Wolbachia virus blocking.

Possible effects of ancestry-related oxysterol-binding protein-like 10 genetic polymorphisms on dengue virus replication and anti-dengue immune response

PurposeOxysterol-binding protein-like 10 (OSBPL10) gene has been associated with reduced susceptibility to severe dengue in individuals of African descent. The aim of this study was to determine the possible effect of OSBPL10 on dengue virus (DENV) replication as well as the impact of African and European haplotypes of six OSBPL10 small nuclear polymorphisms (SNPs) on dengue multiplication and innate immune response. MethodsWe conducted gene knockdown experiments targeting OSBPL10 in THP-1 and Huh-7D12 cell lines, followed by a DENV-2 replication assay. Extracellular viral load was determined using qRT-PCR. To investigate the impact of SNPs haplotypes on viral replication and gene expression we cultured peripheral blood mononuclear cells (PBMC) from individuals with homozygous African and European haplotypes of OSBPL10 with DENV-2. Individual genotyping was performed using High Resolution Melt (HRM) analysis. The level of viral replication was assessed through plaque assay, while RT-PCR was employed to determine the expression levels of RXR-α, IFN-γ, IL-10 and IL-8 genes. ResultsIn vitro OSBPL10 knockdown significantly reduced DENV-2 replication. Individuals carrying European haplotypes showed higher DENV titers along with elevated levels of RXR-α and IL-8 mRNA compared to those carrying African haplotypes, who exhibited lower viral loads alongside increased IFN-γ and IL-10 expression. ConclusionsOur findings further explore the role of OSBPL10 in DENV multiplication, immune response to infection. The European haplotypes of OSBPL10 appear to increase DENV replication and promote RXR-α and IL-8 mRNA expression which correlates with the suppressive effect of these mediators on type I IFN, promoting viral replication and a deficient antiviral response. In contrast, the African haplotype showed a reduction in DENV replication and enhanced IFN-γ and IL-10 mRNA expression, which could be related to the better management of dengue infection and the low frequency of severe disease in this ethnic groupe.

7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

There are a limited number of effective vaccines against dengue virus (DENV) and significant efforts are being made to develop potent anti-virals. Previously, we described that platelet-chemokine CXCL4 negatively regulates interferon (IFN)-α/β synthesis and promotes DENV2 replication. An antagonist to CXCR3 (CXCL4 receptor) reversed it and inhibited viral replication. In a concurrent search, we identified CXCR3-antagonist from our compound library, namely 7D, which inhibited all serotypes of DENV in vitro. With a half-life of ~2.85 h in plasma and no significant toxicity, 7D supplementation (8 mg/kg-body-weight) to DENV2-infected IFNα/β/γR−/−AG129 or wild-type C57BL6 mice increased synthesis of IFN-α/β and IFN-λ, and rescued disease symptoms like thrombocytopenia, leukopenia and vascular-leakage, with improved survival. 7D, having the property to inhibit Sirt-1 deacetylase, promoted acetylation and phosphorylation of STAT3, which in-turn increased plasmablast proliferation, germinal-center maturation and synthesis of neutralizing-antibodies against DENV2 in mice. A STAT3-inhibitor successfully inhibited these effects of 7D. Together, these observations identify compound 7D as a stimulator of IFN-α/β/λ synthesis via CXCL4:CXCR3:p38:IRF3 signaling, and a booster for neutralizing-antibody generation by promoting STAT3-acetylation in plasmablasts, capable of protecting dengue infection.

Baicalin suppresses type 2 dengue virus-induced autophagy of human umbilical vein endothelial cells by inhibiting the PI3K/AKT pathway

To investigate the effect of type 2 dengue virus (DENV-2) infection on autophagy in human umbilical vein endothelial cells (HUVECs) and the mechanism mediating the inhibitory effect of baicalin against DENV-2 infection. Cultured HUVECs with DENV-2 infection were treated with different concentrations of baicalin, and the changes in autophagy of the cells were detected using transmission electron microscopy. Lyso Tracker Red staining was used to examine pH changes in the lysosomes of the cells, and the expressions of ATG5, beclin-1, LC3, P62, STX17, SNAP29, VAMP8, and PI3K/AKT signaling pathway-related proteins were detected by Western blotting. DENV-2 replication in the cells were evaluated using RT-qPCR. The differentially expressed proteins in DENV-2-infected HUVECs were identified by proteomics screening. Treatment with baicalin did not significantly affect the viability of cultured HUVECs. Proteomic studies suggested that the PI3K-AKT pathway played an important role in mediating cell injury induced by DENV-2 infection. The results of RT-qPCR demonstrated that baicalin dose-dependently inhibited DENV-2 replication in HUVECs and produced the strongest inhibitory effect at the concentration of 50 μg/mL. Transmission electron microscopy, Lyso Tracker Red staining, RT-qPCR, and Western blotting all showed significant inhibitory effect of baicalin on DENV-2-induced autophagy in HUVECs. DENV-2 infection of HUVECs caused increased cellular expressions of LC3 and P62 proteins, which were significantly lowered by treatment with LY294002 (a PI3K inhibitor). Baicalin inhibits DENV-2 replication in HUVECs and suppresses DENV-2-induced cell autophagy by inhibiting the PI3K/AKT signaling pathway.

CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication.

The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.

Mosquito E-20-Monooxygenase Gene Knockout Increases Dengue Virus Replication in Aedes aegypti Cells.

E-20-monooxygenase (E20MO) is an enzymatic product of the shade (shd) locus (cytochrome p450, E20MO). Initially discovered in Drosophila, E20MO facilitates the conversion of ecdysone (E) into 20-hydroxyecdysone (20E) and is crucial for oogenesis. Prior research has implicated 20E in growth, development, and insecticide resistance. However, little attention has been given to the association between the E20MO gene and DENV2 infection. The transcriptome of Ae. aegypti cells (Aag2 cells) infected with DENV2 revealed the presence of the E20MO gene. The subsequent quantification of E20MO gene expression levels in Aag2 cells post-DENV infection was carried out. A CRISPR/Cas9 system was utilized to create an E20MO gene knockout cell line (KO), which was then subjected to DENV infection. Analyses of DENV2 copies in KO and wild-type (WT) cells were conducted at different days post-infection (dpi). Plasmids containing E20MO were constructed and transfected into KO cells, with pre- and post-transfection viral copy comparisons. Gene expression levels of E20MO increased after DENV infection. Subsequently, a successful generation of an E20MO gene knockout cell line and the verification of code-shifting mutations at both DNA and RNA levels were achieved. Furthermore, significantly elevated DENV2 RNA copies were observed in the mid-infection phase for the KO cell line. Viral RNA copies were lower in cells transfected with plasmids containing E20MO, compared to KO cells. Through knockout and plasmid complementation experiments in Aag2 cells, the role of E20MO in controlling DENV2 replication was demonstrated. These findings contribute to our understanding of the intricate biological interactions between mosquitoes and arboviruses.

Host serine protease ACOT2 assists DENV proliferation by hydrolyzing viral polyproteins.

Dengue fever is a mosquito-borne tropical disease caused by the dengue virus (DENV). The replication of DENV relies on the processing of its genome-encoded polyprotein by both viral protease NS3 (NS3pro) and host proteases. However, the impact of host proteases on DENV proliferation is not well understood. In this study, we utilized fluorophosphonate-based probes (FPs) to investigate the up-regulation of host serine proteases during DENV infection in detail. Among the identified proteases, acyl-CoA thioesterase 2 (ACOT2), an enzyme that hydrolyzes acyl-CoA molecules to generate fatty acids and free CoA, exhibited cleavage activity against DENV polypeptide substrates. Enzymatic assays and virological experiments confirmed that ACOT2 contributes to DENV propagation during the replication stage by cleaving the viral polyprotein. Docking models provided insights into the binding pocket of viral polypeptides and the catalytic mechanism of ACOT2. Notably, this study is the first to demonstrate that ACOT2 functions as a serine protease to hydrolyze protein substrates. These findings offer novel insights into DENV infection, host response, as well as the potential development of innovative antiviral strategies.IMPORTANCEDENV, one of the major pathogens of Dengue fever, remains a significant public health concern in tropical and subtropical regions worldwide. How DENV efficiently hijacks the host and accesses its life cycle with delicate interaction remains to be elucidated. Here, we deconvoluted that the host protease ACOT2 assists the DENV replication and characterized the ACOT2 as a serine protease involved in the hydrolysis of the DENV polypeptide substrate. Our results not only further the understanding of the DENV life cycle but also provide a possibility for the usage of activity-based proteomics to reveal host-virus interactions.

Inhibition of cellular activation induced by platelet factor 4 via the CXCR3 pathway ameliorates Japanese encephalitis and dengue viral infections

BackgroundActivated platelets secrete platelet factor 4 (PF4), which contributes to viral pathogenesis. Recently, we reported the proviral role of PF4 in replication of closely related flaviviruses, Japanese encephalitis virus (JEV) and dengue virus (DENV). ObjectivesThis study aimed to investigate the detailed mechanism of PF4-mediated virus replication. MethodsPF4−/− or wild-type (WT) mice were infected with JEV, and host defense mechanisms, including autophagic/interferon (IFN) responses, were assessed. WT mice were pretreated with the CXCR3 antagonist AMG487 that inhibits PF4:CXCR3 pathway. This pathway was tested in PF4−/− monocytes infected with DENV or in monocytes isolated from patients with DENV infection. ResultsPF4−/− mice infected with JEV showed reduced viral load and improved brain inflammation and survival. PF4−/− mice synthesized more IFN-α/β with higher expression of phosphorylated IRF3 in the brain. PF4 treatment decreased IRF-3/7/9 and IFN-α/β expression and suppressed autophagic LC3-II flux and lysosomal degradation of viral proteins in JEV-infected cells. PF4 increased the expression of P-mTOR, P-p38, and P-ULK1Ser757 and decreased expression of LC3-II. Decreased autophagosome-lysosome fusion in turn promoted DENV2 replication. The above processes were reversed by AMG487. Uninfected PF4−/− monocytes showed elevated LC3-II and autophagosome-lysosome fusion. Microglia of JEV-infected PF4−/− mice exhibited elevated LC3-II inversely related to viral load. Similarly, monocytes from PF4−/− mice showed reduced infection by DENV2. In patients with DENV infection, higher plasma PF4 and viral load were inversely correlated with LC3-II, LAMP-1, and lysosomal degradation of DENV-NS1 in monocytes during the febrile phase. ConclusionThese studies suggest that PF4 deficiency or inhibition of the PF4:CXCR3 pathway prevents JEV and DENV infection. The studies also highlight the PF4:CXCR3 axis as a potential target to develop treatment regimens against flaviviruses.

Suppression of dengue virus replication by the French maritime pine extract Pycnogenol®

Dengue virus (DENV) is mainly found in the tropics and infects approximately 400 million people annually. However, no clinically available therapeutic agents specific to dengue have been developed. Here, we examined the potential antiviral effects of the French maritime pine extract Pycnogenol® (PYC) against DENV because we previously found that the extract exerts antiviral effects on hepatitis C virus, which belongs to the Flavivirus family. First, we examined the efficacy of PYC against DENV1, 2, 3, and 4 serotypes and determined that it had a dose-dependent suppressive effect on the viral load, especially in the supernatant. This inhibitory effect of PYC may target the late stages of infection such as maturation and secretion, but not replication. Next, we examined the efficacy of PYC against DENV infection in type I interferon (IFN) receptor knockout mice (A129). As the propagation of DENV2 was the highest among the four serotypes, we used this serotype in our murine model experiments. We found that PYC significantly inhibited DENV2 replication in mice on day 4 without significantly decreasing body weight or survival ratio. We further examined the mechanism of action of PYC in DENV2 infection by characterizing the main PYC targets among the host (viral) factors and silencing them using siRNA. Silencing long noncoding-interferon-induced protein (lnc-IFI)-44, polycystic kidney disease 1-like 3 (Pkd1l3), and ubiquitin-specific peptidase 31 (Usp31) inhibited the replication of DENV2. Thus, the results of this study shed light on the inhibitory effects of PYC on DENV replication and its underlying mechanisms.

The Antiviral Activity of Varenicline against Dengue Virus Replication during the Post-Entry Stage.

Dengue virus (DENV) poses a significant global health challenge, with millions of cases each year. Developing effective antiviral drugs against DENV remains a major hurdle. Varenicline is a medication used to aid smoking cessation, with anti-inflammatory and antioxidant effects. In this study, varenicline was investigated for its antiviral potential against DENV. This study provides evidence of the antiviral activity of varenicline against DENV, regardless of the virus serotype or cell type used. Varenicline demonstrated dose-dependent effects in reducing viral protein expression, infectivity, and virus yield in Vero and A549 cells infected with DENV-1 and DENV-2, with EC50 values ranging from 0.44 to 1.66 μM. Time-of-addition and removal experiments demonstrated that varenicline had a stronger inhibitory effect on the post-entry stage of DENV-2 replication than on the entry stage, as well as the preinfection and virus attachment stages. Furthermore, cell-based trans-cleavage assays indicated that varenicline dose-dependently inhibited the proteolytic activity of DENV-2 NS2B-NS3 protease. Docking models revealed the formation of hydrogen bonds and van der Waals forces between varenicline and specific residues in the DENV-1 and DENV-2 NS2B-NS3 proteases. These results highlight the antiviral activity and potential mechanism of varenicline against DENV, offering valuable insights for further research and development in the treatment of DENV infection.

Synergistic correlation between host angiogenin and dengue virus replication

ABSTRACT DENV infection poses a major health concern globally and the pathophysiology relies heavily on host-cellular machinery. Although virus replication relies heavily on the host, the mechanistic details of DENV–host interaction is not fully characterized yet. Here, we are focusing on characterizing the mechanistic basis of virus-induced stress on the host cell. Specifically, we aim to characterize the role of the stress modulator ribonuclease Angiogenin during DENV infection. Our results suggested that the levels of Angiogenin are up-regulated in DENV-infected cells and the levels increase proportionately with DENV replication. Our efforts to knockdown Angiogenin using siRNA were unsuccessful in DENV-infected cells but not in mock-infected control. To further investigate the modulation between DENV replication and Angiogenin, we treated Huh7 cells with Ivermectin prior to DENV infection. Our results suggest a significant reduction in DENV replication specifically at the later stages as a consequence of Ivermectin treatment. Interestingly, Angiogenin levels were also found to be decreased proportionately. Our results suggest that Angiogenin modulation during DENV infection is important for DENV replication and pathogenesis.

A cellular screening platform, stably expressing DENV2 NS5, defines a novel anti-DENV mechanism of action of Apigenin based on STAT2 activation

Type I interferon (IFN–I) evasion by Dengue virus (DENV) is key in DENV pathogenesis. The non-structural protein 5 (NS5) antagonizes IFN-I response through the degradation of the signal transducer and activator of transcription 2 (STAT2). We developed a K562 cell-based platform, for high throughput screening of compounds potentially counteracting the NS5-mediated antagonism of IFN-I signaling. Upon a screening with a library of 1220 approved drugs, 3 compounds previously linked to DENV inhibition (Apigenin, Chrysin, and Luteolin) were identified. Luteolin and Apigenin determined a significant inhibition of DENV2 replication in Huh7 cells and the restoration of STAT2 phosphorylation in both cell systems. Apigenin and Luteolin were able to stimulate STAT2 even in the absence of infection. Despite the “promiscuous” and “pan-assay-interfering” nature of Luteolin, Apigenin promotes STAT2 Tyr 689 phosphorylation and activation, highlighting the importance of screening for compounds able to interact with host factors, to counteract viral proteins capable of dampening innate immune responses.

Antiviral Activity of Propyl Gallate against Replication of Dengue Virus Serotype 2: In Vitro and In Silico Study

Introduction: Dengue fever (DF) and dengue hemorrhagic fever (DHF) are infectious diseases caused by dengue virus (DENV) with high mortality rates. The mainstay of treatment for DENV infection is supportive, since there were no commercial specific antiviral drug for DENV. Propyl gallate has potential to be an antivirus for DENV. However, the mechanism is still unknown. This study aims to identify the activity of propyl gallate in DENV-2 replication in vitro and analyze the binding energy of propyl gallate towards NS3 and NS5 protein in silico.&#x0D; Methods: We used DENV serotype 2 New Guinea C and Vero cells for in vitro study. Focus and MTT assay was conducted to measure inhibition percentage and to measure viability percentage. Furthermore, in silico was conducted to identify the binding energy and inhibition constant of propyl gallate towards NS3 dan NS5 protein.&#x0D; Results: The percentage inhibition of pre and post infection was 4,34±7,53% and 30,7±4,88% with viability of 94,64±0,4% and 95,31±3,38%, respectively. The binding energy of propyl gallate with NS5, NS3 protease, and NS3 helicase were -3,49 kcal/mol, -2,47 kcal/mol, and -3,72 kcal/mol.&#x0D; Conclusion: Propyl gallate has high inhibition activity towards DENV-2 adhesion-replication in vitro with low binding energy to NS5 and NS3 in silico.&#x0D;

N-Butanol Extract of Glycyrrhizae Radix et Rhizoma Inhibits Dengue Virus through Targeting Envelope Protein.

At present, about half of the world's population is at risk of being infected with dengue virus (DENV). However, there are no specific drugs to prevent or treat DENV infection. Glycyrrhizae Radix et Rhizome, a well-known traditional Chinese medicine, performs multiple pharmacological activities, including exerting antiviral effects. The aim of this study was to investigate the anti-DENV effects of n-butanol extract from Glycyrrhizae Radix et Rhizome (GRE). Compounds analysis of GRE was conducted via ultra-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). The antiviral activities of GRE were determined by the CCK-8 assay, plaque assay, qRT-PCR, Western blotting, and the immunofluorescence assay. The DENV-infected suckling mice model was constructed to explore the antiviral effects of GRE in vivo. Four components in GRE were analyzed by UHPLC-MS/MS, including glycyrrhizic acid, glycyrrhetnic acid, liquiritigenin, and isoliquiritigenin. GRE inhibited the attachment process of the virus replication cycle and reduced the expression of the E protein in cell models. In the in vivo study, GRE significantly relieved clinical symptoms and prolong survival duration. GRE also significantly decreased viremia, reduced the viral load in multiple organs, and inhibited the release of pro-inflammatory cytokines in DENV-infected suckling mice. GRE exhibited significant inhibitory activities in the adsorption stage of the DENV-2 replication cycle by targeting the envelope protein. Thus, GRE might be a promising candidate for the treatment of DENV infection.

Discrepant Activation Pattern of Inflammation and Pyroptosis Induced in Dermal Fibroblasts in Response to Dengue Virus Serotypes 1 and 2 and Nonstructural Protein 1.

Four serotypes of dengue virus (DENV-1 to DENV-4) cause mild to severe disease in humans through infected mosquito bites. Dermal fibroblasts were found to be susceptible to DENV, and this may play a critical role in establishing the initial infection stage. However, the cellular response induced by the different DENV serotypes in dermal fibroblasts during the early stage of infection remains unclear. To determine this, normal human dermal fibroblast WS1 cells were infected with DENV-1 or DENV-2. Compared with the response elicited by DENV-1 infection, DENV-2 induced a stronger innate inflammatory response and cell death in the WS1 cells. However, DENV-1 activated a higher level of pyroptosis signaling than did DENV-2, which was associated with higher virion production. Caspase-1 inhibitor Ac-YVAD-cmk and imipramine, an antidepressant drug, reduced DENV-mediated caspase-1 and interleukin 1β (IL-β) cleavage in the pyroptosis pathway. Ac-YVAD-cmk and imipramine downregulated DENV virion production in WS1 cells. Furthermore, DENV-1 and DENV-2 NS1 proteins promoted diverse activation levels of cell death, inflammatory response, and activation of caspase-1 and IL-β in dermal fibroblasts at different time points. Collectively, these data suggest that DENV-1, DENV-2, and their nonstructural protein 1 (NS1) induce discrepant activation patterns of inflammation and pyroptosis in dermal fibroblasts. The pyroptosis caused by virus and NS1 may facilitate DENV replication in dermal fibroblasts. IMPORTANCE Skin fibroblasts are the primary cells of DENV infection through mosquito bites. Establishing a successful infection in dermal fibroblasts might be critical for dengue disease. However, the cellular response induced by DENV in dermal fibroblasts remains unclear. In this in vitro study, we found that DENV-2 and DENV-1 showed different time course patterns of virus replication and inflammation in dermal fibroblasts. We demonstrated that DENV-1 and DNEV-2 and their viral protein NS1 activate the cellular pyroptosis response to regulate virus replication in dermal fibroblasts. This finding suggests that pyroptosis activation in the DENV primary inoculation site plays a role in the establishment of a DENV infection.

Dengue virus induced autophagy is mediated by HMGB1 and promotes viral propagation

Dengue virus (DENV) exploits various cellular pathways including autophagy to assure enhanced virus propagation. The mechanisms of DENV mediated control of autophagy pathway are largely unknown. Our investigations have revealed a novel role for high-mobility group box1 protein (HMGB1) in regulation of cellular autophagy process in DENV-2 infected A549 cell line. While induction of autophagy by rapamycin treatment resulted in enhanced DENV-2 propagation, the blockade of autophagy flux with bafilomycin A1 suppressed viral replication. Furthermore, siRNA-mediated silencing of HMGB1 significantly abrogated dengue induced autophagy, while LPS induced HMGB1 expression counteracted these effects. Interestingly, silencing of HMGB1 showed reduction of BECN1 and stabilization of BCL-2 protein. On the contrary, LPS induction of HMGB1 resulted in enhanced BECN1 and reduction in BCL-2 levels. This study shows that the modulation of autophagy by DENV-2 is HMGB1/BECN1 dependent. In addition, glycyrrhizic acid (GA), a potent HMGB1 inhibitor suppressed autophagy as well as DENV-2 replication. Altogether, our data suggests that HMGB1 induces BECN1 dependent autophagy to promote DENV-2 replication.