DENV Infection Research Articles

Dengue Virus Infection: Immune Response and Therapeutic Targets.

Flavivirus infection, especially dengue virus infection caused by DENV, is known to be a significant health concern globally owing to the high incidence and mortality rate. The expanding and increasing disease burden calls for the need to develop an effective treatment and prevent the event of fatal complications, including dengue hemorrhagic fever/dengue shock syndrome. The DENV-induced immune response has been described as paradoxical because it has a protective role in viral clearance but, at the same time, causes more severe infection through viral-specific immunity. This is further complicated by high homology and cross-reactivity between different serotypes of DENV, causing a more severe disease presentation during secondary infection by a heterologous serotype. This serotype complexity poses a challenge for the development of a universal flavivirus vaccine. This review highlights the significance of high motility group box 1 (HMGB1) and nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) inflammasome activation pathways in initiating an inflammatory response through the downstream activation of nuclear factor κB and proinflammatory cytokine Interleukin (IL)-1B, IL-18 release in DENV infection. It also discusses the role of NLRP3 in activating cellular apoptosis and pyroptosis leading to systemic failure, especially in peripheral tissues. Over the decades, there has been much progress in understanding the immunopathogenesis of DENV infection. Researchers have been studying key pathogenic molecules for potential therapeutic targets including HMGB1 and NLRP3 inflammasome inhibitors, which is explored in this review. Ultimately, although there is not yet an effective antiviral or vaccine for DENV, immunomodulators continue to pave the way to decrease disease severity in infected individuals.

Autochthonous dengue outbreak in Lombardy, Italy: enhanced surveillance and vector control measures

Abstract Description of the problem On 18 August 2023, the first autochthonous case of dengue was registered in Lombardy, in a person living in a village in Lodi province. The case, a man with no history of travels prior to symptoms onset, developed flu-like symptoms on 3 August. After initial suspicion of WNV infection, he was confirmed to have a DENV-1 infection. Following the notification, measures to control the spread of the disease were activated, including alert of healthcare professionals, vector control activities, entomological monitoring and active case finding among household contacts and people living close to the case. Moreover, a serological screening was proposed to population of the entire municipality, along with a survey collecting data about travels abroad and symptoms. Results Forty-one confirmed and six probable autochthonous cases were identified. The last case developed symptoms on 20 October. Among confirmed cases, 24 were identified after admission to Emergency Department, 16 through community screening and one at the time of blood donation. 38/41 confirmed cases were symptomatic, with fever, rash and retro-orbital pain being the most common symptoms. Mean age was 55,5 years (3-88), while male: female ratio was 2:1. Hospitalisation was required for 7/41 cases. Community screening was offered from 25 August to 8 October and 416 individuals participated. Among them 10 tested positive for DENV IgG and 27 for DENV IgM (16 were subsequently confirmed as DENV cases, 6 remained probable and 5 were ruled out as non-cases). The primary case of the outbreak was not identified. Vector control activities and entomological monitoring were conducted on 21 August within 200-m radius area around the residence and places visited by index case. Subsequent interventions were conducted in all public areas and sensitive sites of the municipality. Lessons Enhancement of surveillance through community screening allowed identification of several additional autochthonous cases. Key messages • Strengthening of surveillance, vector control activities and entomological monitoring is crucial to prevent and control future outbreaks in non-endemic areas with stable presence of Aedes mosquitoes. • To minimize the risk of introduction and spread of dengue virus in non-endemic areas, it is essential to raise awareness among travellers and general population about preventive and control measures.

Serotype-specific epidemiological patterns of inapparent versus symptomatic primary dengue virus infections: a 17-year cohort study in Nicaragua.

Dengue is the most prevalent mosquito-borne viral disease and a major public health problem worldwide. Most primary infections with the four dengue virus serotypes (DENV1-4) are inapparent; nonetheless, whether the distribution of symptomatic versus inapparent infections by serotype varies remains unknown. Here, we present (1) the evaluation of a DENV1-4 envelope domain III multiplex microsphere-based assay (EDIII-MMBA) to serotype inapparent primary infections and (2) its application leveraging 17 years of prospective sample collection from the Nicaraguan Pediatric Dengue Cohort Study (PDCS). We analysed primary DENV infections in the PDCS from 2004 to 2022 detected by inhibition ELISA (iELISA) or RT-PCR. First, we evaluated the performance of the EDIII-MMBA for serotyping with samples characterised by RT-PCR or focus reduction neutralisation test. Next, we analysed a subset of inapparent primary DENV infections in the PDCS with the EDIII-MMBA to evaluate the epidemiology of inapparent infections. Remaining infections were inferred using stochastic imputation, taking year and neighbourhood into account. Infection incidence and percentage of inapparent, symptomatic, and severe infections were analysed by serotype. Between Aug 30, 2004, and March 10, 2022, a total of 5931 DENV-naive participants were followed in the PDCS. There were 1626 primary infections (382 symptomatic, 1244 inapparent) detected by iELISA or RT-PCR over the study period. The EDIII-MMBA demonstrated excellent overall accuracy (100%, 95% CI 95·8-100) for serotyping inapparent primary DENV infections when evaluated against gold-standard serotyping methods. Of the 1244 inapparent infections, we analysed 574 (46%) using the EDIII-MMBA. We found that the majority of primary infections were inapparent, with DENV3 exhibiting the highest likelihood of symptomatic (pooled odds ratio compared with DENV1: 2·13, 95% CI 1·28-3·56) and severe (6·75, 2·01-22·62) primary infections, whereas DENV2 was similar to DENV1 in both analyses. Considerable within-year and between-year variation in serotype distribution between symptomatic and inapparent infections and circulation of serotypes undetected in symptomatic cases were observed in multiple years. Our study indicates that case surveillance skews the perceived epidemiological footprint of DENV. We reveal a more complex and intricate pattern of serotype distribution in inapparent infections. The substantial differences in infection outcomes by serotype emphasises the need for vaccines with balanced immunogenicity and efficacy across serotypes. National Institute of Allergy and Infectious Diseases (National Institutes of Health) and Bill & Melinda Gates Foundation. For the Spanish translation of the abstract see Supplementary Materials section.

Dengue virus surveillance in Nepal yields the first on-site whole genome sequences of isolates from the 2022 outbreak

BackgroundThe 4 serotypes of dengue virus (DENV1-4) can each cause potentially deadly dengue disease, and are spreading globally from tropical and subtropical areas to more temperate ones. Nepal provides a microcosm of this global phenomenon, having met each of these grim benchmarks. To better understand DENV transmission dynamics and spread into new areas, we chose to study dengue in Nepal and, in so doing, to build the onsite infrastructure needed to manage future, larger studies.Methods and resultsDuring the 2022 dengue season, we enrolled 384 patients presenting at a hospital in Kathmandu with dengue-like symptoms; 79% of the study participants had active or recent DENV infection (NS1 antigen and IgM). To identify circulating serotypes, we screened serum from 50 of the NS1+ participants by RT-PCR and identified DENV1, 2, and 3 – with DENV1 and 3 codominant. We also performed whole-genome sequencing of DENV, for the first time in Nepal, using our new on-site capacity. Sequencing analysis demonstrated the DENV1 and 3 genomes clustered with sequences reported from India in 2019, and the DENV2 genome clustered with a sequence reported from China in 2018.ConclusionThese findings highlight DENV’s geographic expansion from neighboring countries, identify China and India as the likely origin of the 2022 DENV cases in Nepal, and demonstrate the feasibility of building onsite capacity for more rapid genomic surveillance of circulating DENV. These ongoing efforts promise to protect populations in Nepal and beyond by informing the development and deployment of DENV drugs and vaccines in real time.

Preliminary survey of three mosquito-borne viruses using a self-established multiplex RT-qPCR assay in Chinese blood donors

BackgroundMosquito-borne pathogens pose a significant threat to both human health and blood safety. The primary mosquito-borne viruses that present this threat are Zika virus, Chikungunya virus, and Dengue virus. At present, there are limited efficacious vaccines or therapeutic drugs for the prevention and treatment of these viral infections. Blood donors can remain asymptomatically infected and unfortunately, screening for these three viruses in Chinese blood donors are not mandatory, leaving the residual risk to transfusion recipients uncertain. Objective: To address this, we developed a single-tube multiplex RT-qPCR assay for ZCD detection and was preliminarily employed to screen a total of 10,566 blood donations in Nanning Blood Center in order to assess the prevalence risk of these pathogens in blood donors. Results: None of the blood samples was reactive for ZCD by nucleic acid test (NAT). One out of 173 donations (1/173, 0.58 %) was IgG positive for ZIKV and 14 (14/173, 8.4 %) were IgG positive for DENV. None of these 173 donations was IgG positive for Chikungunya virus. These findings suggest that the prevalence of ZCD infection in blood donors in Nanning is very low although past DENV infection (IgG positive) was relatively common. Conclusion: A single-tube multiplex RT-qPCR assay for simultaneous detection of ZCD viruses was successfully established and applied for screening in blood donors. The residual risk of ZCD infection through transfusion is currently low in Nanning, China. The NAT assay for ZCD will serve as a technical reserve in response to future epidemic or pandemic of mosquito-borne pathogens.

Computational identification of Vernonia cinerea-derived phytochemicals as potential inhibitors of nonstructural protein 1 (NSP1) in dengue virus serotype-2.

Dengue virus (DENV) infection, spread by Aedes aegypti mosquitoes, is a significant public health concern in tropical and subtropical regions. Among the four distinct serotypes of DENV (DENV-1 to DENV-4), DENV-2 is associated with the highest number of fatalities worldwide. However, there is no specific treatment available for dengue patients caused by DENV-2. This study aimed to identify inhibitory phytocompounds in silico in Vernonia cinerea (V. cinerea), a widely used traditional medicinal plant, for treating DENV-2 associated illnesses. The chemical structures of 17 compounds from V. cinerea were sourced from the Indian Medicinal Plants, Phytochemistry, and Therapeutics (IMPPAT) database. These compounds underwent geometry optimization, were screened against nonstructural protein 1 (NSP1) of DENV-2, and further validated through molecular dynamics simulations (MDS). Baicalein, an established drug against DENV-2, was used for validation in molecular screening, MDS, and MM-GBSA analyses. Among these compounds, Beta-amyrin, Beta-amyrin acetate, Chrysoeriol, Isoorientin, and Luteolin showed promising potential as inhibitors of the NSP1 of DENV-2, supported by the results of thermodynamic properties, molecular orbitals, electrostatic potentials, spectral data and molecular screening. Besides, these compounds adhered to the Lipinski's "rule of 5", showing no hepatotoxicity/cytotoxicity, with mixed mutagenicity, immunotoxicity, and carcinogenicity. Furthermore, final validation through MDS confirmed their potential, demonstrating stable tendencies with significant inhibitory activities against NSP1 of DENV-2 over the control drug Baicalein. Among the screened compounds, Chrysoeriol emerged as the most promising inhibitor of NSP1 of DENV-2, followed by Luteolin and Isoorientin. Taken together, our results suggest that Chrysoeriol is the best inhibitor of NSP1 of DENV-2, which could be evaluated as a therapeutic agent or a lead compound to treat and manage DENV-2 infections.

Detection and differentiation of antinuclear antibodies in serum of dengue suspected patients with or without systemic autoimmune disease in Kolkata, India

ABSTRACT The pathophysiology of dengue may be influenced by antibodies released during infection. Several autoimmune diseases are accompanied by antinuclear antibodies (ANAs) but 8–10% of the general population have positive ANA tests. To test the hypothesis that an ANA-positive test indicates an immune dysregulated state that modifies the risk for certain clinical disorders in people with or without an autoimmune disease, we examined the various ANA profiles and their relationships to various autoimmune disorders, as well as the severity of these relationships, in patients infected with dengue fever. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) methods were used. Indirect immunofluorescence assay (IIFA) and line immunoassay (LIA) were performed to detect and differentiate the ANAs among dengue infected patients. Out of 135 dengue virus-positive patients, 94.07% were positive by ELISA and 5.93% positive by RT-PCR method. ANAs by IIFA and LIA were detected in 54.8% and 18.5% of the dengue positive patients, respectively, and 10.3% and 7.1% of the 126 dengue negative patients, respectively. This study showed that dengue was associated with an increased risk of autoimmune myositis and mixed connective tissue disease (MCTD), a rare complication of dengue. The risk of other autoimmune diseases did not seem to increase after DENV infection.

The ApoA1-mimetic peptide 4F blocks flavivirus NS1-triggered endothelial dysfunction and protects against lethal dengue virus challenge

Flavivirus infections result in a variety of outcomes, from clinically inapparent infections to severe, sometimes fatal cases characterized by hemorrhagic manifestations and vascular leakage leading to shock (dengue), meningomyeloencephalitis (West Nile), and congenital abnormalities (Zika). Although there are approved vaccines against several flaviviruses, potentially enhancing cross-reactive immune responses have complicated the development and implementation of vaccines against dengue and Zika viruses, and no specific therapeutics currently exist. The flavivirus nonstructural protein 1 (NS1) is a promising antiviral target because it is a conserved multifunctional virulence factor that directly triggers vascular leak. We previously showed that interactions between NS1 and the ApoA1 lipoprotein modulate DENV infection. Here, we evaluated the potential of the ApoA1-mimetic peptide, 4F, to interfere with endothelial dysfunction mediated by the NS1 protein of dengue, Zika, and West Nile flaviviruses. In an in vitro model consisting of human endothelial cell monolayers, 4F inhibited NS1-induced hyperpermeability, as measured by a transendothelial electrical resistance assay, and prevented NS1-triggered disruption of the endothelial glycocalyx layer. We also demonstrate that treatment with 4F inhibited NS1 interaction with endothelial cells. Finally, we show that 4F protects against lethal DENV challenge in a mouse model, reducing morbidity and mortality in a dose-dependent manner. Our data demonstrate the potential of 4F to inhibit flavivirus NS1-mediated pathology and severe dengue disease in mice and suggest that 4F can also serve as a molecular tool to probe different NS1 functions in vitro and in vivo.

Quantification of the interaction forces between dengue virus and dopamine type-2 receptor using optical tweezers

BackgroundDengue virus (DENV) causes the most significant mosquito-borne viral disease with a wide spectrum of clinical manifestation, including neurological symptoms associated with lethal dengue diseases. Dopamine receptors are expressed in central nervous system, and dopamine antagonists have been reported to exhibit antiviral activity against DENV infection in vivo and in vitro. Although identification of host-cell receptor is critical to understand dengue neuropathogenesis and neurotropism, the involvement of dopamine receptors in DENV infection remains unclear.ResultsWe exploited the sensitivity and precision of force spectroscopy to address whether dopamine type-2 receptors (D2R) directly interact with DENV particles at the first step of infection. Using optical tweezers, we quantified and characterized DENV binding to D2R expressed on Chinese hamster ovary (CHO) cells. Our finding suggested that the binding was D2R- and DENV-dependent, and that the binding force was in the range of 50–60 pN. We showed that dopamine antagonists prochlorperazine (PCZ) and trifluoperazine (TFP), previously reported to inhibit dengue infection, interrupt the DENV-D2R specific binding.ConclusionsThis study demonstrates that D2R could specifically recognize DENV particles and function as an attachment factor on cell surfaces for DENV. We propose D2R as a host receptor for DENV and as a potential therapeutic target for anti-DENV drugs.Graphical abstract

Prevalence of dengue, Zika, and chikungunya virus infections among mosquitoes in Asia: A systematic review and meta-analysis

BackgroundDengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) continue to pose significant public health risks. This study aims to assess the prevalence of these arbovirus infections in field-caught mosquitoes across Asia. MethodsStudies published after the year 2000 on DENV, ZIKV, and/or CHIKV infections in Asian mosquitoes were identified from Embase, Scopus, PubMed, and Ovid. A random-effects model estimated the pooled prevalence, defined as the overall prevalence from included studies, adjusted for variability among the studies. Meta-regression models were used to evaluate the association between predictors and their prevalence. ResultsA total of 2529 articles were retrieved; 57 met the inclusion criteria. Pooled prevalence of DENV, ZIKV, and CHIKV infections in Asian mosquitoes were 5.85%, 2.15%, and 1.26%, respectively. Subgroup analysis revealed varying DENV prevalence across regions: East Asia (3.32%), South Asia (5.26%), and Southeast Asia (6.92%). Univariate regression analysis demonstrated significant associations between mosquito capture site and DENV prevalence (P < 0.001), and between study region and ZIKV prevalence (P = 0.005). However, no significant predictors were identified for CHIKV prevalence. ConclusionOur findings provide reference pooled summary estimates of arbovirus infections in mosquitoes, offering crucial insight into the regional disease burden and - guidance in the development and implementation of arbovirus surveillance in mosquitoes.

Effects of the oxoaporphine alkaloid hernandonine on dengue virus. Evidence for its mechanisms of action

BackgroundDengue, caused by the dengue virus (Orthoflavivirus dengue, encompassing DENV types 1-4), is a member of the Flaviviridae family. The symptoms of dengue range from subclinical or mild manifestations to potentially fatal complications. The management of severe dengue is exceptionally challenging due to the absence of effective antiviral medications. In this context, natural products, whether in the form of pure compounds or standardized plant extracts, have emerged as a promising source for the development of novel antiviral therapeutics. Hernandonine, an oxoaporphine alkaloid found in Hernandia nymphaeifolia (C. Presl) Kubitzki. serves both as a metabolite and an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase. PurposeThis study investigated the ability of hernandonine to inhibit DENV infection and explored its potential mechanisms. Study designTo assess the in vitro anti-DENV activity, cells or induced pluripotent stem cell (iPSC)-derived cerebral organoids were exposed to hernandonine before or after infection with DENV. Along with hernandonine, the endocytosis modulators, genistein, wortmannin, Methyl-β-cyclodextrin (MβCD) and lovastatin, were used in the assays. MethodsThe DENV infectivity and virion production in cells or cerebral organoids treated with compounds were determined. Various methods, including cell and cerebral organoids imaging, protein and gene detection were conducted to explore their antiviral mechanisms. ResultsThe results revealed notable antiviral properties of hernandonine, particularly in inhibiting DENV during the early stages of infection. Mechanistic analysis demonstrated that, akin to genistein, wortmannin, methyl-β-cyclodextrin (MβCD), and lovastatin, hernandonine exerted an influence on cholesterol-rich lipid rafts. It also restrained the pseudopodial movement ability of cells, potentially through the downregulation of cytoskeleton and endocytosis regulatory genes or protein expression. Moreover, hernandonine's virucidal activity was demonstrated. Hernandonine's inhibition of DENV infection was further validated in a disease-relevant iPSC-derived cerebral organoids model, a novel DENV-2 infection system worthy of further application. ConclusionThis study evidenced the potential of hernandonine as a novel candidate in the fight against DENV infection.

Dengue virus: pathogenesis and potential for small molecule inhibitors.

Dengue, caused by dengue virus (DENV), is now endemic in nearly 100 countries and infection incidence is reported in another 30 countries. Yearly an estimated 400 million cases and 2200 deaths are reported. Effective vaccines against DENV are limited and there has been significant focus on the development of effective antiviral against the disease. The World Health Organization has initiated research programs to prioritize the development and optimization of antiviral agents against several viruses including Flaviviridae. A significant effort has been taken by the researchers to develop effective antivirals against DENV. Several potential small-molecule inhibitors like efavirenz, tipranavir and dasabuvir have been tested against envelope and non-structural proteins of DENV, and are in clinical trials around the world. We recently developed one small molecule, namely 7D, targeting the host PF4-CXCR3 axis. 7D inhibited all 4 serotypes of DENV in vitro and specifically DENV2 infection in two different mice models. Although the development of dengue vaccines remains a high priority, antibody cross reactivity among the serotypes and resulting antibody-dependent enhancement (ADE) of infection are major concerns that have limited the development of effective vaccine against DENV. Therefore, there has been a significant emphasis on the development of antiviral drugs against dengue. This review article describes the rescue effects of some of the small molecule inhibitors to viral/host factors associated with DENV pathogenesis.

Immunization of recombinant NS3 protein (protease region) of dengue virus induces high levels of CTLA-4 and apoptosis in splenocytes of BALB/c mice.

DENV infection outcomes depend on the host's variable expression of immune receptors and mediators, leading to either resolution or exacerbation. While the NS3 protein is known to induce robust immune responses, the specific impact of its protease region epitopes remains unclear. This study investigated the effect of recombinant NS3 protease region proteins from all four DENV serotypes on splenocyte activation in BALB/c mice (n = 5/group). Mice were immunized with each protein, and their splenocytes were subsequently stimulated with homologous antigens. We measured the expression of costimulatory molecules (CD28, CD80, CD86, CD152) by flow cytometry, along with IL-2 production, CD25 expression, and examined the antigen-specific activation of CD4 + and CD8 + T cells. Additionally, the expression of IL-1, IL-10, and TGF-β1 in splenocytes from immunized animals was assessed. Apoptosis was evaluated using Annexin V/PI staining and DNA fragmentation analysis. Stimulation of splenocytes from immunized mice triggered apoptosis (phosphatidylserine exposure and caspase 3/7 activation) and increased costimulatory molecule expression, particularly CD152. Low IL-2 production and low CD25 expression, as well as sustained expression of the IL-10 gene. These results suggest that these molecules might be involved in mechanisms by which the NS3 protein contributes to viral persistence and disease pathogenesis.

Impact of pre-existing cross-reactive antibodies on cyclic dengue outbreaks in the hyperendemic region of Bali, Indonesia

The four serotypes of the dengue virus (DENV) cause a range of diseases ranging from mild fever to severe conditions. Understanding the immunological interactions among the four serotypes is crucial in comprehending the dynamics of serotype shifting during outbreaks in areas where all four serotypes co-circulate. Hence, we evaluated the neutralizing antibody and antibody-dependent enhancement responses against the four DENV serotypes using acute-phase plasma samples collected from 48 laboratory-confirmed dengue patients during a dengue outbreak in Bali, Indonesia in 2022. Employing single-round infectious particles to exclusively investigate immunogenicity to the structural surface proteins of DENV, which are the targets of antibodies, we found that individuals with a probable prior history of DENV-1 infection exhibited increased susceptibility to secondary DENV-3 infection, attributed to cross-reactive antibodies with limited neutralizing activity against DENV-3 (geometric mean 50 % neutralization titer (GMNT50) = 47.6 ± 11.5). This susceptibility was evident in vitro, with a mean fold enhancement of 28.4 ± 33.9. Neutralization titers against DENV-3 were significantly lower compared to other serotypes (DENV-1 GMNT50 = 678.1 ± 9.0; DENV-2 GMNT50 = 210.5 ± 8.7; DENV-4 GMNT50 = 95.14 ± 7.0). We demonstrate that prior immunity to one serotype provides limited cross-protection against the other serotypes, influencing the dominant serotype in subsequent outbreaks. These findings underscore the complexity of dengue immunity and its implications for vaccine design and transmission dynamics in hyperendemic regions.

Dengue severity by serotype and immune status in 19 years of pediatric clinical studies in Nicaragua.

Dengue virus, a major global health threat, consists of four serotypes (DENV1-4) that cause a range of clinical manifestations from mild to severe and potentially fatal disease. This study, based on 19 years of data from the Pediatric Dengue Cohort Study and Pediatric Dengue Hospital-based Study in Managua, Nicaragua, investigates the relationship of serotype and immune status with dengue severity. Dengue cases were confirmed by molecular, serological, and/or virological methods, and sudy participants 6 months to 17 years old were followed during their hospital stay or as ambulatory patients. We enrolled a total of 15,266 participants, of whom 3,227 (21%) were positive for DENV infection. Of 2,630 cases with serotype result by RT-PCR, 557 corresponded to DENV1, 992 to DENV2, 759 to DENV3 and 322 to DENV4. Severe disease was more prevalent among secondary DENV2 and DENV4 cases, while similar disease severity was observed in both primary and secondary DENV1 and DENV3 cases. According to the 1997 World Health Organization (WHO) severity classification, both DENV2 and DENV3 caused a higher proportion of severe disease compared to other serotypes, whereas DENV3 caused the greatest percentage of severity according to the WHO-2009 classification. DENV2 was associated with increased odds of pleural effusion and low platelet count, while DENV3 was associated with both hypotensive and compensated shock. These findings demonstrate differences in dengue severity by serotype and immune status and emphasize the critical need for a dengue vaccine with balanced effectiveness against all four serotypes, particularly as existing vaccines show variable efficacy by serotype and serostatus.

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.

In Silico Analysis of Active Compounds from Allium tuberosum as Drug Candidate for Inhibitor DENV-3 Envelope Protein

Dengue fever has become a global health issue, the development of dengue vaccine has not yet been established. Medicinal plants are an ideal alternative for DENV infection drugs. The purpose of this study was to determine in silico the potential of active compounds from Allium tuberosum as envelope protein inhibitors of DENV-3. The method of this research is to do docking analysis of compounds with DENV-3 envelope protein and analysis of amino acid residues using MVD, pharmacokinetic analysis using SwissADME, toxicity analysis using ProTox-II. The best docking value for the potential activity to inhibit the receptor DENV-3 is the thymidine compound (RS: -81.1245 kcal/mol). The highest activity of thymidine is the most promising as a drug candidate, as evidenced by the toxicity analysis which is predicted to have non-carcinogenic, non-mutagenic, inactive properties against hepatotoxicity, cytotoxicity and immunotoxicity parameters, as well as pharmacokinetic analysis that fulfills 6 parameters of lipopolicity, molecular weight, polarity. , insolubility, insaturation, and flexibility which indicate the drug candidate of thymidine is safe for its bioavailability. The conclusion from the results of this study is that one compound has the ability as an antiviral, binding score with DENV-3 is good, and is safe in terms of pharmacokinetics and toxicity, namely thymidine compound.

Inhibition of dengue virus infection in vitro by fucoidan and polysaccharide extract from marine alga Sargassum spp.

Dengue virus (DENV) infection poses a global health threat, leading to severe conditions with the potential for critical outcomes. Currently, there are no specific drugs available whereas the vaccine does not offer comprehensive protection across all DENV serotypes. Therefore, the development of potential antiviral agents is necessary to reduce the severity risk and interrupt the transmission circuit. The search for effective antiviral agents against DENV has predominantly focused on natural resources, particularly those demonstrating diverse biological activities and high safety profiles. Cyanobacteria and algae including Leptolyngbya sp., Spirulina sp., Chlorella sp., and Sargassum spp., which are prevalent species in Thailand, have been reported for their diverse biological activities and high safety profiles. However, their anti-DENV activity has not been documented. In this study, the screening assay was performed to compare the antiviral activity against DENV of crude polysaccharide and ethanolic extracts derived from 4 species of cyanobacteria and algae in Vero cells. Polysaccharide extracts from Sargassum spp. were the most effective in inhibiting DENV-2 infection under co-infection conditions, where the virus was exposed to the extract at the time of infection. Treatment of the extract significantly reduced the ability of DENV to bind to the host cells to 47.87 ± 3.88 % while treatment upon virus binding step had no antiviral effect suggesting the underlaying mechanism of the extract on interfering virus binding step. Fucoidan, a key bioactive substance in Sargassum polysaccharide, showed to reduce DENV-2 infection to 26.59 ± 5.01 %, 20.46 ± 6.58 % under the co-infection condition in Vero and A549 cells, respectively. In accompanied with Sargassum polysaccharide, fucoidan disturbed the virus binding to the host cells. These findings warrant further development and exploration of the Sargassum-derived polysaccharide, fucoidan, as a promising candidate for combating DENV infections.

Genomic Variation in Dengue Virus Non-Structural Protein 5 (NS5).

Objective: To identify the most frequently repeated mutations in the NS5 protein in DENV RNA isolated from dengue patients in the province Punjab of Pakistan.Methodology: The study was conducted at Aziz Fatimah Hospital and Allied Hospital Faisalabad during the 2022 dengue outbreak. The temporal sampling method was applied while selecting confirmed dengue patients from both hospitals, and written consent was taken. 120 blood samples were selected in total and after quantification only 23 samples were found fit for whole genome sequencing. Data was uploaded to Torrent Suite Server 4.10 for analysis. Epi Data Analysis, a WHO-developed software program, was used to calculate and summarize mutation frequencies. Excel analysis was used to check for flaws in the data.Results: There were a total of 199 mutations across the entire nonstructural protein 5 (NS5) sequence. NS5 mutations were found in 9 genomic isolates, with Sample (S) 17 having the highest number.Conclusion: The presence of large number of mutations in the structure of NS5 protein may be used in designing specific anti-viral against it, for better management of DENV infection in local settings in future.

Serotype-Specific Epidemiological Patterns of Inapparent versus Symptomatic Primary Dengue Virus Infections: A 17-year cohort study in Nicaragua.

Dengue is the most prevalent mosquito-borne viral disease and a major public health problem worldwide. Most primary infections with the four dengue virus serotypes (DENV1-4) are inapparent; nonetheless, whether the distribution of symptomatic versus inapparent infections by serotype varies remains unknown. Here, we present (1) the evaluation of a multiplex DENV1-4 envelope domain III multiplex microsphere-based assay (EDIII-MMBA) to serotype inapparent primary infections and (2) its application leveraging 17 years of prospective sample collection from the Nicaraguan Pediatric Dengue Cohort Study (PDCS). First, we evaluated the performance of the EDIII-MMBA with samples characterized by RT-PCR or focus reduction neutralization test. Next, we analyzed 46% (N=574) of total inapparent primary DENV infections in the PDCS with the EDIII-MMBA to evaluate the epidemiology of inapparent infections. Remaining infections were inferred using stochastic imputation, taking year and neighborhood into account. Infection incidence and percentage of inapparent, symptomatic, and severe infections were analyzed by serotype. The EDIII-MMBA demonstrated excellent overall accuracy (100%, 95.8-100%) for serotyping symptomatic and inapparent primary DENV infections when evaluated against gold-standard serotyping methods. We found that a significant majority of primary infections were inapparent, with DENV3 exhibiting the highest likelihood of symptomatic and severe primary infections (Pooled OR compared to DENV1 = 2.13, 95% CI 1.28-3.56, and 6.75, 2.01-22.62, respectively), whereas DENV2 was similar to DENV1 in both analyses. Significant within- and between-year variation in serotype distribution between symptomatic and inapparent infections and circulation of serotypes undetected in symptomatic cases were observed in multiple years. Our study indicates that case surveillance skews the perceived epidemiological footprint of DENV. We reveal a more complex and intricate pattern of serotype distribution in inapparent infections. The significant differences in infection outcomes by serotype emphasizes the need for vaccines with balanced immunogenicity and efficacy across serotypes.