Optimization of expression conditions of dengue virus NS2B-NS3 protease in E.coli and determination of enzyme activity / 中国生物制品学杂志

@#Objective To express dengue virus(DENV)NS2B-NS3 protease in E.coli,optimize the expression conditions and determine the enzyme activity,so as to lay a foundation of screening and discovering of lead compounds targeting DENV.Methods Codon-optimized NS2B-NS3 gene was inserted into pET-28a vector to construct recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3,which was transformed E.coli Rosetta(DE3)competent cells and induced by IPTG to express NS2B-NS3 protease. The optimal expression conditions of NS2B-NS3 protease in E.coli were determined by optimizing induction length,induction temperature and IPTG concentration. NS2B-NS3 protease was isolated and purified by HisTrap~(TM) affinity chromatography column and measured for the protease activity by fluorescence resonance energy transfer(FRET)assay.Results The recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3 was constructed correctly as identified by restriction analysis(NheⅠ/XhoⅠ)and sequencing. The optimal expression conditions of NS2BNS3 protease in E.coli were as follows:induction temperature of 20 ℃,induction length of 10 h and IPTG concentration of0. 2 mmol/L. The purified NS2B-NS3 protease showed a purity of more than 90% with a exhibited a of 20 mg/L,which bound to mouse monoclonal antibody against His-tag specifically and had good hydrolytic activity with a specific activity of 16. 111 U/mg,a K_m of 16. 46 μmol/L and a k_(cat) of 0. 028/s.Conclusion DENV NS2B-NS3 protease with high purity and activity was successfully prepared,which laid an experimental foundation of the establishment of high-throughput screening model for inhibitors targeting NS2B-NS3 protease.

Construction of CeRNA network of differential LncRNAs in THP-1 cells infected by Dengue virus type Ⅲ and its antibody dependent enhancement / 中国生物制品学杂志

@#ObjectiveTo establish models of Dengue virus type Ⅲ（DENV-3,DV-3）infection and antibody dependent enhancement(ADE)infection at the acute monocytic leukemia cells(THP-1),investigate the differential expression of long non-coding RNAs(LncRNAs),map the competitive endogenous RNA(CeRNA)regulatory network and predict the translation function of LncRNAs.MethodsThe culture supernatant was harvested 6 d after C6/36 cells were infected with DENV-3,the virus titer was determined by CCID50,and the type and full-length genome amplification were identified by PCR;The DENV-3 standard plasmid was amplified,identified by PCR,and the standard curve was drawn;THP-1 cells were divided into negative control group(THP-1),direct infection group(DV-3),ADE group and blank control group[1640(-)]. After 48 h of infection,the total RNA was extracted and the copy number of intracellular virus nucleic acid was measured;Through the whole transcriptome sequencing technology,the CeRNA regulatory network was constructed for the top five up-regulated and down-regulated LncRNAs in THP-1 vs DENV3,THP-1 vs ADE,DENV3 vs ADE groups,and the functions of their coding proteins were analyzed.ResultsC6/36 cells infected with DENV-3 for 3 d showed obvious cell fusion,vacuoles and abscission;The virus had a titer of about 1. 0 × 104. 64PFU/mL and was identified as DENV-3 by PCR specific primers,of which the complete gene sequence was obtained;The number of viral nucleic acid copies in ADE group was significantly higher than those in DV-3 group and blank control group;In THP-1 vs DENV-3,the expression of cytohesin interacting protein(CYTIP)was predicted to be up-regulated;In THP-1 vs ADE,the expression of kinesin family5A(KIF5A)was predicted to be down-regulated;In DENV-3 vs ADE,the expression of cluster differentiation antigen 9(CD9)and insulin like growth factor 2(IGF2)was predicted to be up-regulated. All of these differential LncRNAs had open reading frames(ORFs). Except Lnc-SH3BP1 and Lnc-RPL41,all of the other LncRNAs had internal ribosome binding site(IRES).ConclusionIn DENV-3 infection of THP-1 cells and ADE infection mediated by DENV-3,the expression of LncRNAs has changed significantly,and may regulate the process of infection through a variety of biological functions,which is helpful for a deeper understanding of the mechanism of ADE infection.

In silico screening of plant peptides against the envelope protein of dengue virus

@#Peptide therapeutics are found to be an emerging and attractive class of treatment due to their highly specific and safe nature. Hence twenty plant peptides were subjected to screening by molecular docking against the envelope protein of the dengue virus using Clus Pro, Patch Dock, and HADDOCK servers. Physicochemical parameters, allergenicity, and toxicity profile of the plant peptides were estimated by Protparam analysis, AllergenFP, and ToxinPred web servers. Six potential compounds namely Ginkbilobin, Cycloviolin-D, Circulin-B, Circulin-A, Cycloviolacin-013, and Circulin-C showed the highest binding energy with both nonallergenic and nontoxic properties. They also exhibited desirable half-lives extending to 30 hrs except for Ginkbilobin, which showed the least half-life of 4.4 hours and non-polar activity. The residues of Ala-4 of Ginkbilobin; Arg-30 of Cycloviolin D; Arg-29 of Circulin A and C interacted with the Try 101 of the domain II of Envelope protein, implying the possible inhibition of the insertion process of the trimeric E protein during fusion with the host cells. Thus, the identified plant peptides could serve as potential leads upon further subjection to in vitro studies.

Analysis of gene expression profile and construction of ceRNA regulatory network after DENV-2 infection with HUVECs / 西安交通大学学报(医学版)

【Objective】 To establish a co-expression lncRNA-mRNA ceRNA network and explore the potential molecular mechanism of lncRNA in dengue fever. 【Methods】 DENV-2-infected and normal pHUVEC were sequenced and screened for differentially expressed lncRNA and mRNA by gene microarray technology. Differentially expressed mRNA was analyzed by protein-protein interaction (PPI), and significantly related co-expressed lncRNA-mRNA was screened by Pearson’s correlation coefficient. The microRNA (miRNA) that bound to co-expressed lncRNA-mRNA was predicted by the database. The ceRNA network of co-expressed lncRNA-mRNA was constructed by Cytoscape software. Finally differentially expressed mRNAs and co-expressed lncRNA-mRNA were analyzed by GO and KEGG enrichment, and co-expressed lncRNA-mRNA was verified by RT-qPCR. 【Results】 At 48 h and 72 h after infection, 105 and 51 differentially expressed mRNAs were obtained, respectively, while 59 and 29 differentially expressed lncRNAs were obtained, respectively. Furthermore, at the two time intervals, there were 10 differential mRNAs and 5 differential lncRNAs, respectively. PPI analysis of differential mRNAs showed that isocratic values of interleukin 6 (IL6), interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), and 2’-5’-oligoadenylate synthetase 2 (OAS2) were relatively high. The pairing results of lncRNA-mRNA co-expression analysis with the highest correlation coefficients at 48 h and 72 h after infection were XLOC_001966-SMTNL1 and XLOC_001966-ESR2, respectively. According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the functions of differentially expressed mRNA and co-expressed lncRNA-mRNA were mainly involved in virus epidemic prevention response, immune response, and signal transduction, as well as the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, type I interferon, and cytokine receptor interaction. RT-qPCR revealed that lncRNA XLOC-I2-8991 was upregulated in the co-expressed lncRNA-mRNA, whereas all the other lncRNA and mRNA were downregulated. 【Conclusion】 This study initially revealed the potential lncRNA-mRNA co-expression network during dengue virus infection, and found that co-expressed lncRNA-mRNA was mainly enriched in the immune regulation and signal transduction pathways during virus infection. The findings will help further exploration into the infection mechanism of DENV-2.

Phosphoproteomic analysis of human umbilical venous endothelial cells with DENV-2 infection / 南方医科大学学报

OBJECTIVE@#To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection.@*METHODS@#The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs.@*RESULTS@#A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01).@*CONCLUSION@#DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.

Primary infection of BALB/c mice with a dengue virus type 4 strain leads to kidney injury

BACKGROUND Dengue is a disease caused by dengue virus (DENV-1 through -4). Among the four serotypes, DENV-4 remains the least studied. Acute kidney injury is a potential complication of dengue generally associated with severe dengue infection. OBJECTIVES The goal of this study was to investigate the alterations caused by experimental dengue infection in the kidney of adult BALB/c mice. METHODS In this study, BALB/c mice were infected through the intravenous route with a DENV-4 strain, isolated from a human patient. The kidneys of the mice were procured and subject to histopathological and ultrastructural analysis. FINDINGS The presence of the viral antigen was confirmed through immunohistochemistry. Analysis of tissue sections revealed the presence of inflammatory cell infiltrate throughout the parenchyma. Glomerular enlargement was a common find. Necrosis of tubular cells and haemorrhage were also observed. Analysis of the kidney on a transmission electron microscope allowed a closer look into the necrotic tubular cells, which presented nuclei with condensed chromatin, and loss of cytoplasm. MAIN CONCLUSIONS Even though the kidney is probably not a primary target of dengue infection in mice, the inoculation of the virus in the blood appears to damage the renal tissue through local inflammation.

Rendimiento diagnóstico de la prueba rápida para la detección del antígeno NS1 y anticuerpos IgM e IgG contra el virus del dengue / Diagnostic performance of the rapid test for the detection of NS1 antigen and IgM and IgG anti-antibodies against dengue virus

Objetivos. Determinar el rendimiento diagnóstico de la prueba rápida SD dengue DUO (Inyecta) para la detección de NS1, IgM e IgG en comparación con la prueba de ELISA. Materiales y métodos. Es una evaluación de prueba diagnóstica que incluyó 286 muestras de suero de pacientes con sintomatología atribuible a dengue de zonas endémicas del Perú. Las muestras se analizaron por ELISA y la prueba rápida SD dengue DUO (Inyecta) para IgM, NS1 e IgG en el Instituto de Investigación Nutricional en Lima. Resultados. La sensibilidad de la prueba rápida fue de 68% para NS1 e IgM, y 86% para IgG, mejorando este parámetro a 75% y 81% para NS1 e IgM, respectivamente, en los tres primeros días. La especificidad para los tres analitos fue mayor a 87%. La concordancia de los resultados obtenidos medidos por el coeficiente Kappa para los tres analitos fue buena y no se encontró reacción cruzada con otros arbovirus. Conclusiones. La prueba rápida SD Dengue DUO permite detectar con una adecuada sensibilidad y especificidad NS1, IgM e IgG. La sensibilidad para IgM y NS1 aumenta cuando se detecta en los tres primeros días de síntomas, por lo que se recomienda su implementación en los centros de primer nivel de atención para un diagnóstico temprano y oportuno.

Objectives . To assess the diagnostic performance of the SD dengue DUO rapid test (Inyecta) for the detection of NS1, IgM and IgG in comparison to the ELISA test. Materials and methods . This is a diagnostic test evaluation that included 286 serum samples from patients with symptomatology attributable to dengue from endemic areas of Peru. The samples were analyzed by ELISA and the SD dengue DUO rapid test (Inyecta) for IgM, NS1 and IgG at the Instituto de Investigación Nutricional in Lima. Results . The sensitivity of the rapid test was 68.0% for NS1 and IgM, and 86.0% for IgG, improving to 75.0% and 81.0% for NS1 and IgM, respectively, during the first three days. The specificity for all three analytes was greater than 87.0%. The concordance of the results, measured by the Kappa coefficient for the three analytes, was good and no cross-reaction with other arboviruses was found. Conclusions . The SD dengue DUO rapid test allows detection of NS1, IgM and IgG with adequate sensitivity and specificity. Sensitivity for IgM and NS1 increases when detected during the first three days of symptoms. Therefore, we recommend its implementation in primary care centers for early and timely diagnosis.

Development of monoclonal antibodies against prM of Dengue virus 4 / Desenvolvimento de anticorpos monoclonais para prM de Dengue vírus 4

Introduction: dengue is a most common mosquito-borne viral disease in the Americas and tropical countries. Objective: in this work, mice were hyperimmunized with DENV 4 antigen to produce monoclonal antibodies (mAbs). Methodology: DENV 4 (GenBank KC806069) was inoculated in C6/36 cell monolayers cultivated in Leibovitz's 15 medium supplemented with 5% fetal bovine serum and incubated at 28 oC. The virus stock was submitted to concentration and ultracentrifugation and stored at -80 oC until use (VC DENV 4). Balb/c mice were injected intraperitoneally with 50µg of DENV-4 and successive intraperitoneal injections of 25 µg of VCDENV 4 with Freund's incomplete adjuvant were performed. The spleen cells were fused to SP2/0 myeloma cells with PEG 1540 and distributed in 96-well microplates with Iscove's modified medium with Hipoxantina­Aminopterina­Timidina. Hybridoma screening by indirect ELISA showed positive results for six mAbs, and their characterization was performed by Western blotting and Indirect Immunofluorescence (IFI) techniques. Results: the six mAbs showed strong recognition of prM (24/29 kDa), and minor reaction to E protein (66 kDa), E/E protein dimer (105 kDa), and NS1 (49 kDa) protein in two mAbs. The use of mAbs anti-prM as a diagnostic tool using IFI has been demonstrated to detect DENV-4 antigen in infected cells or tissues. Conclusion: DENV 4 generate mAbs with strong reactivity to prM with potential use to confirm the presence of DENV 4 antigen in tissues or infected cells.

Introdução: a dengue é uma doença viral transmitida por mosquitos comumente das Américas e países tropicais. Objetivo: neste trabalho, camundongos foram hiperimunizados com antígeno DENV 4 para produzir anticorpos monoclonais (mAbs). Metodologia: DENV 4 (GenBank KC806069) foi inoculado em monocamadas de células C6 / 36 cultivadas em meio Leibovitz 15 suplementado com 5% de soro fetal bovino e incubadas a 28oC. O estoque viral foi submetido à concentração, ultracentrifugação e armazenado a -80 oC (VC DENV 4). Camundongos Balb / c foram injetados intraperitonealmente com 50 µg de VC DENV-4 e injeções intraperitoneais sucessivas de 25 µg de antigeno com adjuvante incompleto de Freund. As células do baço foram misturadas a células SP2/0 com PEG 1540 e distribuídas em microplacas de 96 poços com meio Iscove Modificado em presença de Hipoxantina ­ Aminopterina ­ Timidina. A triagem de hibridomas por ELISA indireto apresentou resultados positivos para seis mAbs, e sua caracterização foi realizada por técnicas de Western blotting e Imunofluorescência Indireta (IFI). Resultados: os seis mAbs mostraram forte reconhecimento de prM (24/29 kDa) e reação menor à proteína E (66 kDa), dímero de proteína E / E (105 kDa) e proteína NS1 (49 kDa) em dois mAbs. O uso de mAbs anti-prM como uma ferramenta de diagnóstico utilizando IFI demonstrou eficacia em detectar o antígeno DENV-4 em células ou tecidos infectados. Conclusão: o mAbs produzidos para DENV 4 demonstraram uma forte reatividade contra prM, e poderiam ser uma ferramenta de uso potencial no diagnóstico de DENV 4 .

Evidence of dengue virus in eviscerated specimens of panophthalmitis secondary to dengue fever: A possible cause-effect phenomenon

Purpose: To report a retrospective series of three cases of infectious panophthalmitis post?dengue fever with ex vivo confirmation of dengue virus ribonucleic acid (RNA) in the tissues of the eye. Methods: Four eyes of three patients, who were diagnosed with panophthalmitis following dengue fever and who underwent evisceration, were included. All demographic and clinical data were recorded. The eviscerated samples were subjected to direct microscopy, culture for bacteria, fungi, and parasites, and molecular virology (dengue virus [DENV] NS1?specific reverse transcription loop?mediated isothermal amplification (RT?LAMP) assay). Results: The time from the development of dengue fever to the occurrence of ocular symptoms was 4.33 ± 1.15 (median 5) days. DENV NS1 RNA, suggestive of the presence of the dengue virus, was confirmed in all evisceration specimens (uveal tissue, cornea). All the patients recovered completely from dengue fever and on follow?up had healthy eviscerated sockets. Conclusion: Demonstration of the DENV RNA in the eviscerated specimens of panophthalmitis following dengue fever implicates the DENV in the pathophysiology of the ocular infection.

Dengue in India: An Overview

Background: Dengue fever has become a major public health concern in our country, causing significant morbidity and mortality. Because there is no definite drug or commercially available vaccine for dengue, prevention is the only option. As a result, early reporting of dengue cases is required in order to implement preventive measures before the disease spreads to epidemic proportions. Therefore, healthcare providers should notify every dengue incident to local authorities in the current format, including the District Health Officer or Chief Medical Officer of district concerned and the Municipal Health Officer of municipality concerned every week (daily during the transmission period). Materials and Methods: The data on dengue (2015-2021 till Oct.) was available at the National Vector Borne Disease Control Programme under the Ministry of Health & Family Welfare, Government of India, and has been used in this paper. Results and discussion: During the last two decades (2000-2009 and 2010-2019), significant geographical spread of the dengue has been experienced in India with the repeated outbreaks, and an 11 fold increase in number of cases. Despite an increase in the incidence of dengue fever, the case fatality rate in India has decreased from 3.3% in 1996 to 0.4% in 2010 to 0.1% in 2019. Early diagnosis and timely referral play a critical role in bringing down Case Fatality Rate (CFR). Conclusion: Dengue is a manifestation arising from the process of increasing vector density and adaptation to human habitation, as well as human lifestyle transformation, unplanned developmental activities exacerbated by climate change.

Natural DENV-2 NS2B/NS3 protease inhibitors from Myristica cinnamomea King

@#The NS2B/NS3 protease is crucial for the pathogenesis of the DENV. Therefore, the inhibition of this protease is considered to be the key strategy for the development of new antiviral drugs. In the present study, malabaricones C (3) and E (4), acylphenols from the fruits of Myristica cinnamomea King, have been respectively identified as moderate (27.33 ± 5.45 μM) and potent (7.55 ± 1.64 μM) DENV-2 NS2B/NS3 protease inhibitors, thus making this the first report on the DENV-2 NS2B/NS3 protease inhibitory activity of acylphenols. Based on the molecular docking studies, compounds 3 and 4 both have π-π interactions with Tyr161. While compound 3 has hydrogen bonding interactions with Gly151, Gly153 and Tyr161, compound 4 however, forms hydrogen bonds with Ser135, Asp129, Phe130 and Ile86 instead. The results from the present study suggests that malabaricones C (3) and E (4) could be employed as lead compounds for the development of new dengue antivirals from natural origin.

Detection of dengue virus serotype 3 in Cajamarca, Peru: Molecular diagnosis and clinical characteristics

Objective: To describe and molecularly characterize an outbreak of dengue virus (DENV) infection in Cajamarca, an Andean region in Peru. Methods: A total of 359 serum samples from patients with acute febrile illness were assessed for the presence of DENV via RT-PCR, ELISA NS1, IgM and IgG in Cajamarca, Peru from January 2017 to June 2017. The evaluation of the different diagnostic tests and their applicability was performed. Results: Dengue virus was detected in 24.7% of samples by RTPCR. Meanwhile, serological analysis detected 30.3% positive cases via ELISA NS1 antigen, 16.7% via ELISA IgG and 9.7% via ELISA IgM. Most of the cases corresponded to DENV-3 (77.5%). The use of RT-PCR performed better in primary infections (P<0.01), while detection of ELISA IgM performed better in secondary infections (P<0.01). The combination of NS1 and IgM performed better than the other assays in detecting primary (92.5%) and secondary infections (96.6%). The most frequent symptoms associated with fever were headaches, myalgias, and arthralgias across all groups. Conclusions: We report an important outbreak of dengue infection caused by DENV-3 in Cajamarca, Peru. Our findings encourage the use of NS1 antigen and IgM co-detection. These findings demonstrate an increasing expansion of DENV-3 in Peru and highlight the importance of molecular diagnosis and serotype characterization among the clinically defined dengue cases to strengthen the Peruvian epidemiological surveillance.

Antiviral activity of ethanolic extract of Nilavembu Kudineer against dengue and chikungunya virus through in vitro evaluation

BackgroundCurrently, no vaccines or modern drugs are available for dengue and chikungunya and only symptomatic relief is provided to the patients. Siddha medicine, a traditional form of indigenous medical system uses specific polyherbal formulations for the treatment of such infections with considerable success. One such polyherbal formulation for the treatment of chikungunya and dengue is Nilavembu kudineer (NVK). The mechanistic details of this drug as an antiviral for chikungunya virus (CHIKV) and dengue virus (DENV) is poorly understood.ObjectivesThe current study was undertaken to study the efficacy of NVK as an antiviral formulation against CHIKV and DENV.Materials and methodsCytotoxicity assays (MTT) were performed to determine the role of NVK as an antiviral during chikungunya and dengue infections in the following conditions-i). post infection, ii). during active infections and iii) protective, not allowing virus infection.ResultsIt was observed that NVK provides protection against CHIKV and DENV-2 during active infection as well can help to prevent virus infection in the cells and it mainly depends on the cellular availability of drugs for maximum protection against both the infections.ConclusionOur study establishes that extraction protocols are important to ensure maximum efficacy of NVK along with the time of addition of the drug during CHIKV and DENV infections in the cells. This study provides insights to the possible mode of action of NVK in in vitro condition during CHIKV and DENV infection.

Research progress of laboratory diagnostic methods of dengue virus / 中华疾病控制杂志

Dengue virus(DENV) has been identified by World Health Organization as a major public health problem in tropical and subtropical regions. In recent years, dengue outbreaks have become more and more frequent in the world. In 2019, dengue outbreaks of varying degrees have occurred in the Philippines, Thailand, Bangladesh, Myanmar and Chongqing City in China. The laboratory diagnostic method of DENV is of great significance to the prevention and control of dengue epidemic. Therefore, the methods and strategies of DENV laboratory diagnosis are reviewed in this paper. By reviewing the traditional diagnostic methods and looking forward to the emerging diagnostic strategies, this paper aims to provide a reference to select the appropriate laboratory diagnostic scheme for the outbreak of dengue.

An in silico investigation of phytochemicals as potential inhibitors against non-structural protein 1 from dengue virus 4

Dengue fever has emerged as a big threat to human health since the last decade owing to high morbidity with considerable mortalities. The proposed study aims at the in silico investigation of the inhibitory action against DENV4-NS1 of phytochemicals from two local medicinal plants of Pakistan. Non-Structural Protein 1 of Dengue Virus 4 (DENV4-NS1) is known to be involved in the replication and maturation of viron in the host cells. A total of 129 phytochemicals (50 from Tanacetum parthenium and 79 from Silybum marianum) were selected for this study. The tertiary structure of DENV4-NS1 was predicted based on homology modelling using Modeller 9.18 and the structural stability was evaluated using molecular dynamics simulations. Absorption, distribution, metabolism, excretion and toxicity (ADMET) along with the drug-likeness was also predicted for these phytochemicals using SwissADME and PreADMET servers. The results of ADMET and drug-likeness predictions exhibited that 54 phytochemicals i.e. 25 from Tanacetum parthenium and 29 from Silybum marianum showed effective druglikeness. These phytochemicals were docked against DENV4-NS1 using AutoDock Vina and 18 most suitable phytochemicals with binding affinities ≤ -6.0 kcal/mol were selected as potential inhibitors for DENV4-NS1. Proposed study also exploits the novel inhibitory action of Jaceidin, Centaureidin, Artecanin, Secotanaparthenolide, Artematin, Schizolaenone B, Isopomiferin, 6, 8-Diprenyleriodictyol, and Anthraxin against dengue virus. It is concluded that the screened 18 phytochemicals have strong inhibition potential against Dengue Virus 4.

Influences of interaction between dengue virus type 2-infected human umbilical vein endothelial cells and macrophages on major inflammatory cytokines / 中华微生物学和免疫学杂志

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells ( HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate periph-eral blood mononuclear cells ( PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor ( M-CSF) to prepare macro-phages. The purity of CD14+CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-in-fected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pre-treating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2. 66±0. 53, P<0. 05) and then de-creased. The purity of macrophages detected by flow cytometry was (89. 16±2. 07) ％. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16. 10±0. 17 and 29. 76±0. 58, while the expression levels at 24 h in the un-infected group were 1. 46±0. 67 and 1. 60±0. 54, respectively. Expression of IL-6, IL-8, TNF-αand IL-1βat mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-αand IL-1β expression at 24 h were 45. 82±3. 72, 52. 34±1. 69 (12 h), 8. 94±1. 75 and 30. 96±1. 44 in the infected macrophages, and 1. 16±0. 22, 1. 15±0. 21, 1. 11±0. 09 and 1. 47±0. 31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infec-ted HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-cul-ture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0. 01) and that of IL-6, IL-8, TNF-αand IL-1βin macrophages (P<0. 01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-αand IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent.

Phylogenetic analysis of Dengue-2 serotypes circulating in mangroves in Northern Cordoba, Colombia

Abstract INTRODUCTION: In this study, we aimed to identify DENV-2 subtypes in Aedes aegypti pools collected between 2011 and 2017 in a rural area of Northern Cordoba, Colombia ("La Balsa"). METHODS: RT-PCR was performed to analyze the capsid/pre-membrane region (C-PrM). Sequencing and phylogenetic bayesian inference using reference DENV-2 sequences were performed. RESULTS: Twelve pools that tested positive for DENV-2 were characterized based on the C-PrM region and grouped under the Asian/American clade. CONCLUSIONS: This study is the first to report the DENV-2 Asian-American subtype in a rural area of Cordoba region, which is associated with severe dengue and local epidemics.

Dengue outbreaks in Taiwan, 1998-2017: Importation, serotype and temporal pattern

Objective:To ascertain the role of imported cases and serotypes on dengue outbreaks in Taiwan which have been sporadic yet highly volatile during the past two decades, exhibiting record-breaking magnitude in recent years.Methods:Confirmed case and serotype data from Taiwan Centers for Disease Control during 1998-2017 were fully examined, with fitting of weekly and daily case data of each city/county to a mathematical model to pinpoint the waves of cases and their locations. Moreover, we quantify the timing of turning point and transmission potential of each wave and determine its circulating serotype, to ascertain any pattern or connection between the variations in circulating serotypes and the magnitude/transmissibility of outbreak.Results:While the number of imported case increased steadily during past two decades, the yearly number of indigenous cases fluctuated wildly. Moreover, while yearly percentages of serotypes for imported cases remains steady, that of indigenous cases does not exhibit any clear pattern. There was at least one wave of reported cases somewhere in Taiwan every year from 1998 to 2015, except in 2016-2017. The effective reproduction number R for all waves in all locations ranged from 1.14 to 2.87, with the exception of two Tainan waves, in 2010 (3.95) and 2015 (6.84). Four major outbreaks of over 2000 cases reveal circulation of one dominant serotype.Conclusions:Correlation between imported cases and indigenous outbreak prove to be difficult to ascertain, even with the availability of serotype data. However, although there had been occasional co-circulation of serotypes in one location, and for some years with different serotypes circulating in different locations, all major outbreaks of over 2 000 cases during the past two decades are due to circulation of mainly a single serotype, perhaps indicating greater transmission potential with one dominating serotype.

Dengue outbreaks in Taiwan, 1998-2017: Importation, serotype and temporal pattern

Objective: To ascertain the role of imported cases and serotypes on dengue outbreaks in Taiwan which have been sporadic yet highly volatile during the past two decades, exhibiting record-breaking magnitude in recent years. Methods: Confirmed case and serotype data from Taiwan Centers for Disease Control during 1998-2017 were fully examined, with fitting of weekly and daily case data of each city/county to a mathematical model to pinpoint the waves of cases and their locations. Moreover, we quantify the timing of turning point and transmission potential of each wave and determine its circulating serotype, to ascertain any pattern or connection between the variations in circulating serotypes and the magnitude/transmissibility of outbreak. Results: While the number of imported case increased steadily during past two decades, the yearly number of indigenous cases fluctuated wildly. Moreover, while yearly percentages of serotypes for imported cases remains steady, that of indigenous cases does not exhibit any clear pattern. There was at least one wave of reported cases somewhere in Taiwan every year from 1998 to 2015, except in 2016-2017. The effective reproduction number R for all waves in all locations ranged from 1.14 to 2.87, with the exception of two Tainan waves, in 2010 (3.95) and 2015 (6.84). Four major outbreaks of over 2000 cases reveal circulation of one dominant serotype. Conclusions: Correlation between imported cases and indigenous outbreak prove to be difficult to ascertain, even with the availability of serotype data. However, although there had been occasional co-circulation of serotypes in one location, and for some years with different serotypes circulating in different locations, all major outbreaks of over 2 000 cases during the past two decades are due to circulation of mainly a single serotype, perhaps indicating greater transmission potential with one dominating serotype.

Advances in research on non-structural protein 3 of dengue virus / 中华微生物学和免疫学杂志

Dengue virus (DENV) is one of the most significant mosquito-borne viral pathogens that spread in the tropical and subtropical areas causing severe diseases in humans such as dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DENV RNA genome encodes three structural proteins and seven non-structural proteins. The non-structural protein 3 (NS3) contains two main functional domains: serine protease and RNA helicase. As a serine protease, NS3 together with host cell proteases can directly hydrolyze the ploy-protein that is translated from the viral genome into functional proteins. Functioning as a RNA helicase, it is closely related to the replication and transcriptional translation of the viral genome RNA. In this paper, the structure and functions of DENV NS3 and the research progress in related antiviral drugs were reviewed systematically.