Dissertations / Theses: 'Virus de la dengue'

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Tiew, Kok-Chuan. "Dengue virus protease inhibitors." Thesis, Wichita State University, 2011. http://hdl.handle.net/10057/6117.

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Dengue virus (DENV) is a major health threat that affects 2.5 billion people, or 40% of the world’s population. However, there are no approved antiviral drugs or vaccines to treat Dengue infection. This thesis describes the design, synthesis and discovery of a new class of inhibitors of DENV NS3 protease. Structure-activity relationship studies have been carried out in order to delineate the structural elements responsible for the activity of this series of compounds. A lead compound suitable for further development has been identified.
Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry.

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Aravapalli, Sridhar. "Dengue virus and West Nile virus protease inhibitors." Diss., Wichita State University, 2013. http://hdl.handle.net/10057/6719.

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Dengue virus and West Nile virus are important mosquito-borne pathogens of Flaviviridae family affecting millions of people worldwide and causing a severe global healthcare threat. However, currently there are no approved effective antiviral drugs or vaccines available for the treatment of virus infection. This thesis describes the design, synthesis and discovery of two novel classes of reversible competitive inhibitors of Dengue Virus and West Nile Virus NS2B/NS3 protease. Structure-activity relationship studies have led to the identification of a low micromolar hit, which will be used in a hit-to-lead campaign to generate lead compounds that display superior ADMET and PK characteristics.
Thesis (Ph.D.)--Wichita State University, Fairmount College of Liberal Arts and Sciences, Dept. of Chemistry

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Tran, Tuan Anh. "Screening against the dengue virus polymerase." Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM4006.

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La dengue, une des maladies les plus largement émergents actuellement, avec 390 millions d'infections chaque année (OMS), est causée par le virus de la dengue contre lequel il n’existe pas de traitements. La protéine NS5 a un rôle important dans le cycle de réplication. Cette protéine se compose d'une méthionine S-transférase d’adénosyl en N-terminal et une ARN polymérase dépendante de l'ARN (RdRp) en C-terminal. Cette NS5 RdRp peut catalyser non seulement la synthèse du brin négatif de l'ARN, utilisé comme matrice pour synthétiser l'ARN brin plus-supplémentaire, mais aussi pour la synthèse d'un ARN complémentaire à partir d'une matrice court e d'ARN sans amorce (de novo). Dans ce travail de thèse, nous présentons la production et le test de l'activité de la protéine NS5, ainsi que du domaine polymérase RdRp pour les quatre sérotypes du virus de la dengue en développant un nouveau test enzymatique, en utilisant comme un réactif fluorescent. L'utilisation de ce réactif fluorescent a également contribué à la détermination des conditions optimisées pour développer un essai de criblage de l'activité polymérase pour identifier des inhibiteurs contre le virus de la dengue. En outre, quatre flavonoïdes, Hinokiflavone, apigénine, la quercétine et Amentoflavone ont montré des valeurs d’IC50 équivalentes contre toutes les constructions NS5 et les domaines polymérase des quatre sérotypes
Dengue fever, one of the most widely emerging diseases nowadays with 390 million infections each year (WHO), is caused by Dengue virus in which no official antiviral reagent or vaccine is available. The NS5 protein has an important role in the replication cycle. This protein consists of a S-adenosyl methionine transferase at N-terminal and a RNA dependent RNA polymerase (RdRp) at C-terminal. This NS5 RdRp can catalyse for not only synthesis of minus-strand RNA to be used as the template to synthesize additional plus-strand RNA but also synthesizing a complement RNA from a short RNA template without primer (de novo). In this research we present the production and activity test for NS5 protein and N-terminal extended sequence 266-900 from NS5 RdRp of all first four serotypes of Dengue virus and a construct of sequence 273-900 using a new enzymatic assay, using Picogreen as fluorescent reagent. Using this fluorescent reagent also helped determining the optimised conditions to develop a screening assay for inhibitors against dengue polymerase activity. In addition, four flavonoids, Hinokiflavone, Apigenin, Quercetin and Amentoflavone showed approximate IC50 values when testing on all NS5 and polymerase protein constructs of all four serotypes

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He, Runtao. "Characterization of dengue virus envelope protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq24745.pdf.

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5

Schaar, Hilde van der. "Cell entry mechanisms of dengue virus." [S.l. : [Groningen : s.n.] ; University Library Groningen] [Host], 2009. http://irs.ub.rug.nl/ppn/318.

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6

Thu, Hlaing Myat. "Virus diversity and the emergence of Dengue." Thesis, Queensland University of Technology, 2004. https://eprints.qut.edu.au/16081/1/Hlaing_Myat_Thu_Thesis.pdf.

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The aims of this study were to investigate the role of the diversity of dengue virus populations in changing patterns of virus transmission and disease. Prior to the commencement of this study, dengue 2 virus (DENV-2) had been associated most frequently with severe disease, so the study commenced with this serotype. Because it was not possible to quantitate diversity in the entire 11 kb of the viral genome, the study focussed on the envelope (E) gene, because the E protein is the major protein on the surface of the virion and thus might be under strong selective pressure from the host immune system and from the requirement to engage specific receptors on host cells. This study was the first direct quantification of the diversity of dengue virus populations in individual hosts. The nucleotide sequences of more than 70 per cent of the E genes in each virus population differed from the consensus nucleotide sequence for the population. In the course of quantitating genetic diversity in DENV-2 virus populations in patients and in mosquitoes, recombinant DENV-2 and both parental virus populations were detected in a single mosquito. This was the first such report. In 2001, just after the commencement of this study, Myanmar had the largest outbreak of dengue on record. Unlike previous outbreaks, 95 per cent of dengue viruses isolated from patients were of a single serotype, DENV-1. Despite the large number of cases of dengue, the proportion of patients with severe dengue was low. In the light of these observations, the direction of this study changed to focus on DENV-1. Phylogenetic analysis of the E genes of DENV-1 collected before and after the 2001 dengue outbreak suggested that some time before 1998, an early lineage of DENV-1 had become extinct and had been replaced by two new lineages. There was no evidence that these changes were due to selection or to recombination within the E protein genes of the old clade of viruses and the newly introduced viruses. A more detailed analysis was undertaken, of the entire genome of 11 human DENV-1 isolates and of 4 from mosquitoes recovered in Yangon between 1971 and 2002, to determine whether the extinction of the pre-1998 lineage of DENV-1 (clade A) and the appearance of the two new lineages (clades B and C) could have been due to selective pressures acting on genes other than E. Evidence of only weak selection was found in the NS5 gene (at amino acids 127,135 and 669) but the resultant amino acid changes did not distinguish all recent viruses from viruses belonging to the extinct clade. The phylogenetic relationships between individual genes from these viruses and between the open reading frames were similar. No evidence was found of recombination that might have given rise to two new clades of virus with enhanced fitness. Collectively, these data suggested that the extinction of clade A viruses and their replacement by the two new clades, between 1998 and 2000 was a stochastic event in an inter-epidemic period when rates of virus transmission were low. This was the first report of such an extinction of a lineage of DENV-1 and its replacement by new lineages. At about the same time as the 2001 outbreak of DENV-1 infection in Myanmar, an outbreak of DENV-1 began in the Pacific. A comparison of the nucleotide sequences of the E genes of viruses from the Pacific with those of viruses from throughout south-east Asia suggested that the outbreak in the Pacific was due to the introduction of multiple genotypes of DENV-1 from Asia and that some of these DENV-1 could have originated in Myanmar. The principal observations from this study are: - (a) Dengue virus populations in individual hosts are extremely heterogenous and may contain a significant proportion of non-infectious genomes. (b) Intra-serotypic recombination between dengue viruses may be far more common than the literature suggests but it may not be detected because of the almost universal use of consensus nucleotide sequences. (c) Significant changes in dengue virus genotypes that occur at single localities may be due to genetic bottlenecks rather than to selection or to recombination. (d) Dengue viruses can be transported more than 10,000 km to cause outbreaks in non-endemic areas.

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7

Thu, Hlaing Myat. "Virus Diversity and the Emergence of Dengue." Queensland University of Technology, 2004. http://eprints.qut.edu.au/16081/.

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The aims of this study were to investigate the role of the diversity of dengue virus populations in changing patterns of virus transmission and disease. Prior to the commencement of this study, dengue 2 virus (DENV-2) had been associated most frequently with severe disease, so the study commenced with this serotype. Because it was not possible to quantitate diversity in the entire 11 kb of the viral genome, the study focussed on the envelope (E) gene, because the E protein is the major protein on the surface of the virion and thus might be under strong selective pressure from the host immune system and from the requirement to engage specific receptors on host cells. This study was the first direct quantification of the diversity of dengue virus populations in individual hosts. The nucleotide sequences of more than 70 per cent of the E genes in each virus population differed from the consensus nucleotide sequence for the population. In the course of quantitating genetic diversity in DENV-2 virus populations in patients and in mosquitoes, recombinant DENV-2 and both parental virus populations were detected in a single mosquito. This was the first such report. In 2001, just after the commencement of this study, Myanmar had the largest outbreak of dengue on record. Unlike previous outbreaks, 95 per cent of dengue viruses isolated from patients were of a single serotype, DENV-1. Despite the large number of cases of dengue, the proportion of patients with severe dengue was low. In the light of these observations, the direction of this study changed to focus on DENV-1. Phylogenetic analysis of the E genes of DENV-1 collected before and after the 2001 dengue outbreak suggested that some time before 1998, an early lineage of DENV-1 had become extinct and had been replaced by two new lineages. There was no evidence that these changes were due to selection or to recombination within the E protein genes of the old clade of viruses and the newly introduced viruses. A more detailed analysis was undertaken, of the entire genome of 11 human DENV-1 isolates and of 4 from mosquitoes recovered in Yangon between 1971 and 2002, to determine whether the extinction of the pre-1998 lineage of DENV-1 (clade A) and the appearance of the two new lineages (clades B and C) could have been due to selective pressures acting on genes other than E. Evidence of only weak selection was found in the NS5 gene (at amino acids 127,135 and 669) but the resultant amino acid changes did not distinguish all recent viruses from viruses belonging to the extinct clade. The phylogenetic relationships between individual genes from these viruses and between the open reading frames were similar. No evidence was found of recombination that might have given rise to two new clades of virus with enhanced fitness. Collectively, these data suggested that the extinction of clade A viruses and their replacement by the two new clades, between 1998 and 2000 was a stochastic event in an inter-epidemic period when rates of virus transmission were low. This was the first report of such an extinction of a lineage of DENV-1 and its replacement by new lineages. At about the same time as the 2001 outbreak of DENV-1 infection in Myanmar, an outbreak of DENV-1 began in the Pacific. A comparison of the nucleotide sequences of the E genes of viruses from the Pacific with those of viruses from throughout south-east Asia suggested that the outbreak in the Pacific was due to the introduction of multiple genotypes of DENV-1 from Asia and that some of these DENV-1 could have originated in Myanmar. The principal observations from this study are: - (a) Dengue virus populations in individual hosts are extremely heterogenous and may contain a significant proportion of non-infectious genomes. (b) Intra-serotypic recombination between dengue viruses may be far more common than the literature suggests but it may not be detected because of the almost universal use of consensus nucleotide sequences. (c) Significant changes in dengue virus genotypes that occur at single localities may be due to genetic bottlenecks rather than to selection or to recombination. (d) Dengue viruses can be transported more than 10,000 km to cause outbreaks in non-endemic areas. Key words: Dengue viruses, diversity, recombination, selection, genetic bottleneck

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Lindegren, Gunnel. "Optimisation of dengue diagnostic tools in order to increase the knowledge of the pathogenesis." Stockholm : Department of Microbiology, Karolinska Institutet, 2008. http://diss.kib.ki.se/2008/978-91-7409-129-8/.

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9

Niberon, Yann. "Hyperspectral Plasmonic Biosensor for Dengue Virus Detection." Electronic Thesis or Diss., Troyes, 2021. http://www.theses.fr/2021TROY0041.

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La résonance des plasmons de surface (SPR en anglais) est une technique utilisée pour détecter certaines liaisons chimiques spécifiques et leurs évolutions dans le temps avec une sensibilité et une spécificité élevée. L’objectif de ce projet est de proposer un biocapteur SPR en hyperspectral et compact pour détecter le virus de la dengue. Basé sur un système statique avec des réflexions sur des surfaces paraboliques et utilisant une source de lumière blanche, dans la configuration de Kretschmann il a été possible de réaliser simultanément une interrogation angulaire et spectrale pour générer une image hyperspectrale donnant en temps réel une cartographie de la réflectance dans les deux axes. L'aspect instantané de la technique permet d'étudier l'évolution des conditions de résonance avec une variation de l'indice de réfraction d'une solution testée en temps réel pour une certaine gamme d'angles incidents dans le spectre visible. Une étude cinétique des liaisons chimiques est réalisée sur le virus de la Dengue de type 2 (DENV-2) au travers de son anticorps Immunoglobuline G (IgG1) associé. Lors de la caractérisation de cette technique, il a été possible d’atteindre une sensibilité pour détecter un décalage d'indice de réfraction d'ordre 10-5 RIU (refractive index unit). La particularité de cette conception pour un biocapteur SPR par rapport au système plus conventionnel est l'aspect tridimensionnel (angulaire, spectral et temporel) déterminant les conditions de résonance sans qu'il soit nécessaire de fixer l'un des trois paramètres : angle d'incidence, longueur d'onde ou temps
Surface plasmon resonance (SPR) is a technic use to detect specific chemical binding and their evolution through time with high sensitivity and specificity. The objective of this project is to propose a compact hyperspectral SPR bio-sensor to detect Dengue virus. Based on a static system with reflection over parabolic surfaces and using white light source, in the Kretschmann configuration it was possible to realise simultaneously an angular and spectral interrogation to generate a hyperspectral image that gives us in real time a mapping of the reflectance along two axes. The real time aspect allows to study the evolution the resonance condition from the variation of the refractive index of a solution tested through time in a certain range of incident angle in the visible spectrum. A kinetic study of chemical bindings was realized with the Dengue virus Type 2 (DENV-2) with its Immunoglobulin G antibody (IgG1). During the calibration of this technic, it was reached a sensitivity to detect a refractive index shift within an order of 10-5 RUI (refractive index unit). The advantage of this SPR bio-sensor design compare to conventional system is the three-dimensional aspect (angular, spectral and temporal) to determine the resonance condition without the need to fix one of the three parameters: incident angle, wavelength and time

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Tison, Laurence. "La dengue." Bordeaux 2, 1995. http://www.theses.fr/1995BOR2P091.

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11

Patramool, Sirilaksana. "Interactions virus (dengue)-vecteurs (aedes) et mise en évidence d'une méthode d'isolement des virus de la dengue et du chikungunya." Thesis, Montpellier 2, 2013. http://www.theses.fr/2013MON20139.

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La dengue et le chikungunya sont deux arboviroses émergentes qui sont transmises à l'homme par la piqûre de moustiques du genre Aedes. Il n'existe ni vaccin ni traitements commercialisés pour ces arboviroses. Il apparaît donc nécessaire de développer de nouvelles stratégies pour isoler les virus circulants et bloquer leur transmission. La compréhension des mécanismes mis en jeu dans les cellules des vecteurs Aedes lors d'une infection par le virus de la dengue (DENV) sont encore très peu étudiés, notamment pour les sérotypes 1 et 3. Par des analyses protéomiques de l'infection d'une lignée cellulaire du moustique Aedes albopictus par ces séroytypes, nous avons démontré qu'en réponse à l'infection, les cellules de moustiques utilisent les mécanismes antioxydants combinés à la production d'énergie pour faire face au virus. Les résultats de notre étude devraient permettre de mieux comprendre l'interaction DENV-vecteur Aedes au niveau cellulaire dans le but de concevoir des stratégies efficaces pour le contrôle du DENV. Nous avons également regroupé dans une revue les connaissances acquises sur les études protéomiques des principaux compartiments des arthropodes vecteurs de maladies humaines. Dans un second volet, nous avons mis en évidence une méthode rapide d'isolement et de concentration des DENV et du chikungunya. Cette technique d'isolement basée sur la capture de virus sur des billes magnétiques enrobées de polymères anioniques permet d'obtenir des particules virales infectieuses. Cette méthode combinée à des approches classiques de détection de virus pourrait non seulement permettre l'identification des échantillons infectés ayant une faible charge virale, mais aussi l'isolement simultanée de particules infectieuses de dengue et de chikungunya à partir d'un seul échantillon
Dengue (DENV) and Chikungunya (CHIKV) viruses are two emerging arboviruses that are transmitted to humans by the bite of Aedes sp. mosquito vectors. Neither vaccines, nor medical treatments, are commercially available for these infections. It is, therefore, necessary to elaborate novel strategies to isolate the circulating viruses and block their transmission.Our understanding of the molecular mechanisms involved, during the infection of the Aedes vector by dengue virus (DENV), especially serotypes 1 and 3, remains very scant. We, therefore, performed a proteomics analysis of an Aedes albopictus cell line, infected by these two DENV serotypes, and showed that the cells use both anti-oxidant and energy-production mechanisms in the fight against the virus. These results should help to improve our knowledge of the interaction of the DENV virus and the Aedes mosquito vector, at the cellular level, with the aim of designing efficient strategies for the control of this virus. We have, in addition, developed a rapid and sensitive isolation technique, based on viral particle adsorption to magnetic beads coated with an anionic polymer. The use of this technique is of great interest, as it permits the rapid and simultaneous detection and isolation of CHIKV and DENV from samples with reduced viral loads

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Hacker, Kari Ema De Silva Aravinda Manu. "Characterization of dengue virus interactions with host cells." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2009. http://dc.lib.unc.edu/u?/etd,2845.

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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.
Title from electronic title page (viewed Jun. 4, 2010). "... in partial fulfillment of the degree of Doctor of Philosophy in the Department of Microbiology and Immunology." Discipline: Microbiology and Immunology; Department/School: Medicine.

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Katri, Patricia. "Modeling the Transmission Dynamics of the Dengue Virus." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_dissertations/417.

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Dengue (pronounced den'guee) Fever (DF) and Dengue Hemorrhagic Fever (DHF), collectively known as "dengue," are mosquito-borne, potentially mortal, flu-like viral diseases that affect humans worldwide. Transmitted to humans by the bite of an infected mosquito, dengue is caused by any one of four serotypes, or antigen-specific viruses. In this thesis, both the spatial and temporal dynamics of dengue transmission are investigated. Different chapters present new models while building on themes of previous chapters. In Chapter 2, we explore the temporal dynamics of dengue viral transmission by presenting and analyzing an ODE model that combines an SIR human host- with a multi-stage SI mosquito vector transmission system. In the case where the juvenile populations are at carrying capacity, juvenile mosquito mortality rates are sufficiently small to be absorbed by juvenile maturation rates, and no humans die from dengue, both the analysis and numerical simulations demonstrate that an epidemic will persist if the oviposition rate is greater than the adult mosquito death rate. In Chapter 3, we present and analyze a non-autonomous, non-linear ODE system that incorporates seasonality into the modeling of the transmission of the dengue virus. We derive conditions for the existence of a threshold parameter, the basic reproductive ratio, denoting the expected number of secondary cases produced by a typically infective individual. In Chapter 4, we present and analyze a non-linear system of coupled reaction-diffusion equations modeling the virus' spatial spread. In formulating our model, we seek to establish the existence of traveling wave solutions and to calculate spread rates for the spatial dissemination of the disease. We determine that the epidemic wave speed increases as average annual, and in our case, winter, temperatures increase. In Chapter 5, we present and analyze an ODE model that incorporates two serotypes of the dengue virus and allows for the possibility of both primary and secondary infections with each serotype. We obtain an analytical expression for the basic reproductive number, R_0, that defines it as the maximum of the reproduction numbers for each strain/serotype of the virus. In each chapter, numerical simulations are conducted to support the analytical conclusions.

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Wangteeraprasert, Apirath. "CD8+ T-cells responses in Dengue virus infection." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/39398.

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Dengue virus, which has four serotypes, has several clinical manifestations including asymptomatic infection, a self-limiting febrile illness termed dengue fever (DF) and a severe form characterized by plasma leakage termed dengue hemorrhagic fever (DHF). The pathogenesis of DHF is not fully understood and many studies have shown that it is more prevalent during secondary infection. In addition to a mechanism termed antibody dependent enhancement (ADE), the role of T-cells in the pathogenesis of dengue also has been investigated. It has been hypothesized that upon secondary infection dengue-specific memory T-cells generated during a previous infection, which are cross-reactive and have low avidity for the current serotype dominate the T-cell response. This phenomenon is called 'Original antigenic sin' and the consequence of this low avidity T-cell response may be ineffective viral elimination leading to increased production of inflammatory cytokines which could cause plasma leakage. To study CD8+ T-cell responses to dengue-peptide variants, HLA-A11-restricted NS3 133-142-specific T-cell clones were generated and their cytotoxicity, proliferation and cytokine production in response to variant epitopes was tested. The results support that the magnitude of T-cell responses is related to the strength of the TCR-peptide-MHC interaction. AG129 mice, which lack both IFN (symbol missing) and IFN (symbol missing) receptors, show susceptibility to dengue virus infection and develop symptoms seen during human infection such as vascular leakage. This allows for investigation of the role of T-cell responses generated towards sequential infections with well defined serotypes. Experiments that would be very difficult to carry out in human dengue patients. Splenocytes from sequentially infected AG129 mice were assayed for their response to whole dengue proteins from serotype 1 and 2 virus. New epitopes were identified and CD8+ T-cell lines and clones were generated and their functions studied using peptide variants. The results showed that dengue-specific cross-reactive memory CTLs displayed better recognition of epitopes encountered during primary immunisa tion as compare to those recognised during secondary immunisation, which supports the idea of original antigenic sin in dengue-specific CD8+ T -cells. In conclusion, this study focuses on cross-reactive dengue-specific CD8+ T-cells and their functions when recognizing heterologous dengue peptides to clarify their role in pathogenesis.

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Twiddy, Sally Susanna. "The molecular epidemiology and evolution of dengue virus." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269490.

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Xu, Yongtao. "Computational design of peptide inhibitors for dengue virus." Thesis, Queen's University Belfast, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603298.

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Fusion process is known to be the initial step of viral infection and hence targeting the entry process is a promising strategy to design antiviral therapy. The self-inhibitory peptides derived from the envelope (E) proteins function to inhibit the protein-protein interactions in the membrane fusion step mediated by the viral E proteins. Thus, they have the potential to be developed into effective antiviral therapy. We developed a Monte Carlo-based computational method with the aim to identify and optimize potential peptide hits from the E proteins. Some novel peptides may inhibit the protein-protein interaction in the Dengue Virus (DENV) or Herpes Simplex Virus-! (HSV -1) fusion process and serve as starting points for the development of antiviral therapy to treat viral infection. Residue-specific all-atom conditional probability discriminatory function (RAPDF) has been justified effective for designing novel peptide inhibitors targeting E proteins in our computational method, The statistical potential has its universality and specificity which is decided by the reference state and the decoy sets. We built new distance-dependant statistical potentials using E, tetratricopeptide and ankyrin repeat proteins respectively. We demonstrated that the specific statistical potentials outperformed the general statistical potentials. Our computational method for identifying self-inhibitory peptides from three types of E proteins has also illustrated E proteins may have some unique features in common. In order to identify the active peptides from non-active peptides, we applied a support vector machine (SVM) approach for envelope peptide inhibitors activity prediction (EAP) based on the physicochemical properties, amino acid composition and statistical scoring function of peptide inhibitors which target E proteins. The results suggest that the rational connecting between properties of peptide inhibitors derived from E proteins and antivirus activity.

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Nguyen, Celina Tien, and Celina Tien Nguyen. "Using Bioorthogonal Chemistry for Investigating Dengue Virus Interactome." Thesis, The University of Arizona, 2017. http://hdl.handle.net/10150/625109.

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A mosquito-borne virus endemic to the tropics and subtropics, dengue (DENV) is the most common arbovirus in the world. Among other obstacles, an incomplete understanding of specific host-virus protein-protein interactions (PPI) challenges the development of vaccines and antiviral therapies for DENV. Previous studies on DENV PPI depend on genetic modifications, which can be tedious and time-consuming to accomplish. Mixing the principles of chemical biology and virology, the work in this thesis aims to take advantage of chemical modifications on DENV to pull down DENV-host PPI. First, this thesis will present the chemical modification of the DENV E glycoprotein via the metabolic incorporation of an unnatural sugar. This modification seemed to produce a morphology change in the viral structure, rendering it an impractical chemical modification. Then, this thesis will introduce an attempt to use bioorthogonal chemical modifications to DENV to pull down an established DENV PPI with an antibody as a proof-of-concept experiment to demonstrate the possibility of using bioorthogonal chemistry for identifying DENV PPI. Currently, this experiment is stalled by the presentation of background labelling. In the future, we hope to optimize the pull-down proof-of-concept and later use the same protocol to identifying PPI in DENV-infected mosquitoes.

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Obeysekera, Maheshi Prabodani. "Dengue Virus Infection of Human Microvascular Endothelial Cells." Thesis, Griffith University, 2016. http://hdl.handle.net/10072/366032.

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Dengue virus (DENV) infection is an important public health problem, being globally the most highly prevalent mosquito-borne viral illness. The World Health Organization (WHO) has estimated 2.5 billion people are at risk of DENV infection and each year 50- 100 million infections are reported, with over 500,000 hospitalisations and 25,000 deaths. DENV consist of four serotypes, each of which are capable of producing a wide spectrum of syndromes varying from mild dengue fever (DF) to more severe dengue haemorrhagic fever (DHF). Vascular leakage resulting from increased micro vessel endothelial permeability is one of the life-threatening complications in DHF. The relationship between DENV infection and the endothelium has previously been investigated both in vitro and in vivo experiments. Nevertheless, the mechanisms by which dengue infection leads to increased vascular permeability remain unclear. Understanding the underlying mechanisms of vascular leakage is required to identify better control strategies for DHF/DSS.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
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19

Mohan, Swathi Groutas William C. "Potential inhibitors of dengue and West Nile virus proteases." Diss., The archival copy of this thesis can be found at SOAR (password protected), 2006. http://soar.wichita.edu/dspace/handle/10057/569.

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Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences., Dept. of Chemistry.
"July 2006." Title from PDF title page (viewed on May 2, 2007). Thesis adviser: William C. Groutas. Includes bibliographic references (leaves 64-66).

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Flanagan, Aleksandra. "Structural and biophysical studies of antibody - Dengue virus interaction." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.558310.

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Dengue virus (DENV), a member of Flaviviriade family, is a mosquito-borne human viral pathogen, causing every year more than 50 million infections, some of which can lead to dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). It co-circulates as four serotypes. Humoral immunity plays a significant role in controlling flavivirus dissemination within an infected host. However, antibodies raised against DENV may be both protective and pathogenic, due to the phenomenon of antibody-dependent enhancement (ADE), which leads to a dramatic increase in the infection of cells bearing Fcy receptors. As such ADE is a major obstacle in the development of a successful vaccine against DENV. The main challenge in the field is to understand the mechanisms of neutralization and enhancement of DENV infection at the molecular level and identify antibody epitopes that minimize ADE. DENV envelope (E) and precursor membrane (prM) proteins are the main targets of antibodies. Each monomer of E consists of three domains (El, Ell and EIII), of which EIII contains critical neutralization determinants. PrM acts as a chaperone for the correct folding of E and undergoes proteolytic cleavage to soluble pr peptide and membrane-associated M during virus maturation. This thesis reports the functional and structural characterization of four types of antibodies in complexes with recombinant antigens, E and prM. The crystal structures of murine antibodies 3H5 and 2C8 complexed with EIII indicate a possible explanation of ADE mediated by strongly neutralizing antibodies specific to EIII. The proposed structural rearrangement of E induced by 3H5 binding, results in neutralization at a significantly lower occupancy of the antibody on the virus than that observed with 2C8 which can bind without distorting the DENV envelope. The occupancy required for neutralization is directly correlated with ADE as low density of antibodies bound at neutralization may fail to reach a threshold to drive efficient Fc-receptor-dependent uptake. The crystal structures of the fully cross-reactive murine antibody 2H12 in complex with EIII from DENV serotypes 1, 3 and 4 revealed that it recognizes a highly conserved epitope, which has limited accessibility on the mature virus and the ability of the antibody to bind the virus is serotype- dependent. 2H12 displayed high affinity to isolated antigen (EIII) yet the position of the epitope in the mature virus hindered efficient neutralization. The structural analyses of human mAbs recognizing El-Ell prove to be challenging. Whilst the Fab fragment of 30E2 formed a stable complex with recombinant E, and yielded crystals, other Fabs did not bind to recombinant E in a monomeric form indicating that they target epitopes on oligomeric forms of E present only in the virions. DENV cross-reactive human antibodies against prM do not neutralize the infection with DENV yet greatly promote ADE. They bind immature and partially mature yet infectious particles and recognize a discontinuous epitope spanning across pr peptide and M. Initial crystallographic studies of Fab- prM complexes provide a platform for further experiments aimed at the elucidation of the specificity of prM-specific antibodies.

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Romero-Vivas, Claudia Maria Elisa. "Studies on Aedes aegypti breeding sites and Dengue virus." Thesis, London School of Hygiene and Tropical Medicine (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298742.

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22

Chan, Ying Kai. "Interplay of Dengue Virus and the Human Immune Response." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467339.

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RIG-I is a key cytosolic sensor of many RNA viruses, including dengue virus (DV), the most significant arboviral pathogen. Upon viral RNA binding, RIG-I signals via the adaptor protein MAVS, located at mitochondria, to induce the expression of interferons (IFNs), proinflammatory cytokines and interferon-stimulated genes (ISGs), thereby establishing an antiviral state. Among the ISGs, the IFITM proteins are critical for antiviral restriction of numerous pathogenic viruses including DV by inhibiting viral entry. Here, we uncover how DV escapes RIG-I-mediated immunity. The NS3 protein of DV binds directly to 14-3-3ε, a mitochondrial-targeting protein that is essential for translocation of RIG-I from the cytosol to mitochondria. Specifically, NS3 blocks 14-3-3ε from forming a “translocon” complex with RIG-I and its upstream activator, TRIM25, thereby inhibiting RIG-I translocation to mitochondria for MAVS interaction and antiviral signaling. Furthermore, RIG-I that fails to translocate to mitochondria is degraded in a lysosome-dependent manner in DV- infected cells. Intriguingly, NS3 binds to 14-3-3ε using a phosphomimetic motif that resembles a canonical phospho-serine/threonine motif found in cellular 14-3-3-interaction partners. We engineer a recombinant DV encoding a mutant NS3 protein deficient in 14-3-3ε binding (DV2KIKP) and find that this mutant virus is attenuated in replication compared to the parental virus. Strikingly, DV2KIKP fails to antagonize RIG-I and elicits high levels of IFNs, proinflammatory cytokines and ISGs in human hepatocytes and monocytes. Taken together, our data reveal a novel phosphomimetic-based mechanism for viral antagonism of innate immunity and provide a foundation for DV vaccine development. DV can infect cells directly, or complex with non-neutralizing antibodies to infect Fc- receptor-bearing cells in a secondary infection, which is associated with severe disease. While it has been shown that IFITMs restrict DV direct infection, it is unknown if the latter process, commonly termed antibody-dependent enhancement (ADE), might bypass IFITM-mediated restriction. Comparison of direct and ADE-mediated DV infection shows that IFITM proteins restrict both infection modes equally, suggesting that upregulation of IFITMs may be a therapeutic strategy. In summary, our work elucidates several molecular aspects of the interplay of DV with the human immune response, which may guide the rational design of vaccines and antivirals.
Medical Sciences

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23

Misbah, Suzana. "Host cellular regulatory networks in dengue virus-human interactions." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/7330/.

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Dengue fever is one of the most important mosquito-borne diseases worldwide and is caused by infection with dengue virus (DENV). The disease is endemic in tropical and sub-tropical regions and has increased remarkably in the last few decades. At present, there is no antiviral or approved vaccine against the virus. Treatment of dengue patients is usually supportive, through oral or intravenous rehydration, or by blood transfusion for more severe dengue cases. Infection of DENV in humans and mosquitoes involves a complex interplay between the virus and host factors. This results in regulation of numerous intracellular processes, such as signal transduction and gene transcription which leads to progression of disease. To understand the mechanisms underlying the disease, the study of virus and host factors is therefore essential and could lead to the identification of human proteins modulating an essential step in the virus life cycle. Knowledge of these human proteins could lead to the discovery of potential new drug targets and disease control strategies in the future. Recent advances of high throughput screening technologies have provided researchers with molecular tools to carry out investigations on a large scale. Several studies have focused on determination of the host factors during DENV infection in human and mosquito cells. For instance, a genome-wide RNA interference (RNAi) screen has identified host factors that potentially play an important role in both DENV and West Nile virus replication (Krishnan et al. 2008). In the present study, a high-throughput yeast two-hybrid screen has been utilised in order to identify human factors interacting with DENV non-structural proteins. From the screen, 94 potential human interactors were identified. These include proteins involved in immune signalling regulation, potassium voltage-gated channels, transcriptional regulators, protein transporters and endoplasmic reticulum-associated proteins. Validation of fifteen of these human interactions revealed twelve of them strongly interacted with DENV proteins. Two proteins of particular interest were selected for further investigations of functional biological systems at the molecular level. These proteins, including a nuclear-associated protein BANP and a voltage-gated potassium channel Kv1.3, both have been identified through interaction with the DENV NS2A. BANP is known to be involved in NF-kB immune signalling pathway, whereas, Kv1.3 is known to play an important role in regulating passive flow of potassium ions upon changes in the cell transmembrane potential. This study also initiated a construction of an Aedes aegypti cDNA library for use with DENV proteins in Y2H screen. However, several issues were encountered during the study which made the library unsuitable for protein interaction analysis. In parallel, innate immune signalling was also optimised for downstream analysis. Overall, the work presented in this thesis, in particular the Y2H screen provides a number of human factors potentially targeted by DENV during infection. Nonetheless, more work is required to be done in order to validate these proteins and determine their functional properties, as well as testing them with infectious DENV to establish a biological significance. In the long term, data from this study will be useful for investigating potential human factors for development of antiviral strategies against dengue.

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Bletchly, Cheryl. "Antigenic and structural analysis of the NS1 glycoprotein of dengue virus /." St. Lucia, Qld, 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16420.pdf.

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Campanelli, Eliane Silveira. "O desenvolvimento de um processo de infecção do Aedes aegypti pelo vírus dengue: caracterização da interação do vírus com uma população de mosquitos autóctones." reponame:Repositório Institucional da FIOCRUZ, 2007. https://www.arca.fiocruz.br/handle/icict/4044.

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(...) Este estudo analisou a competência vetorial de uma população colonizada de A. aegypti ao DENV-2 além da existência da transmissão vertical do vírus por esta população e a capacidade de transmissão vertical destas fêmeas. Fêmeas de A. aegypti foram infectadas com o DENV-2 através de alimentação oral. Para analisar se a infecção artificial funcionou insetos foram dissecados e imunomarcados para o vírus dengue e observados ao microscópio laser confocal. Esta análise mostrou que intestinos, ovários e glândulas salivares apresentaram marcação para o vírus. Para confirmação dos dados de microscopia, fêmeas foram analisadas por RT-PCR e também se observou a positividade da infecção em 75 por cento das amostras testadas. A presença do vírus nos ovários nos levou a testar a capacidade das fêmeas em transmitir o vírus para a sua progênie. Para isso, mosquitos infectados puderam ovipor e as larvas do terceiro e quarto estádios foram separadas e analisadas por RT-PCR. Verificou-se que após a primeira oviposição 100 por cento das fêmeas transmitiram o vírus verticalmente e após uma segunda oviposição duas fêmeas das 25positivas, não transmitiram o vírus à sua progênie. Estes resultados mostram que a metodologia utilizada para infecção artificial foi bem sucedida e demonstrou que as fêmeas dacolônia são susceptíveis à infecção. Isto prova que, além de se infectarem, as fêmeas são também capazes de transmitir o vírus para a sua progênie. Estudos estão em progresso paraque se entenda melhor o processo de invasão do vírus dengue nos mosquitos do gênero Aedes

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Labeau, Athena. "Identification and characterization of cellular factors required for zika virus and dengue virus infection." Thesis, Université de Paris (2019-....), 2019. http://www.theses.fr/2019UNIP7060.

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Les virus de la Dengue (DENV) et Zika (ZIKV) sont deux virus émergents transmis par des moustiques et sont responsables de pathologies sévères chez l’homme. Il n’y a actuellement aucun vaccin efficace, ni traitements antiviraux disponibles contre ces arbovirus. DENV et ZIKV sont des parasites intracellulaires obligatoires qui dépendent entièrement de leur cellule hôte pour se multiplier. Cette dépendance constitue un potentiel talon d’Achille qui pourrait être exploité dans le développement de nouvelles stratégies thérapeutiques. L’objectif général de mon travail de thèse a été de comprendre a été de comprendre les mécanismes par lesquels DENV et ZIKV exploitent les fonctions cellulaires à leur avantage, et d’identifier des facteurs de l’hôte requis à l’initiation de leur cycle infectieux.ZIKV a un tropisme cellulaire pour le cerveau et est responsable de troubles neurologiques sévères tels que les microcéphalies congénitales chez le fœtus. La première partie de mon travail a été de caractériser le rôle du récepteur Axl dans le neurotropisme de ZIKV. Axl est un récepteur à la phosphatidylsérine appartenant à la famille des récepteurs TAM, qui est impliqué dans la reconnaissance et l’élimination des cellules apoptotiques par phagocytose. Ce travail de thèse a montré qu’Axl est abondamment exprimé par les cellules microgliales, les cellules gliales radiales et les astrocytes dans le cerveau en développement de fœtus et est important pour l’infection de ces cellules par ZIKV. Nous décrivons deux rôles distincts joués par Axl durant l’infection par ZIKV. Tout d’abord, Axl, par l’intermédiaire de son ligand Gas6, permet l’attachement et l’endocytose des particules virales dans les cellules gliales. Dans un deuxième temps et simultanément à l’entrée virale, les complexes Gas6-ZIKV agissent comme des « super » agonistes et phosphorylent le domaine intracellulaire à tyrosine kinase d’Axl, ce qui déclenche des cascades de transduction du signal aboutissant à l’inhibition de la réponse immunitaire innée et à une réplication virale optimale. En outre, nous avons identifié deux antagonistes d’Axl, le MYD1 et le R428, et avons montré leurs prometteuses propriétés antivirales en in vitro.La deuxième partie de mon travail a consisté à identifier de façon systématique les gènes de la cellule hôte qui sont importants pour le cycle infectieux de DENV. A l’aide d’un crible CRISPR-Cas9 à l’échelle du génome, nous avons identifié les protéines DPM1 et DPM3 comme de nouveaux facteurs de dépendance de DENV. DPM1 et DPM3 sont deux sous-unités du complexe dolichol-phosphate-mannose synthase (DPMS), localisé à la membrane du réticulum endoplasmique. Ce complexe catalyse la synthèse du dolichol-phosphate-mannose, qui fournit le mannose requis pour les différentes voies de glycosylation des protéines. A l’aide de cellules DPM knockout, nous avons montré que le complexe DPMS facilite l’infection des quatre sérotypes de DENV mais aussi de ZIKV ainsi que d’autres flavivirus apparentés tel que le virus de la fièvre jaune. A l’aide de mutants catalytiques de DPM1, nous avons pu décrire que l’activité catalytique de DPMS est cruciale pour l’infection par DENV. Nous avons montré que le complexe DPMS accomplissait différentes fonctions au cours du cycle infectieux de DENV. Ce complexe est nécessaire au mécanisme d’amplification du génome viral et, est également requis pour la N-glycosylation des protéines virales structurales, qui permet ainsi leur repliement correct.En conclusion, ces travaux ont contribué à améliorer notre compréhension des mécanismes d’entrée et de réplication de deux arbovirus majeurs, que sont ZIKV et DENV. Nos études ont identifié Axl et le complexe DPMS comme des facteurs cellulaires importants pour le cycle infectieux de ZIKV et DENV et suggèrent que ces molécules pourraient constituer de nouvelles cibles pour une intervention antivirales
Dengue virus (DENV) and Zika virus (ZIKV) are two emerging viruses transmitted to humans by mosquitoes and are responsible for severe pathologies. There is currently no efficient vaccine neither antiviral treatment available against these arboviruses. DENV and ZIKV are fully dependent on the host cell for their multiplication. This dependence creates a “Achille’s heel” that may be exploited to develop new approaches to treat these viral infections. The general objective of my PhD is to understand the mechanisms by which DENV and ZIKV exploit the cellular functions for their advantage and to identify the host factors required for the initiation of their viral life cycle.ZIKV displays a cellular tropism for the brain and is responsible for severe neurological disorders such as congenital microcephaly in the fetus. The first part of my work was to characterize the role of the Axl receptor in the ZIKV neurotropism. Axl is a phosphatidylserine receptor belonging to the TAM receptor family, which is involved in the recognition and the removal of the apoptotic cells by phagocytosis. This work showed that Axl is highly expressed by microglial cells, radial cells and astrocytes in developing brain of fetuses and is important for ZIKV infection of these cells. We described two distinct roles played by Axl during ZIKV infection. First Axl, through its ligand Gas6, promotes the adsorption and the endocytosis of viral particles into glial cells. Second and simultaneously to the viral entry, the Gas6-ZIKV complexes act a ‘super agonist” that phosphorylate Axl tyrosine kinase domain to trigger signaling cascades that lead to the inhibition of the innate immune response and optimal viral replication. Furthermore, we identified two antagonists of Axl, the MYD1 and the R428, and showed their promising antiviral properties in vitro.The second part of my work aims to identify the cellular genes required for DENV infection. Using a genome-wide CRISPR-Cas9 screen, we identified the DPM1 and DPM3 proteins as new DENV host dependency factors. DPM1 and DPM3 are two subunits of the dolichol-phosphate-mannose synthase (DPMS) complex. This latter catalyzes the dolichol-phosphate-mannose synthesis, which provides the mannose required for the different glycosylation pathways in the ER lumen. Using DPM knockout cells, we showed that the DPMS complex facilitates the infection of the four DENV serotypes as well as other flaviviruses such as ZIKV and yellow fever virus (YFV). Using DPM1 mutants, we found that the catalytic activity of DPMS is crucial for DENV infection. We showed that DPMS complex plays several functions during the DENV life cycle. This complex is necessary for the replication of the viral genome, and is also required for the N-glycosylation of structural viral proteins, which allows their correct folding.In conclusion, this PhD work provides new insights for our understanding of the entry and replication mechanisms of two major arboviruses. These studies identified Axl and the DPMS complex as important cellular factors required for ZIKV and DENV life cycle and suggest that targeting these molecules may represent new strategies to combat DENV and ZIKV infection

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Descloux, Elodie. "Dynamique moléculaire et épidémique du virus de la dengue dans différents écosystèmes." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX20719.

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Les facteurs déterminant l’épidémiologie et la sévérité de la Dengue restent mal connus. La compréhension de la dynamique des épidémies est indispensable au management de la lutte anti-vectorielle et des futures stratégies vaccinales. Dans un premier travail, la caractérisation moléculaire de 181 souches de Dengue-1 a permis de déterminer l’origine des épidémies en Polynésie Française (2001, 2006) et d’identifier un phénomène de ré-émergence. La fixation d’une mutation (E-S222T) en période endémique pourrait refléter une adaptation du virus au moustique. A l’échelle intra-hôte, la diversité des populations virales était significativement inférieure en cas de forme sévère suggérant une relation entre diversité génétique virale et clinique.Dans un second travail, cette relation a été confirmée (Bolivie, épidémie de Dengue-1, 2009) et l’importance de la composition des populations virales dans le sang capillaire a été soulignée. Dans un troisième travail, nous avons étudié l’influence du climat sur les épidémies de Dengue (Nouvelle Calédonie, 1971-2010) et développé un modèle prédictif de risque épidémique utilisable par les autorités de santé
The determinant factors of Dengue epidemiology and severity remain unclear. Understanding the epidemic dynamics is crucial to manage vector control and vaccine strategies in the future. In a first work, the molecular characterization of 181 Dengue-1 strains allowed to determine the origin of Dengue outbreaks in French Polynesia (2001, 2006) and to identify a phenomenom of re-emergence. A mutation (E-S222T) has been fixed during the endemic period that may reflect viral adaptation to the mosquito. At the intra-host scale, the genetic variability was significantly lower in patients experiencing severe forms. This suggests a relationship between the diversity of viral populations within hosts and the clinical presentation of the disease.In a second work, this connection has been confirmed (Bolivia, 2009 Dengue-1 outbreak) and the importance of the composition of viral populations in the capillary blood has been underlined.In a third work, the influence of climate on Dengue epidemics has been studied (New Caledonia, 1971-2010) and a predictive model of epidemic risk usable by the health authorities has been developed

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Souza, Jerusa Botelho. "Investigação de dengue virus circulantes em Juiz de Fora- MG." Universidade Federal de Juiz de Fora, 2014. https://repositorio.ufjf.br/jspui/handle/ufjf/601.

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A dengue é a arbovirose de maior importância para a saúde pública no Brasil. A cidade de Juiz de Fora vem passando por diversas epidemias de dengue nos últimos anos, com o registro de casos graves e óbitos. Diante deste contexto, este trabalho visou investigar a circulação de Dengue virus (DENV) em Aedes aegypti coletados do ambiente e em amostras clínicas de pacientes com suspeita de dengue por meio de técnicas moleculares e sorológicas. Amostras de Aedes aegypti (larvas e mosquitos) e soro de pacientes passaram pelo processo de extração de RNA total e este material foi utilizado para detecção de DENV por RT-PCR. Amostras clínicas de pacientes que apresentavam até seis dias de febre foram testadas para a proteína NS1 para Dengue virus. Além disso, foi realizada a análise das fichas dos pacientes nas quais foi possível verificar o dia de início da febre, os dados hematológicos (hematócrito, leucometria global e contagem de plaquetas) e sorológicos (IgM e/ou IgG). Aedes aegypti foi coletado em todas as regiões da cidade e dos 163 pools de larvas analisados, o DENV foi detectado em seis (cinco pools positivos para DENV-1 e um pool positivo para DENV-2). Estes pools haviam sido coletados nas regiões Norte, Centro e Sul. Das 166 amostras clínicas analisadas na detecção molecular, seis foram positivas (uma amostra positiva para DENV-1 e cinco amostras positivas para DENV-2). Sessenta amostras clínicas foram testadas para NS1 e 11 (18,33%) foram positivas. A partir da análise das fichas dos pacientes foi possível observar que 132 apresentavam resultados de sorologia. Destes, 39/132 (29,54%) apresentaram IgM, 9/132 (6,81%) apresentaram IgG e 15/132 (11,36%) apresentaram IgM e IgG. Além disso, 109 pacientes mostraram dados hematológicos. Entre estes, 18 (16,51%) pacientes apresentaram hematócrito inferior ao valor de referência, 32 (29,35%) desenvolveram leucometria abaixo de 3.500/mm3 e 24 (22,02%) tiveram contagem de plaquetas inferior ao valor de referência. Este é o primeiro estudo de investigação molecular de DENV em Juiz de Fora e os resultados indicaram a cocirculação dos sorotipos 1 e 2 em Juiz de Fora e a ocorrência da transmissão transovariana de DENV no Aedes aegypti. A combinação dos testes moleculares e sorológicos permitiu a identificação de 50 pacientes na fase aguda da doença e 22 na fase de convalescença. O conhecimento dos tipos virais circulantes no município e o diagnóstico da dengue em pacientes por mais de um teste e/ou parâmetro laboratorial constituem informações valiosas do ponto de vista epidemiológico e na estruturação de políticas públicas que visem o controle da dengue.
Dengue is the most important arboviral disease to public health in Brazil. The city of Juiz de Fora has undergone several dengue epidemics in recent years, with the record of severe cases and deaths. Given that, this work aimed to investigate the circulation of dengue virus (DENV) in Aedes aegypti collected from the natural environment and in clinical samples from patients with clinical symptoms of dengue, using molecular and serological approaches. Samples of Aedes aegypti (larvae and mosquitoes) and serum of patients were used for total RNA extraction, and the total RNA was used for detection of DENV by RT-PCR. Clinical samples from patients who had fever for up to six days were tested for Dengue virus NS1 protein. Furthermore, analysis of patient records was performed in which we could check the day the fever began the hematological data (hematocrit, total leukocyte count, and platelet count) and serologic response (IgM and/or IgG). Aedes aegypti was collected in all regions of the city and from the 163 larvae pools that were analyzed, DENV was detected in six (five pools DENV-1 positive and one pool DENV-2 positive). These pools were collected in the North, Centre and South regions. A total of 166 serum samples were analyzed for molecular detection, being six DENV positive (one serum sample was DENV-1 positive and five serum samples were DENV-2 positive). Sixty clinical samples were tested for NS1 and 11 (18.33%) were positive. From the analysis of patient records was possible to observe that 132 had serology results. Of these, 39/132 (29.54%) were IgM positive, 9/132 (6.81%) were IgG positive and 15/132 (11.36%) were IgM and IgG positive. In addition, from 109 patients hematological data were available. Among these, 18 (16.51%) patients had less than the reference value, 32 (29.35%) developed leukocyte hematocrit below 3.500/mm3 and 24 (22.02%) had platelet counts below the reference value. This is the first study of molecular investigation of DENV in Juiz de Fora, and the results indicated the co-circulation of serotypes 1 and 2 in Juiz de Fora and the occurrence of DENV transovarial transmission in Aedes aegypti. The combination of molecular and serological tests allowed the identification of 50 patients in the acute phase of the disease and 22 in the convalescent phase. The knowledge of viral types circulating in the municipality and the diagnosis of dengue in patients using different techniques constitute valuable information from an epidemiological point of view and help to structure public policies aiming the control of dengue.

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Jacobs, Michael Graham. "Membrane association of dengue 2 virus non-structural protein 1." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325917.

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Li, Mingyuan, and 李明圓. "Molecular dissection of dengue virus egress : involvement of host cellular factors-KDEL receptors." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208011.

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The life cycle of enveloped viruses is a complex process relying on specific interactions with host factors that, in turn, represent potential targets for interfering with viral replication and pathogenesis. Although the molecular identity of cellular receptors involved in virion entry has been established for many viruses, few studies have investigated whether host proteins on intracellular compartments may function as receptors to facilitate viral trafficking and release from infected cells. In particular, viral-host interactions during dengue virus (DENV) egress are still poorly characterized and most cellular targets identified in high-throughput screens have not been mapped to the secretory pathway. DENV structural glycoproteins, pre-membrane (prM) and envelope (E), are sufficient to assemble native Recombinant Subviral Particles (RSPs) in the endoplasmic reticulum (ER). Newly formed RSPs mimic nascent virions and traffic through the secretory pathway, where they are processed into mature particles, before being released from cells. This study demonstrated that DENV requires host KDEL receptors (KDELRs), which cycle between the ER and Golgi apparatus to retrieve resident ER proteins, for vesicular transport from ER to Golgi. Depletion of KDELRs by siRNA reduced egress of both DENV progeny virions and RSPs produced in stable cell lines expressing prM and E. A direct interaction between KDELRs and DENV prM was demonstrated in either prME expressing or DENV infected cells by co-immunoprecipitation (co-IP) experiments. By immunoblotting with specific antibodies we first showed that KDELRs interacted with prM portion. By interfering with RSPs’ maturation, we then obtained conclusive evidence that interaction was restricted to the pr fragment which released from the mature RSPs after cleavage of prM by cellular protease furin. This finding was further confirmed by GST pull down assay, which mapped the interacting domain to the N-terminal 40 residues of the pr fragment, and by mutagenesis experiments, which showed that KDELRs interact with prM through three positively charged amino-terminal residues. Biochemical analysis and immunofluorescence microscopy indicated that mutations impairing KDELRs/prM binding did not affect RSPs formation, and translocation within the ER, whereas they strongly inhibited trafficking from ER to Golgi apparatus and, consequently, their release into the supernatant. Moreover, perturbation of KDELR cycle by siRNA-mediated depletion of class II Arfs, which accumulates KDELRs in the Golgi, phenocopied results obtained with both an interaction-deficient mutant and KDELR knockdowns. Finally, we compared the effect of KDELRs on all four DENV serotypes and found significant reductions of DENV1-3, but not DENV4, in keeping with the co-IP results that demonstrated, using RSP-producing cell lines, that only DENV1-3 prM proteins interacted with KDELRs. Of note, KDELRs depletion did not affect West Nile Virus progeny virus egress, suggesting that KDELRs might not be utilized by all Flavivirus. Taken together, several lines of evidence have been presented to indicate that the loss of interaction with KDELRs reduced DENV transport from ER to Golgi and, consequently, release from infected cells. These findings, therefore, have uncovered a novel function for KDELRs as an internal receptor required for DENV trafficking and identified a rate-limiting molecular step in the late stages of DENV lifecycle.
published_or_final_version
Public Health
Doctoral
Doctor of Philosophy

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31

O'Neill, Shannon M. "The Effect of Changing Environmental Factors on the Resurgence of Dengue Fever and Severe Dengue." Scholarship @ Claremont, 2016. http://scholarship.claremont.edu/cmc_theses/1308.

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Throughout the early twentieth century, dengue fever was considered to be a nonthreatening illness, only infecting visitors of the tropics. However, in the last fifty years, there has been a resurgence of dengue fever; it is now considered to be the most consequential arbovirus, infecting more than 50 million people each year and leaving about half of the world's current population at risk of infection. The purpose of this thesis is to explore the various environmental factors that have contributed to the resurgence of dengue fever that has been seen in the last half century. Most notable of these factors are climate change and the increasing urbanization associated with population growth. Specifically, increasing temperatures and precipitation increases the available habitat for the dengue fever vector, the Aedes mosquito, while concurrently increasing both the longevity of the virus and the mosquito. Furthermore, changing sociodemographic factors associated with urbanization have helped spread the mosquito around the world, as the vector largely relies on human transportation. Finally, substandard housing often associated with insufficient water management systems creates the ideal breeding spots for the dengue vector. The Aedes mosquito is known to be one of the most versatile and one of the toughest mosquitoes in the world, which has allowed it to quickly adapt and succeed in these changing environments. Understanding these factors and their influence on the spread of dengue fever is vital in order to effectively manage current and future outbreaks. This is specifically important in regards to dengue fever and severe dengue as no vaccine or medications currently exists to treat this virus.

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32

Lo, Chung-yan Joanne, and 羅頌恩. "Characterization by electron microscopy of dengue virus egress using dengue recombinant subviral particle (RSPs) as a model." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48330115.

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Dengue is the most common mosquito-borne human disease, leading to 2.5 billion people at risk, 50-100 millions infections each year worldwide and among them, 500 000 severe dengue cases (dengue hemorrhagic fever, DHF/ dengue shock syndrome, DSS) plus more than 20 000 deaths. It can be caused by any of four dengue virus serotypes, which are antigenicly distinct and belong to the Flaviviridae family, genus Flavivirus. However, up till now there is no specific drug and vaccine against dengue. Understanding mechanisms developed by dengue virus to exploit host cells during all stages of the replication cycle is a first step towards the rationale design of anti-viral strategies. Very little is known about the late stages, which consist of assembly, budding and secretion of the virus. It is therefore very important to develop tools in order to study the egress of the virus. In this study, I investigated a stable cell line named Hela-prME that expresses serotype 1 dengue virus (DENV-1) prM and E native structural envelope proteins and constitutively produces dengue recombinant subviral particles (RSPs). Biochemical characterization of DENV-1 RSPs has validated that this cell line is a potential tool to study the dengue viral late-stage. Indeed, the maturation process observed with RSPs is similar to the pathway described for real virus (cleavage of prM fragment, homodimerization of E, acquisition of complex sugars). To better understand and depict the dengue virus late-stage secretion, I combined various electron microscopy (EM) techniques e.g. classical transmission electron microscopy (TEM), negative staining, immunogold labeling on cryo-ultrathin sections (Tokuyashu method) and tomography (ET) with such RSPs tool. The EM results obtained illustrate that electron dense particles and tubules labeled by antibodies directed against E and prM proteins were abundantly found in the lumen of endoplasmic reticulum (ER)-related cisternae of HeLa prME cells. Epositive particles were also found in other structures such as Golgi stacks and vesicles nearby as well as in aggregates with electron dense materials inside and surrounded by membrane. These particles are most likely corresponding to DENV-1 RSPs whereas the tubules may be other structures induced by assembly of prM and E proteins. This study has clearly shown that DENV-1 RSPs assemble in the ER and transport through the secretory pathway before being released. This work further validates the use of dengue RSPs and RSPs-producing cells as a model to study viral egress.
published_or_final_version
Pathology
Master
Master of Philosophy

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33

Duangchinda, Thaneeya. "Study of dengue virus specific T lymphocytes in Thai populations." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433253.

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34

Sessions, October Michael. "Dengue Virus Host Factors." Diss., 2009. http://hdl.handle.net/10161/3203.

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Dengue fever and dengue hemorrhagic fever are estimated to afflict 50-100 million people annually and are caused by one of the four serotypes of dengue virus. Dengue virus is carried and transmitted to humans by mosquitoes of the Aedes genus. Given the broad geographic distribution of Aedes mosquitoes, it has been estimated that nearly half the world's population is at risk of contracting the disease. Currently, no vaccine or specific antiviral treatment is available to combat this emerging menace.

A greater understanding of how dengue virus interacts with its insect and human hosts will facilitate the intelligent design of specific antivirals to combat the disease and enable the selective breeding of mosquitoes resistant to the virus. Although the genomes of the two primary mosquito vectors have been sequenced, the molecular tools necessary for conducting a systematic genetic analysis of host factors required for DEN infection are not yet available. These tools do however exist in the closely related fruit fly, Drosophila melanogaster. By using a strain of dengue virus that was adapted to propagate in fruit fly cells, we completed a full genetic screen for host factors required for efficient dengue virus propagation. When homologues of these host factors were assayed in a human cell line, over half were also shown to be required for efficient viral propagation. This indicates that while the virus is utilizing many of the same pathways in both of its hosts, the interaction with the insect vector has unique features that may contribute to the observed lack of pathogenesis in mosquitoes.

Dissertation

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35

Lin, Po-Yin, and 林柏吟. "Expressing dengue virus 2 E protein for pseudotype formation of murine leukemia virus with dengue virus envelope." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/35241300985270384102.

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碩士
國立交通大學
生化工程研究所
94
Dengue virus (DV), a member of the family Flaviviridae, is an enveloped, single-stranded positive sense RNA virus. Dengue fever and dengue hemorrhagic fever (DF/DHF) are caused by the dengue viruses that represent a global public health problem. In the process of virus entry, the envelope protein (E protein) plays an important role. It is a glycoprotein of 495 amino acids, with a transmembrane domain at the C-terminal. The extracellular domain of E protein is thought to interact with the host cell receptor. There is no discernable change in the host cell that infected by dengue virus. In general, one can only use plaque assay to determine whether the propagation is successful. However, not every cell line infected can produce plaques and even if it can, the plaque formation needs 5~7 days. Those events make research in dengue virus difficult. The objectives of this study were to generate infectious pseudotyped-particles that were assembled by functional dengue virus E proteins onto retroviral core particles. In this assay, mammalian cells are cotransfected with 3 plasmids, one expressing the retroviral proteins—expect the envelope—from murine leukemia virus, another expressing the replication-defective retroviral genome with β-galactosidase gene marker, and the other expressing the E gene from dengue virus. In conclusion: First, the recombinant proteins of DV-2 E protein fused with His-tag can be detected in the E.coli expression system by Western blotting analysis but not in BHK21. Second, the DV E proteins can reach the cell surface because the stable cell lines transfected with the plasmid expressing DV E gene displayed syncytium. Third, in this study, no pseudotype viruses were detected. Further studies are required.

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36

HSIEH, MING-SHU, and 謝明澍. "Development of dengue virus detection and quantification system using dengue protease activity of dengue virus non-structural protein 3." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/4v6e9j.

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博士
國防醫學院
生命科學研究所
106
Titration of viruses is a fundamental procedure used in virology laboratories. It is especially critical for clinical studies, vaccine development, and antiviral drug screening. The gold standard methods to titrate infectious dengue virus (DENV) particles are the plaque assay and immune focus assay. Both methods are accurate but labour intensive and time consuming (requiring an incubation time of >3 days). To address these drawbacks, we designed a novel reporter system, dengue protease activity detection system (DENPADS). In live cells, DENPADS stably expressed two modules, an NS3 sensor module and a Cre/loxp gene reporter switch. The NS3 sensor module was constructed by fusing Cre to C-terminal DENV-2 NS4/N10NS5, with N10NS5 representing the N-terminal first ten amino acids of NS5. An endoplasmic reticulum (ER) transmembrane characterization of NS4/N10NS5 showed that the fused Cre was maintained within ER under normal circumstances. However, when cells were infected with DENV and expressed activated NS3 protease, protease hydrolysis of NS4/N10NS5 at the specific site between NS4 and NS5 led to the release of the Cre-containing fragment. The Cre subsequently translocated into the nucleus and triggered Cre/loxp gene reporter switching. The titer of infectious virus particles correlated with the expression level of the reporter gene. Furthermore, based on this DENPADS system, we developed a new platform that can be used to easily and simultaneously screen for virus inhibition and cytotoxic effects of anti-dengue drugs. DENPADS can also be a useful tool for current dengue research.

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37

Lin, Mong-Ping, and 林孟平. "THE SEROEPIDEMIOLOGY OF DENGUE VIRUS IN TONGKANG AND THE ESTABLISHMENTOF DENGUE VIRUS RAPID DIAGNOSIS SYSTEM." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/69881736811480253118.

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38

Chen, Chun-wei, and 陳俊瑋. "Utilizing highly conserved sequences of dengue virus envelope protein to develop efficacious dengue virus vaccines." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/02469364336245280852.

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碩士
國防醫學院
生物化學研究所
103
Dengue virus has four serotypes. Because one kind of infection by other serotypes if more serotypes viral infections, and it will produce severe symptoms. Therefore, the vaccine must be able to simultaneously neutralize the four serotypes of dengue virus to be effective against the virus. So up to now still no effective vaccine against the dengue virus. The reports found in the paper, there is a conserved sequence in dengue virus membrane protein domain II bc loop sequence(The 73 to 79 amino acids RCPTQGE). It can be induced against four serotypes of the dengue virus neutralizing antibodies. In this study, we inserted a conserved sequence to Japanese encephalitis virus (JEV) infectious clone to investigate conserved sequence of dengue virus protection force. We used mice administered the recombinant virus into mice inducing an immune response, and collected mouse sera to observe whether there has neutralizing antibodies. It was found that the limited antibody titers could identify conserved sequence. Therefore we changed the way, we increased the number of repetitions of the conserved sequences to insert JEV infectious clone and considered whether more effective to induce an immune response.

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39

ChiChiu and 邱琦. "Study on the mechanism of dengue virus replication: the role of proteases during dengue virus replication." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/x63pgv.

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碩士
國立成功大學
醫學檢驗生物技術學系碩博士班
101
Dengue virus (DENV) infection can causes dengue fever and potentially lethal diseases such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Until now, there is no effective vaccine or therapeutic drugs that can be used specifically to block DENV replication in clinic. One of the difficulties of developing anti-DENV drugs is lacking of a suitable platform for compound selection. Thus, we constructed a sequence which contains secreted alkaline phosphatase (SEAP) and dengue NS2B-NS3 cutting site, followed by ligation with vector pEGFP-C1. After transient transfection of pEG(Δ2B3)SEAP into HuH7 and HT29 cells, results showed that the EGFP signals were degraded after few passages of cell culture. According to previous studies, the midgut trypsin activity can affect DENV-2 infection and dissemination in Aedes aegypti. In order to investigate the role of proteases in DENV replication, we used inhibitors that can inhibit trypsin, chymotrypsin or both of them to understand the effect of cellular enzymes on DENV replication. Supplementation of pEG(Δ2B3)SEAP transfected cells with trypsin inhibitor revealed that the dregradation of EGFP signals was inhibited. Furthermore, tosyl phenylalanyl chloromethyl keton (TPCK), which is a chymotrypsin-like protease and NF-κB inhibitor, demonstrated the most significant inhibitory ability. We then used several NF-κB inhibitors to investigate the role of NF-κB during DENV infection. The results revealed that NF-κB not only inhibit DENV-induced autoaphgy, which is acquired for DENV replication, but also decrease the level of DENV-induced cell apoptosis, and the production of macrophage migration inhibitory factor (MIF) were also decreased after inhibition of NF-κB. Taken together, these results suggested that both trypsin-like enzymes and NF-κB activation play important roles in DENV replication, DENV-induced autophagy and apoptosis, and MIF secretion. Combination of protease and NFκB inhibitors may provide an alternative therapeutic strategy for dengue patients.

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Chang, Yu-Shan, and 張玉珊. "Identification of B-Cell Epitopes of Dengue Virus Type 2 from Dengue Virus Immunized Mouse Serum." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/54497205376407502612.

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碩士
國立臺灣大學
病理學研究所
91
Epitope mapping is important for understanding the pathogenesis of viral infectious diseases as well as for developing vaccines and diagnostic reagents. Here we used phage display, a selection technique for protein-protein interaction, to find out and detect dengue virus (DEN) serotype-specific B-cell epitopes. 4G2 monoclonal antibody which reacted with E-protein of all 4 kinds of dengue serotypes was used to purify whole virus from culture supernatant. The virues were then used as antigens to immunize mice (BALB/c) to produce dengue hyperimmunized serum. We used dengue immunized serum as targets to select the immunopositive phage clones from random phage peptide library. Then we used these phage derived peptide motifs to identify dengue specific epitope, and synthesize peptides mimic dengue epitope as a detection agent. At first we found out two consensus motifs on DEN-2, TPQS and DxExRx. When these two motifs were compared with four dengue serotypes, TPQS motif will react with all four dengue serotypes, and all phage clones belong to TPQS motif group had a structure aSxRM (a=Y/F) which can not pile up to dengue genome. To understand the function of this motif, we had designed new peptide P33M (AMYSNRMA). ELISA assay showed low affinity of P33M with all four dengue serotypes, and high titer anti-P33M sera could be collected when immunized mice with P33M. P33M will be examined in the future. The motif DxExRx had been shown to mimic the sequence DLEKRH, corresponding to amino acid residues 341-346 on E-protein and motif TPQS corresponding to amino acid residues 165~168 on E-protein. E-protein is the major virion surface protein and the most important antigen for the virus. To find out the B-cell epitope according to the E-protein and further develop epitope-based antigen for serologic test reagents will be helpful in both vaccine development and prevention of dengue viral disease.

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41

Yang, Chi-Chen, and 楊季真. "Development of Anti-Dengue Virus Drugs." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/39641496026001401867.

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博士
國防醫學院
生命科學研究所
99
Dengue virus (DENV) causes disease globally with an estimated 25 to 100 million new infections per year. At present, no effective vaccine is available and treatment is supportive. There is urgent need to develop therapeutic agents to cure this epidemic disease. The availability of lead compound screening methods deeply affects the success of antiviral drug discovery. In this thesis, I focused on the development of screening methodologies and studies of anti-dengue virus drugs, and the thesis is divided into two parts. Firstly, in order to design tools for anti-dengue studies and screen small molecules against dengue virus replication, I developed an efficient DENV reporter replicon harboring the entire mature core gene sequence and cell culture-adaptive mutations. Two putative cis-acting elements (nt 160-243 and 307-402) in the core gene essential for DENV replication were identified by analyzing the replication efficacy of deletion mutants of DENV replicons. The cell culture-adaptive mutations within NS1 and NS2A of DENV replicon were selected with G418 and found to significantly enhance the stability of RNA replication in BHK21 cells for over 27 passages. I successfully used this replicon to search inhibitors of viral replication by high-throughput screening (HTS) assay, evaluate the effect of inhibitors on DENV replication, and validate drug resistance mutations. Secondly, I identified a potent and selective small-molecule inhibitor, BP2109, of the DENV NS2B/NS3 protease by HTS using recombinant protease complex consisting of central hydrophilic portion of NS2B and N-terminus of protease domain. BP2109 inhibited DENV (serotype 1-4) but not Japanese encephalitis virus (JEV) replication and viral RNA synthesis without detectable cytotoxicity. The compound inhibited recombinant DENV protease with an IC50 value of 15.43 ± 2.12 µM and reduced the reporter expression of the DENV replicon with an EC50 value of 0.17 ± 0.01 µM. Sequencing analyses of several individual clones derived from BP2109-resistant DENV RNAs revealed that two amino acid substitutions (R55K and E80K) are found in the region of NS2B, a cofactor of protease complex. The introduction of R55K and E80K double mutations into the dengue NS2B/NS3 protease and a dengue replicon conferred 10.3- and 73.8-fold resistance to BP2109, respectively. The E80K mutation was further determined as the key mutation conferring dengue replicon drug resistance (61.3-fold) to BP2109, whereas the R55K mutation alone did not affect drug resistance to BP2109. Both R55K and E80K mutations are located at the central hydrophilic portion of NS2B cofactor where extensive interactions with NS3pro domain exist. Thus, our data provide evidence that BP2109 likely inhibits DENV by a novel mechanism. Taken together, the above results provide important insights into the development of anti-dengue virus drug.

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42

Yang, Cheng-Fen, and 楊正芬. "Molecular epidemiology and pathogenesis of dengue virus and chikungunya virus." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/51954439713659220983.

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43

Chih-HuiKao and 高誌暉. "Generation of dengue virus neutralization single chain fragments of variable region (scFv) using dengue virus recombinant envelope protein." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/49781213701495757472.

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碩士
國立成功大學
醫學檢驗生物技術學系碩博士班
98
Dengue virus (DV) is an important mosquito-borne pathogen causing dengue fever (DF), dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) in the tropical and sub-tropical region of the world. Nearly 50% of people in over 100 contries are at risk of DV infection. The surface of DV particle is covered with 90 envelope (E) protein homodimers which contacted with host cells first and were essential for viral infection through receptor-mediated endocytosis. Each monomer of E protein consists of three domains. E protein domain I (DEI) is the central domain located at the center of envelope protein. E protein domain II (DEII) is the dimerization domain contains highly conserved fusion peptide. E protein domain III (DEIII) is a type-specific and immunoglobin-like domain which is also considered a receptor binding and neutralization site. Until today, there is no effective vaccine or treatment against DV infection due to antibodies against DV may enhance DV infection through Fc receptor which is also known as antibody-dependent enhancement (ADE) effect. To avoid this ADE effect, we constructed single chain fragments of variable region (scFv) from DV and DEIII-immunized mice to search for DV specific antibody that can neutralize DV binding and infection. DV purified by polyethylene glycol (PEG) precipitation and sucrose gradient as well as recombinant DEIII expressed in E.coli Rosetta using pET43.1a(+) vector and purified by Ni2+ column were used for immunization. After four times of immunization, antibodies against DV and DEIII were found in both DV and DEIII immunized mice and DV-immunized mouse sera has higher blocking ability against DV binding as detected by flow cytometry. RNA from the spleens of DV-immunized mice was extracted and transcribed to cDNA to establish scFv library. After several rounds of bio-panning, single colonies that had higher affinity to DV were picked up. In our research, we picked up three single scFv that had high affinity to DV and confimed its blocking ability against DV binding by flow cytometry. Among these three scFv clones, anti-DV scFv 5-38 has higher blocking ability against DV binding.

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44

Huang, Kao-Jean, and 黃國珍. "A murine model of dengue virus infection." Thesis, 1998. http://ndltd.ncl.edu.tw/handle/41309194011486201588.

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碩士
國立成功大學
微生物暨免疫學研究所
86
Abstract. Dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) usually occurs in the late stage of dengue virus infection. The clinical symptoms of DHF/DSS are hemoconcentration, increased vasopermeability, edema, thrombocytopenia, and hypotension. These symptoms are very similar to those of early type hypersensitivity (ETH), a unique hypersensitivity in addition to the four traditional ones. Therefore, we proposed that DHF/DSS is an ETH-associated event. In this study, we established a murine model for dengue virus infection. Dengue virus can infect mice and cause illness, such as paralysis about 2-3 weeks post-infection. Different strains of mice varied in their susceptibility to dengue virus infection . A/J strain was more susceptible than BALB/c mice, and strain of AKR or B10.A was intermediate. Dengue viral genome detected by reverse transcriptase - polymerase chain reaction (RT-PCR) was found in brain and liver of infected mouse. Intense cell infiltration was also observed in these organs as well. Thrombocytopenia developed on day 12 post- infection and anti- platelet antibodies can be detected. We also demonstrated that dengue virus can stimulate mouse immune system to generate both early-type hypersensitivity (ETH) and delayed-type hypersensitivity. When the dengue virus is injected intravenously into dengue antigen-primed or dengue virus- infected mice, the mice will develop anaphylactic responses, such as increased vasopermeability, hemoconcentration and thrombocytopenia. Some of the mice will die within half an hour due to the loss of effective blood volume. In conclusion, we provide a murine model for dengue virus infection to study the pathogenesis of DHF/DSS.

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45

Chen, Wei-Chun, and 陳韋均. "Time-Lapsed Visualization of Dengue Virus Infection." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/kd6udz.

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碩士
國立陽明大學
生醫光電工程研究所
97
Recently, fluorescence microscopic techniques with temporal and spatial resolution have been rapidly progressed and widely applied for dynamic tracking of specific bio-molecules. We have established a time-lapsed fluorescence imaging system for the observation of Dengue virus (DENV) infection. Viral motion in host cells can be thought of as the diffusion of small-particle in colloid solution, we utilized a particle tracking program and statistical analysis methods to investigate the behaviors of viral diffusion and the infectious transport pathway in host cells. After viral moving trajectory analysis, the motion behaviors of DENV have been classified into three types, including two-step diffusion, homogeneous diffusion, and complicated multiple-step motion, that show the diverse and complicated behaviors of DENV particles in host cells. Additionally, we treated cells with chlorpromazine to inhibit clathrin-mediated endocytosis, as the results, 70% of tracked DENV particles were blocked to enter host cells and confined on cell membrane with small-scale simple lateral diffusion. In order to unveil the relation between DENV and cell autophagy, we observed the dynamic images of DENV and autophagosome simultaneously, it showed that DENV could be transported to autophagosome after entering host cells, and then DENV fused with autophagosome membrane to remove its envelope structure. The fluorescent particle image tracking platform with temporal and spatial resolution is capable of observation viral motion in host cells, and virus-cell interaction, it’s quite beneficial to understand the physiological mechanism of virus infection for the development of antivirus drug or vaccine.

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46

Chen, Yu-Tien, and 陳煜恬. "Autoantibodies associated thrombocytopenia during dengue virus infection." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/11096656399076452964.

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碩士
國立成功大學
微生物及免疫學研究所
94
Dengue virus is an arthropod borne virus transmitted by Ades egypti and Ades albopictus. The disease spectrum after dengue virus infection includes self-limited dengue fever (DF) and more seriously life-threatening dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS). Thrombocytopenia and plasma leakage are the two major characteristics of DHF/DSS. Several reasons are proposed for the cause of thrombocytopenia: megakaryocytes were suppressed in bone marrow, endothelium cells activation sequestered the adhesive platelets, and platelets were damaged and consumed directly. Molecular mimicry is reported to involve in dengue virus infection because anti-platelet IgM antibodies were detected and express cross-reactivity with viral NS1 proteins. Monocytes are the major target cells during dengue virus infection, and cytokines overproduction was shown. In DHF patients, it was found that red cells, white cells and platelets been phagocytozed by histiocytes in the bone marrow. These characteristics share similarities with hemophagocytic syndrome. In our studies, we found that serum ferritin level was elevated in dengue patients that correlated with the disease severity. Dengue patients contain IgM or IgG anti-platelet antibodies. Incubation of dengue patient’s sera with platelets will lead to platelets activation such as serotonin release; furthermore, the IFN-γ activated macrophages will phagocytoze more antibody-opsonized platelets. Therefore, we propose that thrombocytopenia is mediated by immune-mediated damage; the autoantibodies play a major role in this process.

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47

Weng, Tsui-Ying, and 翁翠霙. "Lactoferrin inhibits enterovirus71 and dengue virus infection." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/70343590865253079485.

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碩士
國立成功大學
醫事技術學系
92
Lactoferrin (LF) is an iron binding glycoprotein present in some biological fluids of mammals, such as milk, saliva, and mucous secretions. The well-studied function of LF is responsible for the transportation of metals in plasma. However, recent studies have revealed that LF plays an important role in non-specific immune system against many bacterial infection as well as viral agents. In this study we have investigated the antiviral activities of LF against enterovirus71 (EV71) which causes hand, food and mouth disease, pulmonary edema and encephalomyelitis as well as dengue virus (DV), which is the most important arthropod-borne human pathogen, and results in severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We found that the infection of EV71 and DV can be inhibited by different doses of bovine LF (IC50 is 34.5 �慊/ml and 67.6 �慊/ml, respectively). LF not only could prevent EV71 and DV infection, but could also decrease virus titer when target cells were treated with LF at IC90 dose at post-infection stage. Using target cell coated-96 wells plate、IFA and immunoprecipitatioin, we found LF could bind directly to target cells. We also used virus and VP1 of EV71 coated-96 wells plate and competition assay, and found LF could also bind directly to virus. Furthermore, using Far-Western blot, we found that LF can bind to VP1 caspid protein of EV71 and the E protein of DV, both proteins are account for the binding of the viruses to the cells. In addition to no-cytotoxicity to the target cell, LF can induce IFN-��n expression of host cell. Not only in vitro, LF can also prevent mice from EV71 infection. Therefore, the antiviral as well as immunostimulatory effects of LF make it a promising candidate for the treatment of diseases induced by EV71 and DV.

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48

Sung, Tzu-Ling, and 宋紫玲. "Genetic Characterization and Quantification of Dengue Virus." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/50549644381868999415.

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碩士
國立臺灣大學
微生物學研究所
89
Dengue virus is a single-stranded positive sense RNA flavivirus that is transmitted by mosquito to human host. Probably due to the non-proofreading nature of the RNA-dependent RNA polymerase, RNA viruses are known to exist as a swarm of viruses that have slightly different genome compositions, the so-called quasispecies. Although the relationship between the degree of sequence diversity and disease severity has been reported in other RNA viruses such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), little is known about dengue virus. The first aim of this study focuses on analyzing the extent of sequence diversity in the capsid gene of DEN-3 viruses from plasma samples of 18 patients, including 10 dengue fever (DF) and 8 dengue hemorrhagic fever (DHF), during an outbreak in southern Taiwan in 1998. Using the approach of RT/PCR and clonal sequencing, the mean diversity of nucleotide sequences of each patient range from 0.05%~0.78%, indicating the existence of dengue viral quasispecies in vivo. In addition, defective viruses were found in 4.6% of total clones sequenced among 3 out of 18 patients（16.7%）. There is no difference between the extent of sequence diversity and disease severity. The second aim of this study examines the differences in dengue virus envelope gene, which is believed to play an important role in virus-cell interaction, between dengue viruses isolated from three different cells. Based on the results of direct sequencing, there is no difference in the envelope gene between plasma dengue virus and dengue isolates. In the third aim, we established a real-time RT-PCR assay to quantify Den-3 virus. The primers and probe were designed to target a highly conserved region in the capsid gene, based on the sequence information obtained from our first aim and two sequences available in the Genebank. We evaluated this assay by studying the replication kinetics of dengue virus, in comparison with the traditional plaque assay and immunofluorescence assay. It is a convenient, sensitive and accurate method of quantification, and has great potential in the future research.

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49

Yang, Shih-Ting, and 楊世婷. "Infection of murine cells by dengue virus." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/uqqe8p.

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碩士
國立成功大學
微生物及免疫學研究所
90
Dengue virus (DV), an arthropod-borne flavivirus, causes a febrile illness or severe hemorrhagic fever for which there is no effective antiviral treatment and no vaccine available. It is still not clearly understood which tissue cells are the natural targets of DV. In addition to monocytes/ macrophages, neuron, hepatocytes, endothelial cells, B lymphcytes and dendritic cells have also been reported as potential targets for DV. Recent studies have demonstrated two subsets of dendritic cells, monocyte-derived dendritic cells and human skin Langerhans cells are targets for dengue virus infection. We are interested whether mouse dendritic cells can be infected by DV. Large numbers of dendritic cells from mouse bone marrow cultures supplemented with Granulocyte/ Macrophage Colony-stimulating Factor (GM-CSF) were generated. We found that bone marrow-derived dendritic cells (BMDC) can be infected with dengue virus. At 24 hours post-infection, negative- and positive-strand viral genome was detected on low density BMDC by RT-PCR, viral antigens were also detected by immunofluorescence analysis. The efficiency of DV infection on BMDC was extremely low, because BMDC express Type 1 Interferon. Furthermore, DV was injected subcutaneously into mouse skin to mimic mosquito bite. The spread of DV from inoculation site to the draining lymph node was characterized. The lymph node dendritic cells were isolated by optiprep gradient and the viral antigen was analyzed by immunofluorescence assays. The efficiency of DV infection on mouse cells was extremely low. A mouse hepatoma cell ML-15a can support dengue virus replication. This DV2 infected-ML-15a cell was inoculated into mouse footpad. DV viral antigens positive cell that express CD86 can be detected in draining lymph node.

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50

Snitko, Mariya. "Identifizierung neuer Dengue Virus Typ-2 Proteaseinhibitoren." Doctoral thesis, 2014. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-112502.

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Weltweit leben ca. 2,5 Mrd. Menschen im Dengue Virus Verbreitungsgebiet. Dengue Virus Infektionen führen zum Dengue Fieber und können bei Re-Infektionen mit anderen Serotypen das sog. Dengue Schocksyndrom mit einer Letalität von 10% verursachen. Momentan stehen jedoch weder Impfstoffe noch antivirale Substanzen zur Verfügung. In der vorliegenden Arbeit sollten DENV2-Proteaseinhibitoren entwickelt werden. Dazu wurde ein in vitro DENV Proteasetest etabliert, für den die DENV Protease in Bakterien exprimiert und anschließend gereinigt wurde. Mit diesem System wurden 144 Verbindungen getestet und Diaryl-Thioether, Thiazole und Zimtsäurederivate als Dengue PIs charakterisiert. Ein Diarythioether (FM 47) wurde an die Proteasestruktur modelliert und nach den Strukturdaten zielgerichtet derivatisiert. Diese Derivate ihibierten die Protease im mikromolaren Bereich und wurden anschließend in einer Zellkultur getestet. Drei Substanzen - HWu 11, HWu 51, HWu 62 - zeigten gute bis sehr gute Hemmung in vivo bei 2,5 μM. Die Charakterisierung der Inhibitoren zeigte eine nicht-kompetitive Hemmung. Die gefundenen Substanzen bilden eine gute Grundlage für die weitere Inhibitorforschung
About 2,5 billion people live in dengue virus endemic area. There is neither a established medical treatment or vaccine for dengue virus infection. The Dengue virus protease represents a prime target for rational drug design. Here, I report the development of a fluorometric dengue virus protease test and the screening of a library of potential dengue virus protease inhibitors, which resulted in the identification of several substances inhibiting the dengue protease with IC50 values in the low micromolar range. Among these, three diaryl thioethers were shown to be potent non-competitive inhibitors, which blocked DENV replication in cell culture in the submicromolar range

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