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1

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.
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Public Health
Doctoral
Doctor of Philosophy
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2

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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3

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.
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Pathology
Master
Master of Philosophy
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4

Warke, Rajas V. "Activation of TNF alpha, IL1-beta and Type-i IFn pathways in human umbilical vein endothelial cells during dengue 2 virus infection." Link to electronic thesis, 2002. http://www.wpi.edu/Pubs/ETD/Available/etd-0424102-141341.

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5

Bhatia, Kanika Devi. "The role of mononuclear phagocytes in dengue immunopathogenesis /." St. Lucia, Qld, 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17170.pdf.

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6

Lambeth, Cassandra Rashida De Silva Aravinda Manu. "Interactions between dengue type 3 viruses and human dendritic cells." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1238.

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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology in the School of Medicine." Discipline: Microbiology and Immunology; Department/School: Medicine.
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7

Serafin, Ina Loretta. "Epitope mapping of the dengue 3 envelope protein." Thesis, Queensland University of Technology, 1999.

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8

Brown, Jennifer L. "A molecular and immunological investigation of cellular responses to dengue virus identification of potentially upregulated host genes and the constructionof a vaccinia virus expressing the dengue 1 Hawaii NS3 protein." Link to electronic version, 2000. http://www.wpi.edu/Pubs/ETD/Available/etd-0330100-124248/.

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9

Matusan, Anita Esther 1973. "Mutational analysis of the proteinase and helicase regions of the Dengue virus type 2 NS3 protein." Monash University, Dept. of Microbiology, 2001. http://arrow.monash.edu.au/hdl/1959.1/8339.

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10

Kudelko, Mateusz Aleksander. "Characterization of a novel role for class-II ADP-ribosylation factorsin the regulation of dengue egress using newly developed recombinantsubviral particles." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46455620.

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11

Choi, Wai-yee Junet. "Serum neopterin for early assessment of severity of severe acute respiratory syndrome and Dengue virus infection." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B32031579.

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12

Enguehard, Margot. "Interaction between chikungunya and dengue viruses during co-infection in Aedes mosquito cells and in Aedes aegypti mosquito." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1161/document.

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Au cours des dernières années, de nombreuses épidémies ont emergé ou ré émergé, et sont causées par des arbovirus (arthropod-borne viruses), des virus transmis à des vertébrés par des insectes piqueurs vecteurs. Avec l'augmentation de la densité humaine dans certaines zones géographiques et le réchauffement climatique qui contribuent à l'expansion géographique des vecteurs, les maladies induites par ces virus (arboviroses) ont un impact de plus en plus important sur la santé humaine et l'économie mondiale. Il est donc déterminant d'augmenter nos connaissances sur les systèmes mis en jeux pour garantir la sécurité sanitaire des populations exposées. Les enjeux actuels reposent aussi bien sur la compréhension des virus que sur la compréhension de l'alternance d'hôtes, directement responsables de l'émergence et la dissémination des agents infectieux. Les moustiques sont des vecteurs majeurs des arbovirus comme la dengue (genre Flavivirus) et le Chikungunya (genre Alphavirus). Transmis par les mêmes moustiques Aedes aegypti et Aedes albopictus, le virus de la Dengue (DENV) est responsable de la plus importante arbovirose en zone tropicale, et le virus Chikungunya (CHIKV) est responsable dans le monde entier de centaines de milliers de cas d'infection, et les épidémies récentes ont touché les pays européens. Ainsi, il a été observé que le moustique Ae. albopictus pouvait porter simultanément CHIKV et DENV, et des cas de co-infections humaines ont été observés en Afrique. Toutefois, bien qu'en théorie les deux virus soient capables d'infecter les mêmes cellules chez l'insecte ou l'homme, il n'y a aucune étude détaillée sur les interactions au niveau cellulaire entre CHIKV et DENV lors de la co-infection d'une cellule. C'est pourquoi il est indispensable d'accroitre nos connaissances sur l'interférence éventuelle entre les virus Chikungunya et Dengue pour l'utilisation de voies cellulaires communes chez les insectes vecteurs et l'hôte humain lors de co-infection
Emergence and geographical extension of dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses increase simultaneous outbreak in an increasing number of countries. To date, no vaccine or cure have yet been developed against these diseases those cause a tremendous impact on human health and in the economy worldwide. During recent simultaneous outbreaks, up to 12% of patients have been diagnosed to be co-infected by CHIKV and DENV. In addition, it was shown that the mosquitoes Aedes albopictus could carry and transmit simultaneously CHIKV and DENV. However, the pathology, as well as the epidemiology of a pathogen, relies on the interactions between several infectious agents present within an organism or a community in the environment. It is crucial to consider to which extent a host infected by a first microorganism is modified and whether its reaction to the infection by a second microorganism is consequently altered. However, there is no extensive report of Alphavirus-Flavivirus or Flavivirus- Flavivirus interactions. Our global objective is to characterize these co-infections in both mosquitoes and humans, at the cell and molecular level. To this aim, we started this project by performing sequential co- infection in different cell lines from Aedes albopictus and Aedes aegypti. We found that the permissiveness and production of DENV is enhanced in presence of CHIKV. On the contrary, there is no effect of DENV pre-infection on subsequent CHIKV co-infection. We generalized the synergistic phenomena and we showed that CHIKV pre-infection also increased the infection by DENV-1, DENV-3 and DENV-4, but also by two others re-emerging Flaviviruses, the Yellow Fever Virus (YFV), and the Zika Virus (ZIKV). Remarkably, we succeeded to establish a mosquito model of co-infection of Aedes aegypti mosquito after by different two feedings at 4 days interval. Using this sequential co-infection, we were able to show that a pre-infection of Aedes aegypti by CHIKV increase the level of DENV-2 RNA in salivary glands compare to mono-infected mosquitos. This phenotype is reminiscent of the phenotype we observed in vitro during successive infections. Altogether, our study paves the way to the characterization of molecular interaction between Flaviviruses and Alphaviruses in mosquito in vitro and in vivo. This study can be crucial for a better understanding of disease and epidemiology during simultaneous outbreaks
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13

Beasley, David Wayne Colin. "Identification of functional epitopes on dengue 1 environs." Thesis, Queensland University of Technology, 1999.

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14

Brown, Jennifer L. "A Molecular and Immunological Investigation of Cellular Responses to Dengue Virus: Identification of Potentially Upregulated Host Genes and the Construction of a Vaccinia Virus Expressing the Dengue 1 Hawaii NS3 Protein." Digital WPI, 2000. https://digitalcommons.wpi.edu/etd-theses/187.

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The purpose of this thesis for the degree of Master of Science was to use molecular and immunological techniques to study cellular responses to dengue virus infection. In the initial study, Differential Display was used to compare mRNA expression in dengue-infected K562 cells and mock-infected cells. Cloning and sequencing were then used to identify cellular genes that were potentially up-regulated in response to Dengue virus infection. These genes included bleomycin hydrolase and a dystrophin homologue. The goal of the later part of this research was to construct a recombinant vaccinia virus expressing the dengue 1 Hawaii NS3 protein. Cytotoxic T-lymphocyte assays and protein gel electrophoresis showed that the NS3 protein was being expressed. This construct was then used to study the cytotoxic T-cell response of a dengue 1 vaccine recipient. The results of this study showed that this individual has dengue 1 NS3 specific T-cells and also that this vaccinia virus can be used for subsequent T-cell studies.
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15

Murphy, Amanda. "Eco-epidemiology of dengue and Ross River viruses across rural and urban environments." Thesis, Queensland University of Technology, 2020. https://eprints.qut.edu.au/204283/1/Amanda_Murphy_Thesis.pdf.

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This research investigated the epidemiology and ecology of two mosquito-borne viruses of public health importance in the Asia-Pacific: dengue and Ross River viruses. Patterns of disease caused by these two viruses were analysed in space and time across rural and urban settings, and some of the influencing demographic, geographic and environmental factors were explored. The outcomes of this research included increased understanding of the complex influences on mosquito-borne virus transmission in different human habitats, and the development of unique field and lab-based approaches for studying how the interactions of mosquitoes, wildlife, humans and their environments can cause disease.
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16

Choi, Wai-yee Junet, and 蔡偉儀. "Serum neopterin for early assessment of severity of severe acute respiratory syndrome and Dengue virus infection." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B32031579.

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17

Choudhury, Md Abu Hasnat Zamil. "Population Dynamics of RNA viruses." Thesis, Queensland University of Technology, 2013. https://eprints.qut.edu.au/60866/1/Md._Choudhury_Thesis.pdf.

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Between 50 and 100 million people are infected with dengue viruses each year and more than 100,000 of these die. Dr Choudhury has demonstrated that populations of dengue viruses in individual patients are genetically and functionally very diverse and that this diversity changes significantly at the time of major outbreaks of disease. The results of his studies may inform strategies which will make dengue vaccines far more effective.
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18

Warke, Rajas V. "Molecular Dissection of the Cellular Reponse to Dengue Virus Infection." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/366.

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The immune response to viral infection involves a complexity of both innate and adaptive pathways at the cellular and the molecular level. There are many approaches to begin to define the pathways at work to control viral pathogenesis. The approach favored in this thesis was to conduct a broad screen of the innate immune response at the gene expression level of infected cells. The innate immune response is critical to the control of viral infections. Type I interferons (IFN), IFNα and IFNβ, are antiviral proteins that are an integral part of the innate immune response. Furthermore, by virtue of their effects on maturation and activation of antigen-presenting cells, IFNs are a pivotal link between the innate and adaptive immune systems. Most cell types produce type-I IFN when exposed to viruses. However, viruses have evolved multiple strategies to suppress IFN production or signaling. It is imperative to understand the virus-host interaction at the molecular level in order to identify as yet unknown mechanisms of the host antiviral response; these additional pathways may be useful in counteracting the viral suppression of IFN. Type-I IFNs regulate expression of at least five hundred genes, suggesting a complex network of signaling pathways. Depending on the cell type different proteins regulate the induction of IFN or the expression of IFN-inducible genes. Identification of proteins that induce selected IFN-inducible genes may provide synergistic activity with or may have an advantage over type-I IFN for anti-viral therapy in the future. Many diseases are untreatable if identified late in their progression. In resource-limited countries, many diseases are diagnosed clinically, which can lead to incorrect or delayed diagnosis and treatment. The identification of biomarkers of disease has the potential to guide the correct therapy in a timely fashion. The objective of this thesis was to identify novel anti-viral therapies and disease biomarkers for dengue virus (DENV) infection. DENV is a mosquito-borne positive-sense single-stranded RNA virus, which causes an estimated 50 million infections annually. Most DENV infections result in a febrile illness called Dengue fever (DF). Less frequently, infections cause Dengue hemorrhagic fever (DHF), a potentially fatal vascular leakage syndrome associated with the production of pro-inflammatory cytokines. At present patients infected with DENV can only be treated by intravenous fluid support to prevent hypovolemia and hypotensive shock. This treatment is less effective in severe cases if the diagnosis is delayed. Identification of therapeutics with both antiviral and immune-modulatory activity may lower patient mortality and reduce the burden of DENV on society. DENV infection is cleared in most individuals after a short period of viremia {Libraty, 2002 #2225}. Based on in vitro and mouse models, type-I and type-II IFN signaling pathways are thought to be critical in the regulation of DENV infection. Higher serum levels of type I and type II IFNs during acute DENV infection in patients lend support to the above hypothesis {Kurane, 1993 #2152; Libraty, 2002 #2225}. To understand the DENV-human host cell interaction at the molecular level, we performed global gene expression analysis on DENV-infected primary human cells using Affymetrix GeneChips (HG-U133A). We studied dendritic cells (DC), monocytes, B cells and human umbilical vein endothelial cells (HUVECs), all of which are known to be permissive to DENV infection. We first identified genes commonly regulated in multiple cell types in response to DENV infection; we hypothesized that understanding this common gene expression profile would identify signaling pathways involved in regulation of viral spread, activation of immune cells or induction of inflammation. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the 23 common response genes, was identified as a key link between type I and type II interferon response genes. Pretreatment of cells with recombinant TRAIL (rTRAIL) inhibited DENV replication in monocytes, B cells, HUVECs and DCs. Using the DC infection model, we showed that this inhibition of viral replication was apoptosis-independent. Type-I IFN receptor (IFNR) blocking experiments showed that signaling through the type-I IFN receptor played an important role in the antiviral activity of exogenous rTRAIL. Furthermore, TRAIL also significantly reduced the expression of mRNA and protein of pro-inflammatory cytokines (TNFα, MIP-1β and IFNα) and chemokines (MCP-2, IP-10 and IL-6) in response to DENV infection. The data that TRAIL inhibits both viral replication and pro-inflammatory cytokine production suggest that TRAIL has therapeutic value in dengue. The endothelial cell is the site of pathology in DENV infection in vivo (vascular permeability and plasma leakage). To understand the direct effect of DENV infection on endothelial cells and its role in the induction of genes regulating vascular permeability, we compared gene expression in DENV-infected HUVECs to that of uninfected cells and cells infected with other RNA and DNA viruses, including flaviviruses (West Nile, yellow fever, and Japanese encephalitis viruses), bunyaviruses (Sin Nombre and Hantaan viruses), Epstein-Barr virus and vaccinia virus. Among the genes confirmed for their differential expression, ST2 (Interkeukin-1 receptor-like-1 protein-IL1RL1) and indoleamine 2,3-dioxygenase (IDO) were identified to be upregulated specifically in response to DENV infection. Higher serum soluble ST2 (sST2) levels were detected in DENV-infected patients than in patients with other febrile illnesses (OFI) at the end of the febrile stage and at defervescence (p=0.0088 and p=0.0004, respectively). In addition, patients with secondary DENV infections had higher serum sST2 levels compared with patients with primary DENV infections (p=0.047 at the last day of fever and p=0.030 at defervescence). Higher levels of IDO activity (pIn conclusion, global gene expression analysis identified novel proteins with promising characteristics for the treatment and/or diagnosis of DENV infection. Although further studies will be needed to validate the clinical utility of TRAIL, sST2, and IDO, these studies demonstrate the utility of this unbiased genomics approach to identify therapies to currently incurable diseases.
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19

Collins, Jacob M. "Transcriptional Regulation of the Interleukin-8 Promoter by Multiple Dengue Viral Proteins: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/616.

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Dengue virus (DENV) causes over 500,000 infections annually with a spectrum of clinical diseases ranging from subclinical infection to dengue, a mild febrile illness, to life-threatening severe dengue. Vascular leakage without endothelial cell damage is the hallmark symptom of severe dengue illness and is proposed to be directly mediated by soluble inflammatory mediators IL-8 and TNFα. IL-8 production occurs in response to DENV infection, is elevated during severe dengue, is proposed to inhibit interferon, and could potentially recruit target cells to sites of infection. We previously showed that expression of DENV NS5 activates the IL-8 promoter, induces IL-8 transcription, and induces IL-8 protein production in HepG2 and HEK293A cell lines. As multiple DENV proteins are reported to interact with important signaling pathways, we hypothesized that other DENV proteins could contribute to the activation of IL-8. We found that plasmids expressing prM-E together, the GPI-linked variant of NS1 (NS1G), the carboxyl-terminal 112 amino acids of NS4B, as well as NS5 each induced expression from an IL-8 promoter-driven reporter plasmid. Expression of NS5 also induced activation of a RANTES promoter construct and TNFα mRNA expression. Further, we found that the carboxyl-terminal polymerase domain of NS5 was sufficient to induce IL-8 secretion but polymerase function was not required. Like NS5, prM-E and NS1G induced luciferase expression from an AP-1-driven reporter plasmid. We further tested whether activation of the IL-8 promoter depended on any single transcription factor within IL-8 using IL-8 promoter-driven plasmids mutated at the AP-1, C/EBP or NF-κB binding sites. We found that activation of the IL-8 promoter by prM-E, NS1G and NS4B did not depend on activation of any single transcription factor. Our data suggested that AP-1 may be both positively and negatively inducing transcription, fitting with previous theories that DENV regulates IL-8 induction. However, we did not observe any differences in activation of AP-1 subunit c-Jun, or the inhibitory subunits Fra-1 or Fra-2 between DENV and mock-infected cells. These data support a model in which multiple DENV proteins activate the IL-8 promoter, provide a potential basis of IL-8 induction by DENV in multiple cell types, and further supports a mechanism by which DENV contributes to severe dengue illness.
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20

Ostrout, Nicholas D. "Vaccinia and Dengue Viruses: Exploring Current Fundamental Issues of Memory T Cells and Utilizing Comparative Quantitative Immunology to Compare Correlates of Protection Following Smallpox Immunization." Cleveland, Ohio : Case Western Reserve University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1205938117.

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21

Tricou, Vianney M. "Dengue diagnostics and therapeutic interventions in Viet Nam." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:46dfff8c-f7d2-4c43-b053-a5438531290a.

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Dengue is a major public health problem that affects tens of millions of people annually in tropical and sub-tropical countries. This acute viral infection happens to be severe and even life threatening but there is still no available drug or vaccine. Previous studies have noted early higher viral burden in patients who develop more severe symptoms suggesting that administration of a potent and safe antiviral may prevent progression to severe dengue. To verify this hypothesis, we have conducted the first RCT directed towards reducing the viral burden in vivo by administrating chloroquine (CQ), a cheap and well-tolerated drug that inhibits DENV in vitro with concentrations achievable in vivo, to 307 Vietnamese adults with suspected dengue (257 of them were laboratory-confirmed cases). Unfortunately, we did not see an effect of CQ on the duration of infection. However in patients treated with CQ, we observed a trend towards a lower incidence of severe forms. We did not find any differences in the immune response that can explain this trend. We also found more adverse events, primarily vomiting, with CQ. In addition, we have explored the relationships between clinical features, antibody responses and virological markers in these patients. We found that the early magnitude of viremia is positively associated with disease severity and there are serotype dependent differences in infection kinetics. We found as well that DENV was cleared faster and earlier in patients with secondary infections. To complete this study, we have also evaluated 2 rapid lateral flow tests for the diagnosis of dengue in a panel of plasma samples from 245 RT-PCR confirmed dengue patients and 47 with other febrile illnesses. Our data suggest that the NS1 test component of these tests are highly specific and have similar levels of sensitivity (~60%). Both NS1 assays were significantly more sensitive for primary than secondary dengue. The IgM parameter in the SD Duo test improved overall test sensitivity without compromising specificity. All these findings are of major importance for further anti-viral drug testing.
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22

Mathew, Anuja. "Human T Cell Responses to Dengue Virus Infections: CD8+CTL and Acute Immunosuppression: a Dissertation." eScholarship@UMMS, 1999. https://escholarship.umassmed.edu/gsbs_diss/18.

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There are four serotypes of dengue virus designated dengue 1, 2, 3 and 4 (D1, D2, D3 and D4) and epidemiological studies indicate that a severe complication of dengue virus infection - dengue hemorrhagic fever (DHF) is more likely to occur following a secondary infection. DHF is hypothesized to be immunologically mediated and may be triggered by virus-specific T cells. It is also likely that dengue virus-specific cytotoxic T lymphocytes (CTLs) are important for recovery from dengue virus infections. An analysis of the immune response during acute illness and when the patient has recovered from the infection (immune state) is therefore important as it will provide insights into the immunopathological nature of the disease. This thesis initially examines the CD8+CTL responses in volunteers who have received live attenuated dengue vaccines and then investigates acute and immune T cell responses in children following natural infection with dengue. When this project was initiated, there was little available information on the human CD8+ T cell responses to dengue viruses. PBMC from one donor had generated memory CD8+CTL to the nonstructural protein NS3 of dengue virus. Memory CD8+CTL responses were therefore analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult American volunteers who had received monovalent live-attenuated candidate vaccines of the 4 dengue serotypes. All the donors had specific T cell proliferation to dengue viruses and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural proteins NS3 and NS1.2a and the structural protein E were recognized by CD8+CTLs from six, five and three donors respectively. All donors recognized either NS3 or NS 1.2a. In a donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using PBMC of two donors were serotype-specific whereas all other donors had serotype-cross reactive responses. For one donor, CTLs specific for E, NSl.2a and NS3 proteins were all HLA-B44 restricted. For the three other donors tested the potential restricting alleles for recognition of NS3 were HLA-B38, A24 and/or B62 and B35. These results indicate that the CD8+CTL responses of humans after immunization with a single serotype of dengue virus are diverse and directed against a variety of proteins. The nonstructural proteins NS3 and NSl.2a appear to be immunodominant and should be considered when designing subunit vaccines for dengue. Previously T cell responses had not been examined in people who have had natural infections with dengue. The HLA diversity between North American Caucasians and populations where dengue is a serious health problem, calls for the analysis of immune responses in people who have been infected with natural circulating strains of the virus. We examined the memory cytotoxic T lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue infections. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for the NSl.2a and NS3 proteins respectively. All CTL lines derived from both patients were crossreactive with other serotypes of dengue virus. The CD8+ NS1.2a specific lines from patient KPP94-037 were HLA-B57 restricted and the CD8+ NS3 specific lines from patient KPP94-024 were HLA-B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA-DR7 restricted. A majority of the CD8+CTLs isolated from patient KPP94-024 were found to recognize a.a. 221-232 on NS3. These results demonstrate that after symptomatic secondary natural dengue infections in Thai patients, CTLs are mainly directed against nonstructural proteins and are broadly crossreactive. The data correlate with our observations that nonstructural proteins are immunodominant proteins in volunteers who received dengue vaccines. We were interested in examining CTL responses in children during their acute illness and comparing them to memory CTLs obtained from the same children a year or more after the infection. A detailed analysis on samples from nine patients during their acute illness failed to generate any dengue virus-specific CTL responses. We therefore decided to determine if cell mediated responses are altered during acute dengue infection. Decreased proliferative responses to mitogens and recall antigens have been observed in PBMC obtained during several acute human viral infections. All responses of PBMC during acute illness were compared to the same patients PBMC obtained at least 6 months after their infection. Proliferative responses to PHA, anti-CD3, tetanus toxoid and dengue antigens were significantly decreased in PBMC obtained during the acute infection. The proliferative responses to PHA were restored by the addition of gamma-irradiated autologous immune or allogeneic PBMC. Cell contact with the irradiated PBMC was necessary to restore proliferation. Non-T cells from the acute PBMC of dengue patients did not support proliferation of T cells from control donors in response to PHA, but T cells from the PBMC of patients with acute dengue proliferated if accessory cells from a control donor were present. Addition of anti-CD28 antibodies restored anti-CD3-induced proliferation of the PBMC of some patients. The percentage of monocytes was reduced in the acute sample of PBMC of the dengue patients. Addition of IL-2 or IL-7, but not IL-4 or IL-12 also restored proliferation of acute PBMC stimulated with anti-CD3. The results demonstrate that both quantitative and qualitative defects in the accessory cell population during acute dengue illness result in a depression of in vitro T cell proliferation. The data generated from this project shed light on the nature of the immune responses during acute natural dengue infections. It strengthens the existing data on the human memory CD8+CTL responses to dengue viruses and validates the observations by examining memory CTL responses after natural dengue infection in patients from Thailand. In addition, we demonstrate a profound defect in lymphoproliferative responses during dengue illness.
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23

Nightingale, Zachary Davis. "Distinct Behaviors of Infected and Bystander Dendritic Cells Following Exposure to Dengue Virus: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/361.

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Dengue viruses (DV) are re-emerging mosquito-borne pathogens for which four distinct lineages, grouped based on serology and referred to as serotypes 1-4 (DIV-D4V), have been described. Epidemiological data imply that re-infection with a "heterologous" serotype, i.e, one other than that to which the individual was originally exposed, enhances the risk for development of severe disease, dengue hemorrhagic fever (DHF). The hallmark of DHF is a transient capillary leakage syndrome of rapid onset, temporally associated with the resolution of fever and viremia. In its most grave form, the vascular permeability phenomenon in DHF may progress to dengue shock syndrome (DSS), which is often fatal in the absence of appropriate medical care. Despite the fulminant nature of vascular leakage during DHF/DSS, this phenomenon does not appear to be due to direct cytopathic effects of DV. Rather, inappropriate reactivation and/or regulation of dengue-specific memory are the prevailing theorized (immunopathological) etiologies. Traditional vaccine development techniques have proven insufficient for DV, since any vaccine must offer complete protection against all four serotypes to avoid enhanced pathology on natural viral challenge. Understanding the underlying mechanisms that contribute to dengue disease, particularly the development of dengue-specific memory, is therefore of critical importance. Dengue immunopathology and the specific aspects of immunological memory that determine disease severity are heatedly debated. Previous research in our lab has suggested that T cell responses contribute to the severity of dengue illness. Clinical data indicate enhanced immune activation in more grave cases of DV infection, and serotype cross-reactive T cells from multiple individuals are present after both primary and secondary dengue infections. However, little is known about the conditions under which T cells are primed and dengue-specific memory is generated. Dendritic cells (DCs) are bone marrow-derived cells that play a central role in directing activity within the immune system. DCs shape quantitative and qualitative aspects of adaptive immunity, and therefore the intrinsic characteristics of host memory to a pathogen. DCs are essential in generating primary immune responses, due to their particular effectiveness in stimulating naïve T cells. DCs also play important roles in the reactivation of memory to an infectious agent, and as reservoirs for the dissemination of invading microorganisms. Exposure to pathogens or their products initiates a series of phenotypic and functional changes in DCs, termed maturation. DC maturation involves a coordinated response of immunomodulatory surface molecule elaboration and cytokine production, culminating in antigen presentation to, and co-stimulation of, T cells specific for the invading agent. The DC response is ostensibly tailored to facilitate effective elimination by regulating effective downstream interactions of the DC with T cells. A number of viruses have evolved to infect DCs and alter their functional behavior, facilitating their own survival within the host, and the herd. DV readily infects DCs both in primary cell cultures and in vivo. However, reports on the effects of DV infection on DC maturation vary both with regard to some of the cytokines produced, and the phenotypes of infected versus bystander cells. Although DCs appear to be activated following DV exposure, responses on the single-cell level appear to depend on the infection state of the cell, hypothetically driven by intracellular virus-mediated effects. Therefore, downstream responses to these divergent populations - i.e., actively infected cells versus uninfected bystander cells - are likely to be the consequence of at least two modes of DC behavior. Because DCs play a pivotal role in adaptive immune development, and because the resulting memory response appears to be critical in affecting disease pathology after heterologous DV re-infection, I sought to explore the phenomena of DC maturation in response to dengue exposure, and to begin to answer the question of how active infection alters the functional capabilities of DCs. Notably, primary dengue infection is generally well-controlled with minimal pathology. Therefore, this thesis addresses the hypothesis that DV infection of DCs results in cellular activation and stimulation of antiviral immunity, despite virus-mediated alteration of DC maturation. In order to address this hypothesis, I examined both DV infection-dependent and independent effects on DC functional responses including surface molecule regulation secretory activity, and CD4 T cell allostimulatory priming. DCs derived from human peripheral blood monocytes were readily infected with multiple strains of DV. DV infection of DCs derived from separate donors was dose-dependent, with substantial variability in DC susceptibility to infection. Exposure to live DV activated surface molecule expression in DCs, similar to the effects of defined maturation stimuli including a combination of TNF-α and IFN-α, or LPS. In addition, UV-inactivated DV induced expression of cell surface molecules, albeit to a lesser extent than did live virus demonstrating inherent stimulatory properties of DV particles. Using intracellular staining for DV envelope (E) protein, I detected increased surface molecule expression on both infected DCs and uninfected bystander DCs from the same culture, as compared to mock-infected DCs. These data indicate that activation was not prevented in cells undergoing active viral replication. However, the degree of surface molecule induction depended on the infection state of the cell. Infected DCs had enhanced PD-L2 and MHC II expression relative to uninfected bystander cells, while PD-L1, CD80, CD86, and MHC I expression were suppressed with active infection. Therefore, intracellular DV replication altered the process of cell surface molecule regulation within these cells. DV infection of DCs also resulted in the secretion of a broad array of cytokines and chernokines. These included the antiviral cytokine IFN-α, inflammatory cytokines TNF-α, IL-6, and IL-1α, and inflammatory chemokines IP10, MCP-1, MIP-1α, and RANTES. DV infection did not induce DC production of the IL-12 p70 heterodimer, and secretion of the immunosuppressive cytokine IL-10 was low in most experiments. Similar to the results seen with surface molecule induction, UV inactivation of DV reduced, but did not eliminate, cytokine and chemokine responses. At the single-cell level, TNF-α and IP10 production profiles of infected DCs and uninfected bystander DCs were distinct. DV infection in DCs reduced production of IP10, but stimulated TNF-α as compared to uninfected bystander cells in the same culture. Blocking experiments demonstrated that IFN-α/β produced by DCs in response to infection actively inhibited viral protein expression and drove IP10, but not TNF-α, production. DV infection of DCs did not consistently suppress DC stimulation of allogeneic CD4 T cell proliferation. In cases where infection enhanced DC stimulatory function, T cell proliferation was less pronounced than that induced by DCs activated with exogenous TNF-α plus IFN-α. Increasing multiplicity of infection (MOI) of DCs with DV resulted in increasing DC infection rates, but a statistically significant trend at the highest MOIs for decreased T cell alloproliferation, suggesting that direct infection of DCs reduces their CD4 T cell priming function. MOI-dependent reduction in DC stimulatory function depended on replication-competent virus. Increased MOIs during DV infection of DCs did not cause an elevation in detectable IL-10 in supernatants derived from T-DC co-cultures. In addition, increased DV MOI of DCs was not associated with increased levels of either IL-13 or IFN-γ in supernatants from T-DC co-culture, suggesting that actively infected DC do not skew CD4 T cells towards a specific Th phenotype. These data demonstrate that DV infection induces functional maturation of DCs that is modified by the presence of virus through both IFN-dependent and independent mechanisms. However, the allostimulatory phenotype of DCs was not universally enhanced, nor was it skewed towards antiviral (Th1)-type responses. These data suggest a model whereby dengue infection during primary illness results in controlled immune stimulation through activation of bystander DCs, and the generation of mixed Th-type responses. Direct DV infection of DCs appears to attenuate activation of, and potentially clearance by, antiviral mechanisms. During secondary infection, reduced IP10 production and enhanced TNF-α secretion by infected cells coupled with MHC I downregulation and enhanced PD-L2 expression, would subvert both Th1 CD4 T cell recruitment and result in CD8 T cell suppression and death. Furthermore, DV-specific effects on DCs would allow for continued viral replication in the absence of effective clearance. These DV-mediated effects would modify T cell memory responses to infected DC, and potentially facilitate the expansion of pathologic T cell subsets. Contributing to this pathological cascade, antibody-dependent enhancement of infection in monocytic cells and macrophages would shift antigen presentation and cytokine production paradigms, increasing the risk of DHF.
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24

Friberg-Robertson, Heather L. "CD8+ T Cell Serotype-Cross-Reactivity is a Predominant Feature of Dengue Virus Infections in Humans: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/513.

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The four serotypes of dengue virus (DENV 1-4) have a significant and growing impact on global health. Dengue disease encompasses a wide range of clinical symptoms, usually presenting as an uncomplicated febrile illness lasting 5-7 days; however, a small percentage of infections are associated with plasma leakage and bleeding tendency (called dengue hemorrhagic fever, DHF), which can result in shock. Epidemiological studies indicate that severe dengue disease most often occurs during secondary heterotypic DENV infection. Additionally, plasma leakage (the hallmark of DHF) coincides with defervescence and viral clearance, suggesting that severe disease arises from the immune response to infection rather than a direct effect of the virus. A number of studies have found increased levels of markers of immune cell activation in patients with DHF compared to patients with the less severe form of disease (DF). These markers include IFNγ, TNFα, soluble CD8, soluble IL-2 receptor, soluble TNF receptor, and CD69, which support a role for T cells in mediating immunopathology. Because of the high homology of DENV 1-4, some degree of serotype-cross-reactivity is seen for most T cell epitopes. A high percentage of DENV-specific T cells recognize multiple DENV serotypes, as demonstrated by peptide-MHC (pMHC) tetramer binding and in vitro functional assays performed on PBMC from subjects vaccinated with an experimental DENV vaccine or naturally-infected subjects with secondary (>1) DENV infection. This thesis sought to address several gaps in the literature, specifically whether T cell responses differ in primary versus secondary (natural) infection. We studied the frequency, phenotype, and function of DENV-specific T cells. We demonstrated substantial serotype-cross-reactivity of antigen-specific T cells generated in response to naturally-acquired primary as well as secondary DENV infection. The frequency of A11-NS3133 epitope-specific T cells during acute infection did not correlate with disease severity. However, the peak frequency occurred earlier in primary infection while the frequency of CD45RA+ T cells declined quicker in secondary infection, suggesting the expansion of DENV-specific memory T cells. DENV-immune T cells exhibited different functional capabilities that were dependent on the particular serotype of infection. Specifically, DENV-1 or -3 stimulation of A11-NS3133 epitope-specific T cell lines resulted in robust function that included IFNγ production, whereas DENV-2 stimulation resulted in limited function that often included MIP-1β but not IFNγ production. These data support a role for T cells in DENV infection and offer new insights into their potential contribution to dengue pathology.
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25

Ekwudu, O'mezie. "Diversity of arthropod-borne viruses and implications for Wolbachia-based biocontrol." Thesis, Queensland University of Technology, 2019. https://eprints.qut.edu.au/127159/1/O%27mezie_Ekwudu_Thesis.pdf.

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This research examines the impact of genetic diversity in dengue virus (DENV) on incubation period in vector mosquitoes, and on Wolbachia, a novel vector biocontrol agent. It also extends the DENV study to other flaviviruses (Zika and West Nile - Kunjin) and alphaviruses (Barmah Forest, Ross River and Sindbis) to provide evidence as to whether a heat-resistant strain of Wolbachia, wAlbB, can be used to suppress the transmission of these mosquito-borne viruses. The research outcomes show that viral genetic variation is reflected in transmission kinetics of DENV. Data from the Wolbachia experiments provide the groundwork for future in vivo mosquito trials.
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26

Chepkorir, Edith. "Assessing the risk of Transmission of Yellow Fever and Dengue viruses by Aedes (Stegomyia) mosquito populations in Northern Kenya." Thesis, University of Pretoria, 2019. http://hdl.handle.net/2263/75861.

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East Africa has been experiencing an increase in the occurrence of emerging infectious diseases such as yellow fever (YF) and dengue (DEN). Increasing frequency of YF activity in East Africa constitutes a re-emergence that was not detected for over 40 years. Additionally, DEN outbreaks have also increased in frequency and continue to be detected in Kenya and in neighboring countries like Tanzania, Somalia, Djibouti, Eritrea and South Sudan. The renewed vigor of YF and dengue fever (DF) re-emergence in East Africa presents a new challenge to public health in spite of the availability of a safe and effective vaccine for YF. However, there is need to understand the potential for YF and DEN transmission along the border areas of Kenya, because Kenya is classified among countries with medium to high risk for YF transmission. This classification was mainly based on historical data, proximity to countries reporting recent YF outbreaks, the presence of non-human primates known reservoirs for these viruses, unrestricted human movement and presence of potential vector mosquito species. Both YF and DEN share a similar niche in the ecosystem and are associated with Aedes mosquito species of the subgenus Stegomyia. While the factors leading to the re-emergence of these diseases are poorly understood, a better epidemiologic understanding relating to disease ecology including presence of potential vectors, their host blood feeding preferences, the vector competence in transmission of these viruses and evidence of virus circulation in human population, will guide assessment of disease risk in the target areas and help to prevent or mitigate severe outbreaks in this region.
Thesis (PhD)--University of Pretoria,2020
National Institute of Health Sciences L'oreal- UNESCO for women in science
Medical Virology
PhD in Medical Virology
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27

Medin, Carey L. "Chemokine Induction by Dengue Virus Infection: Mechanisms and the Role of Viral Proteins: a Dissertation." eScholarship@UMMS, 2005. https://escholarship.umassmed.edu/gsbs_diss/30.

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The focus of this thesis is the role of dengue virus in the induction of chemokines. Dengue virus (DENV) occurs as four distinct serotypes, called DENV 1,2,3,and 4. Symptomatic DENV infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DHF is characterized by increased capillary permeability resulting in decreased plasma volume, which may be accompanied by hemorrhagic manifestations. Many factors including T cell cross reactivity, viral burden, antibody dependent enhancement and induction of chemokines and cytokines have been reported in DHF and may play a role in the pathogenesis of DENV infection. Cytokines have been shown to modulate endothelial cell permeability [1-3]. Recent studies have shown that DENV-infected endothelial cells secrete the chemokine, interleukin (IL)-8 in vitro [4]. In addition, the permeability of an endothelial cell monolayer was found to be increased by interleukin-8 (IL-8) in vitro[5]. This thesis examines the effects of DEN2V infection on the induction of chemokines, and specifically, which DEN2V viral protein(s) are involved in the induction of IL-8. The chemokine induction profile following DEN2V infection was initially assessed in various cell lines that may represent potential targets in vivo, including monocytes, liver cells and endothelial cells. We hypothesized that distinct profiles of chemokine secretion can be induced by DEN2V infection of various cell types in vitro. We found RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) and IL-8 were induced in two of the five cell lines. DEN2V infection of primary monocyte-derived dendritic cells induced RANTES and IL-8 along with macrophage inflammatory protein-1α (MIP-1α), MIP-1β and monocyte chemoattractant protein-1 (MCP-1) but at an earlier time post infection than in the cell lines. These results showed that DEN2V infection induces distinct chemokine profiles in many cell types. In addition, monocytic-derived DCs can secrete chemokines upon infection with DEN2V. Characterization of the signaling pathways induced by DEN2V revealed that DEN2V induction of chemokines in human embryonic kidney (HEK293A) cells is mainly through the nuclear factor kappaB (NFκB) pathway, as previously reported for endothelial cells and 293T cells [4,6]. Alternatively, the liver cell line (HepG2) activated mainly activator protein (AP)-l. In addition, DENV infection can induce the activation of the interferon-stimulated response element (ISRE) driven promoter. IL-8 has been shown to have multiple effects on the immune system ranging from recruiting cells to the site of infection to countering the antiviral effects of type I interferon (IFN) [7,8]. Previous reports have shown that viral proteins can induce chemokines such as seen with IL-8 induction with the nonstructural protein 5A (NS5A) and core proteins from hepatitis C virus [9,10]. We hypothesized that protein(s) from DENV could induce chemokine production. The expression of DENV proteins was analyzed for effects on IL-8 and RANTES production in HEK293A cells. The effects of viral replication on IL-8 and RANTES induction were also analyzed using a DENV replicon that contains genes for the capsid protein and the nonstructural proteins. Transfection of plasmids expressing NS5 or the DEN2V replicon induced the expression and secretion of IL-8 but not RANTES. We attributed the lack of RANTES induction to the inability of NS5 or the DEN2V replicon to induce transcription from the ISRE driven promoter. We also found that NS5 and the DEN2V replicon induced IL-8 mainly through the CCAAT/enhancer binding protein (c/EBP) and AP-1 pathways. The profile of transcription factor activation is different from what was seen with DENV infection of HEK293A cells and suggests that the transient expression of the NS5 protein and the replication and/or translation of the DEN2V genome use different pathways than viral infection to induce IL-8. In addition, we found that the expression of prM-E, known to produce virus-like particles, could induce IL-8 secretion and activate transcription from the IL-8 promoter. As with the expression of NS5, RANTES was not induced. Analysis of the transcription factors involved in IL-8 induction using luciferase reporter constructs indicated that expression of prM-E induced transcription of IL-8 through NFκB, AP-1 and c/EBP, similar to what was seen with DEN2V infection of HEK293A cells. These results suggest that production of virions or virus-like particles induce IL-8 but that another mechanism in the viral life cycle is responsible for the induction of RANTES expression by DEN2V infection. We were also interested in the effects of drugs that have been used previously to inhibit cytokine or chemokine production on chemokine induction during DEN2V infection. We hypothesized that pharmacological inhibitors of cytokines will inhibit secretion of chemokines in DEN2V infected cells. We found that the pharmacological inhibitors SB203580 and rolipram enhanced chemokine production in a DEN2V infected liver cell line (HepG2), whereas dexamethasone had the same effect in a kidney epithelial cell line (HEK293A). We conclude that drugs that inhibit signaling pathways involved in cytokine production in other experimental systems can have variable effects on chemokine induction in different cell types during DEN2V infection. The data generated in this thesis extend our understanding of how DEN2V manipulates the host cell during viral infection to produce chemokines and perhaps enhance viral propagation and dissemination through the induction of IL-8. In addition, this study provides insight into the variable effects pharmacological drug treatment may have on disease progression during DENV infection. These results increase our understanding of DENV pathogenesis and may be helpful in finding better strategies for treatment and prevention.
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28

Pickett, Brett E. "The contribution of different mechanisms of viral sequence variation to the evolution of positive-sense single-stranded RNA viruses." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2010. https://www.mhsl.uab.edu/dt/2010p/pickett.pdf.

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29

Girmann, Mirko [Verfasser], and Jürgen [Akademischer Betreuer] May. "Seroprevalence of Antibodies against Chikungunya, Dengue and Rift Valley Fever Viruses after Febrile Illness Outbreak, Madagascar / Mirko Girmann. Betreuer: Jürgen May." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2014. http://d-nb.info/1051435625/34.

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30

Luppino, Plinio Luis. "Detecção do vírus dengue pela técnica de aglutinação do látex modelo experimental." Faculdade de Medicina de São José do Rio Preto, 2007. http://bdtd.famerp.br/handle/tede/21.

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Made available in DSpace on 2016-01-26T12:51:14Z (GMT). No. of bitstreams: 1 plinioluisluppino_tese.pdf: 1297185 bytes, checksum: 89ac3b7475f1bfd00a6808afd1ba661e (MD5) Previous issue date: 2007-06-04
Dengue is the arthropod-borne transmitted viral disease of highest worldwide prevalence in mortality and morbidity. The proportion is pandemic ranging 1.6 million of infected patients yearly. Clinical presentation associated to epidemiological factors such as dengue prevalence in the patient s origin have been the only mean for early diagnosis. Laboratorial diagnosis, the conclusive, requires several days when there is viral isolation. Serological methods depend on high level of specific antibodies, and molecular methods are not available for the majority of laboratories of diagnosis and routine. The purpose of this study was to develop an agglutination method using latex to detect dengue virus, using biological samples of mice infected with dengue 1 Mochizuki strain by intracerebral via, and anti-dengue 1 specific antibodies from immunized mice. According to the results, this method was feasible for the dengue viruses diagnosis in positive samples of experimental animals. It provides further approaches for rapid detection of dengue in susceptible populations during the first days of the disease.
A dengue é a doença viral, transmitida por artrópode, de maior prevalência mundial em morbidade e mortalidade. Alcança proporções pandêmicas, estimando-se em 1,6 milhões de doentes anualmente. Manifestações clínicas características, associadas a fatores epidemiológicos, como prevalência da dengue na região de origem do paciente, têm sido os únicos instrumentos de diagnóstico precoce. O diagnóstico laboratorial, que é definitivo, demanda vários dias, quando realizado o isolamento viral. Métodos sorológicos dependem de níveis elevados de anticorpos específicos e os métodos moleculares não estão disponíveis para a maioria dos laboratórios de diagnóstico e rotina. Este estudo teve como objetivo desenvolver método de aglutinação do látex para a detecção do vírus dengue, utilizando amostras biológicas de camundongos infectados por via intracerebral com dengue 1, cepa Mochizuki e anticorpos específicos anti-dengue 1, obtidos de camundongos imunizados. Os resultados obtidos demonstraram a viabilidade deste método para diagnóstico do vírus Dengue em amostras positivas de animais de experimentação, abrindo novas perspectivas para o diagnóstico precoce da dengue na população susceptível, durante os primeiros dias de sintomas.
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31

Sayce, Andrew Cameron. "Iminosugars as dengue virus therapeutics : molecular mechanisms of action of a drug entering clinical trials." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:8d4da0ce-bfa6-447d-a280-630479f898af.

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Iminosugars are a class of small molecules defined by substitution of a sugar’s ring oxygen with nitrogen. Various chemical modifications of these basic structures (e.g. alkyl chain addition off of the ring nitrogen) have been developed during the last several decades. These molecules have been considered as therapeutics for a number of pathologies including viral infection, congenital disorders of glycosylation (of both glycoproteins and glycolipids), and diabetes. This thesis focuses on the application of a small subset of iminosugars, known as deoxynojirimycin derivatives, as therapeutics against dengue virus induced pathology. Dengue virus infection predominates in tropical climates, but autochthonous infection has recently emerged in areas of both southern Europe and the southern United States. With 390 million people infected annually, dengue is the most prevalent arthropod-borne viral infection worldwide, and the possibility of severe pathology including haemorrhage, shock, and/or death, necessitates development of effective antiviral therapies. Although the molecular mechanisms responsible for progression to severe dengue disease are not completely understood, there is considerable evidence for the role of both the innate and the adaptive immune responses in development of life-threatening complications. Excessive activation of the innate immune response, a phenomenon known as cytokine storm, has been hypothesised to explain development of symptoms related to vascular permeability, whereas the adaptive immune response has been implicated in severe disease through two hypotheses – the antibody dependent enhancement and original antigenic sin hypotheses. The evidence regarding each of these potential mechanisms of severe pathology is discussed throughout this thesis principally with respect to how iminosugar treatment could alter any detrimental effects of the immune response to dengue virus infection. The principal aim of this thesis is to consider the potential of deoxynojirimycin iminosugars as antiviral therapeutics in dengue infection with a focus on how these molecules exert their antiviral effects in primary human cells. I first consider the contributions of glycoprotein inhibition and glycolipid inhibition on production of infectious dengue virus. These experiments suggest that inhibition of glycoprotein folding is responsible for inhibition of infectious dengue virus production. I next consider the impact of treatment of a promising clinical candidate iminosugar, N9-methoxynonyl-deoxynojirimycin (MON-DNJ), on the primary human macrophage transcriptome. In uninfected macrophages as well as macrophages infected with dengue virus or treated with lipopolysaccharide to model bacterial sepsis, iminosugar treatment results in activation of the unfolded protein response and inhibition of several elements of the inflammatory response including signalling by the cytokines IFN-γ and TNF-α, and the inflammatory cascade mediated by NF-κB. Activation of the unfolded protein response as a result of treatment with MON-DNJ can be confirmed by analysis of phosphorylated (activated) NFE2L2, a transcription factor that functions principally to control oxidative stress in response to ER stress signals. Modulation of the inflammatory response of macrophages to dengue infection and bacterial sepsis is confirmed by analysis of secreted cytokines. As predicted by my transcriptomic experiments, levels of TNF-α and IFN-γ produced in response to dengue or lipopolysaccharide are reduced by treatment with MON-DNJ. Finally, I attempted to extend these observations to an animal model of dengue infection with a particular focus on TNF receptor and ligand superfamily members. Unfortunately, heterogeneity of cells types from tissue samples as well as limitations of the animal model complicate interpretation of these findings. Nevertheless, this thesis demonstrates that MON-DNJ is an effective dengue antiviral therapeutic and that this therapeutic activity may be related to both reduction of infectious virus as a consequence of inhibition of glycoprotein processing and as a result of changes to the host’s response to the pathogen. These results have been used in part to justify recently initiated clinical trials of MON-DNJ as a dengue antiviral therapy.
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32

Bashyam, Hema Sundara. "Serotype Cross-Reactive CD8+ T Cell Response to Heterologous Secondary Dengue Virus Infections in Humans: a Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/258.

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The generation of memory T cells following primary exposure to a pathogen is a critical feature of the vertebrate immune system which has evolved as a protective mechanism in order to defend the host against repeated assaults by the patnogen. Memory T cells are long-lived, undergo rapid proliferation upon re-activation, mediate a robust secondary response and clear the pathogen much more efficiently. These aspects have made the generation of memory T cells an attractive goal for the production of both prophylactic and therapeutic vaccines. However, the degeneracy of the T cell receptor, whereby a given T cell recognizes more than one epitope, allows the T cell to be modulated by epitope variants which could be self-ligands, ligands related to the original epitope but altered in sequence, or completely unrelated epitopes. Experiments in both mice and humans show that such cross-reactive stimulation of memory T cells results in complete, partial, or no activation of T cells, and in some cases, even alters the functional identity of the T cell (for example, T helper 1 cells start secreting IL-4, IL-5 and become part of a T helper 2 response). In the context of secondary infection of immune organisms with pathogens containing mutated or related T cell epitopes, such alterations at the cellular level translate into drastic changes in the overall clinical outcome of the infection. Thus, the presence of cross-reactive T cells in the memory population implies that the protective or pathologic nature of the secondary immune response is a consequence of the host's infection history. Although several murine models of heterologous infection resulting in altered pathological outcome have been studied, the exact immune correlates of protection versus immunopathology are still unclear. This thesis addresses this issue in dengue virus infections in humans. Dengue fever (DF) and Dengue Hemorrhagic Fever (DHF) are two disease manifestations caused by infections of humans by the dengue viruses. These are a group of 4 serologically distinct flaviviruses (D1-4) which often co-circulate among endemic populations. While primary infection with any of the four serotypes can result in the more severe clinical disease characterized by DHF, epidemiological data from several outbreaks show that 80% - 90% of DHF cases occur among individuals with secondary infection. This implies that prior immunity to dengue is actually a risk factor for developing severe disease. In these DHF cases, there are increased numbers of CD69+ CD8+ T cells in circulation, with increases observed in the frequency of epitope-specific T cells, and the serum levels of several T cell produced cytokines, chemokines, and immune receptors are highly elevated. Since the four serotypes share 65% - 75% amino acid sequence homology, the possibility that unconserved T cell epitope sequences stimulated cross-reactive responses was borne out in in vitroexaminations. In these studies, peripheral blood mononuclear cells (PBMC) and cloned T cells from both vaccinated and infected donors contained large populations of memory T cells that were cross-reactive for heterologous viral serotypes in proliferation and CTL assays. These data suggest that the severity of disease seen in DHF patients can be attributed to an immunopathologic secondary response during heterologous infection, and highlight a role for serotype cross-reactive T cells in this process. This thesis addresses the hypothesis that the recognition of the natural variants of dengue virus T cell epitopes by serotype cross-reactive CD8+ T cells of a dengue-immune donor results in an altered secondary response profile, with the changes reflected in both the quantitative and qualitative nature of the response. In order to compare the functional profile of the secondary response of dengue-immune PBMC re-activated with heterologous serotypes, we focused on a panel of 4 donors who were vaccinated with live attenuated monovalent vaccines corresponding to D1, D2, or D4 serotypes. We screened a panel of peptides predicted to bind to HLA-A*0201 for cytokine responses and identified 4 novel epitopes that were highly immunogenic in all four donors. Direct ex vivo stimulation of donor PBMC with the heterologous sequences of these epitopes also showed sizeable serotype cross-reactive T cell populations. CFSE- and intracellular staining for cytokines and chemokines showed that these cross-reactive T cells not only expanded but also produced IFNγ, TNFα, and MIP-1β. Multi-parameter staining revealed functionally diverse populations comprised of single cytokine (IFNγ+, TNFα+, MIP-1β+, double cytokine (IFNγ+TNFα+, IFNγ+MIP-1β+, TNFα+MIP-1β+, and triple cytokine (IFNγ+TNFα+MIP-1β+ secreting sub-sets. Stimulation with the epitope variants altered the magnitude of the overall response as well as the relative sizes of these sub-sets. The patterns of responses revealed the effects of epitope immunogenicity, infection history and donor-specific variability. All 4 donors showed the highest cytokine response to a -single epitope (NS4b 2353). The same two peptide variants (D2 NS4a 2148 and D3 NS4b 2343) induced the highest response in all 4 donors regardless of the serotype of primary dengue infection. Interestingly, the epitope variants which showed the highest immunogenecity in our donors corresponded to the D2 and D3 serotypes which have been documented as being more virulent as well as a viral risk factor for DHF. In one donor, the response to all peptide variants was dominated by the same cytokine sub-sets. These data suggested that the dengue-immune memory T cell repertoire was functionally diverse and underwent alterations in size after secondary stimulation. Therefore, we also investigated the effect of epitope variants on dengue-specific CD8+T cell clones isolated from vaccinated and infected donors in order to determine if epitope variants induced altered functional outcomes at the clonal level. The epitope variants functioned either as strong agonists (particularly the D2 and D3 sequences), partial agonists, or null ligands. Some variants were able to induce cytolysis but not other effector functions at low concentrations. The variant ligands also influenced the hierarchy of cytokine responses within each clone. The third part of this thesis focused on the characterization of the frequency and phenotypic profile of epitope-specific CD8+ T cells in patients with DHF and DF at different times in the disease course in order to better understand the kinetics of the response and delineate any differences between the immune profile of severe vs. moderate disease. Tetramer staining for a previously identified HLA-B*07 restricted epitope was combined with staining for activation markers (CD69, CD38, HLA-DR), homing receptors (CCR7, CD62L), and programmed death receptor 1 (PD-1). The DHF subjects had early T cell activation with higher frequencies of tetramer+CD69+ cells as compared to DF subjects, in whom T cell frequencies peaked around the time of defervescence. While each subject had a unique phenotypic profile of tetramer+ cells, there was a difference between DF and DHF subjects in terms of CCR 7 expression; all subjects expressed low levels of CCR7 during acute illness but only the DHF subjects did not show upregulation of CCR7 on tetramer+ cells during convalescence. These data suggest that there is a sustained alteration in memory phenotype in those who recovered from severe dengue disease. A majority of the tetramer+cells also expressed PD-1 during acute illness but not during convalescence. Double-staining with variant tetramers allowed us to directly visualize serotype cross-reactivity of the epitope-specific population, and showed that secondary stimulation did induce the expansion of cells with low avidity for that secondary serotype and higher avidity to the variant. Furthermore, the ratios of these sub-sets changed during the course of the response. Taken together, these studies suggest that the immune response to heterologous secondary dengue infection is mediated by a heterogeneous population of serotype-cross reactive T cells that have different functional avidities to epitope variants and is influenced by the serotype of the secondary infection as well as the prior infection history of the individual. The preferential expansion of clones which secrete IFNγ but not inflammatory MIP-1β or TNFα or a repertoire characterized by a higher ratio of cytolytic to cytokine producing clones could limit immune mediated damage while efficiently clearing the virus. This information will be useful in the design of vaccine strategies aimed at inducing protective cross-reactive responses against all 4 dengue serotypes while preventing immunopathological outcomes following secondary infection.
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33

Alves, Beatriz Santos Capela. "Caracterização das proteinas humanas Mov34 e PACT e analise da sua interação com o RNA do virus da dengue." [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317180.

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Orientador: Nilson Ivo Tonin Zanchin
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-11T18:49:26Z (GMT). No. of bitstreams: 1 Alves_BeatrizSantosCapela_D.pdf: 5512305 bytes, checksum: 707ea6299bc24ddc6fb459520d79aeee (MD5) Previous issue date: 2008
Resumo: O combate à dengue atualmente está limitado praticamente aos esforços de eliminação do mosquito transmissor, o Aedes aegypti, porém esta estratégia não tem se mostrado eficiente. O desenvolvimento de novos instrumentos de combate à dengue requer, portanto, maior conhecimento sobre a biologia do vírus com relação à sua interação com seus hospedeiros. O genoma do vírus é constituído por um RNA simples-fita de polaridade positiva e possui duas regiões não traduzidas (5¿ e 3¿ UTR). A região 5¿UTR viral possui organização similar à dos mRNAs eucarióticos, diferentemente da região 3¿UTR que é longa e não possui cauda de poli(A). Em vez disso, na região 3¿UTR encontram-se estruturas conservadas entre os diferentes Flavivirus, dentre elas a estrutura 3¿ stem-loop (3¿SL) que é indispensável para a replicação do RNA viral. O objetivo do nosso estudo foi identificar novas proteínas humanas capazes de interagir com a estrutura 3¿SL do RNA do vírus da dengue. Dados da literatura descrevem que a proteína Mov34 de camundongo interage com 3¿SL do vírus da encefalite japonesa. Devido à alta similaridade entre as proteínas ortólogas humana e de camundongo, bem como das respectivas estruturas 3¿SL dos vírus da dengue e da encefalite japonesa, foi testada a interação entre a Mov34 humana com o 3¿SL do vírus da dengue. Porém, em nenhuma das condições testadas foi possível obter evidência de interação da Mov34 humana com 3¿SL dos vírus da dengue e da encefalite japonesa. Para a identificação de novas proteínas que são capazes de interagir com a estrutura 3¿SL do RNA do vírus da dengue foi utilizado o ensaio de triplo-híbrido de levedura. A proteína humana PACT, conhecida como proteína celular ativadora de PKR, foi isolada neste ensaio utilizando 3¿SL como isca. PKR é uma quinase ativada por PACT ou RNA dupla-fita. A ativação de PKR leva a um estado antiviral adquirido pela fosforilação do fator de iniciação da tradução eIF2a e conseqüente inibição da tradução. Além disso, PKR está envolvida em outras vias de transdução de sinal e na resposta celular à proteínas desenoveladas. A ação antiviral de PACT é evidenciada pela ação de proteínas dos vírus influenza A e herpes simplex tipo 1 que inibem a ativação de PKR por PACT e por RNA dupla-fita. A interação direta de PACT com 3¿SL do RNA do vírus da dengue foi confirmada por ensaio de UVcrosslinking PACT possui três domínios de interação com RNA dupla-fita, sendo que os dois domínios N-terminais são responsáveis pela sua interação com o 3¿SL. Foi identificada uma região específica do 3¿SL, o stem-loop superior, onde PACT interage com maior afinidade. Além disso, foi mostrado que PACT endógena de células HEK293 é capaz de interagir com o 3¿SL biotinilado. Para caracterizar a função desta interação durante a infecção viral, foi desenvolvida uma linhagem celular com inibição da expressão de PACT através da técnica de RNA de interferência. Com esta linhagem poderemos analisar a importância da interação entre PACT e o RNA do vírus da dengue quanto à ativação e/ou inibição de PKR durante a infecção viral
Abstract: The combat to the dengue virus is basically limited to the efforts in eliminating the transmitter mosquito, the Aedes aegypti. But this strategy is not very efficient. The development of new instruments of combat to dengue virus requires improved knowledge about the virus biology and its relation to hosts. The dengue virus genome is a single-stranded RNA of positive polarity flanked by a 5¿ untranslated region (UTR) of ~100 bases and a highly structured 3¿ UTR of ~450 bases. As many other viruses, dengue encodes the enzymes required for its genome replication, but relies completely on the host translational machinery to synthesize its proteins. The essential difference between host cellular mRNAs and dengue virus genome RNA involves the 3¿UTR, which instead of a polyadenylate tail contains highly conserved structural elements, including the 3' stem-loop (3¿SL), located at the 3' terminus of the 3'UTR of many flaviviruses that is essential for their replication. The aim of this study is to identify new human proteins capable of interacting with dengue virus RNA 3¿SL structure. Literature data describe that the murine Mov34 protein interacts with Japanese encephalitis virus 3¿SL. Giving the high similarity between the human and murine ortholog proteins, as well as the conservation of the Flavivivirus RNA 3¿SL structure, we tested the interaction between the human Mov34 and the dengue virus 3¿SL. However, no interaction was detected under the conditions used in this work. In addition, the yeast three-hybrid system was used to screen for novel proteins that interact with the dengue virus 3¿SL. Human PACT, known as the cellular protein activator of PKR, was identified as a putative 3¿SL-interacting protein. PKR is an interferon-inducible, PACT or double-stranded RNA activated protein kinase. Activated PKR phosphorylates the translation initiation factor eIF2a, inhibiting translation of cellular and viral RNAs, leading to a cellular antiviral state. PACT and doublestranded RNA activation of PKR is inhibited by influenza A and herpes simplex type 1 virus proteins during viral infection, indicating that PACT plays a role in the cellular antiviral state. Direct interaction between PACT and 3¿SL was confirmed by UV-crosslinking assays. PACT contains three doublestranded RNA interaction motifs, but only the two N-terminal motifs are responsible for 3¿SL interaction. A 3¿SL specific region, the top stem-loop, was identified to interact with PACT with higher affinity. Furthermore, HEK293 cells endogenous PACT interacts with biotin-labeled 3¿SL. To further characterize PACT-3¿SL interaction during dengue virus infection, a cell line with low expression of PACT was developed using the RNA interference technique. This cell line will be used to determine the propagation rate of dengue virus which is expected to reveal the importance of PACT either for the cell antiviral state or for dengue virus proliferation
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular
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34

Beaumier, Coreen Michele. "Cross-Reactive Memory CD4+ and CD8+ T Cells Alter the Immune Response to Heterologous Secondary Dengue Virus Infections in Mice: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/350.

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Dengue virus (DENV) infects 50-100 million people worldwide every year and is the causative agent of dengue fever (DF) and the more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). There are four genetically and immunologically distinct DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). Evidence suggests that an increased risk for DHF/DSS during secondary infection with a heterologous DENV serotype is due to an immunopathological response mediated by serotype-cross-reactive memory T cells from the primary infection. Furthermore, epidemiological studies have shown that the sequence of infection with different DENV serotypes affects disease severity. Though much has been learned from human studies, there exist uncontrollable variables that are intrinsic in this system such as genetic factors and unknown infection histories. These factors can skew experimental results, making interpretations difficult. Therefore, a murine model to study the immunologic aspects of sequential dengue infections would be an asset to the field of dengue research. To examine the effect of sequential infection with different DENV serotypes on the CD8+ T cell response, we immunized Balb/c mice with a primary DENV infection on day 0 and subsequently challenged with a heterologous secondary DENV infection on day 28. We tested all possible sequences of infection with the four serotypes. We analyzed the T cell response to two previously defined epitopes on the DENV E (Ld-restricted) and NS3 (Kd-restricted) proteins. Using ELISPOT and intracellular cytokine staining, we measured the frequency of T cells secreting IFNγ and TNFα in response to stimulation with these epitopes during three phases: acute primary, acute secondary, and the memory phase after primary infection. We found that the T cell response in heterologous secondary infections was higher in magnitude than the response in acute primary infection or during the memory phase. We also found that the hierarchy of epitope specific responses, as measured by IFNγ secretion, was influenced by the sequence of infections. The adoptive transfer of immune serum or immune splenocytes suggested that memory T cells from the primary infection responded to antigens from the secondary infection. In vitroexperiments with T cell lines generated from mice with primary and secondary DENV infections suggested the preferential expansion of crossreactive memory T cells. In testing all of the different possible sequences of infection, we observed that two different sequences of infection (e.g., DENV-2 followed by DENV-1 versus DENV-2 followed by DENV-3) resulted in differential CD8+ T cell responses to the NS3 peptide even though both secondary infection serotypes contain the identical peptide sequence. To investigate this phenomenon, we examined the role of CD4+ T cell help on the memory CD8+ T cell response. We found that CD4+ T cell cytokine responses differ depending on the sequence of infection. In addition, it was also shown that crossreactivities of the CD4+ T cell response are also sequence-dependent. Moreover, denguespecific memory CD4+ T cells can augment the secondary CD8+ T cell response. Taken together, we demonstrated that this serotype sequence-dependent phenomenon is the result of differential help provided by cross-reactive memory CD4+T cells. The findings in this novel mouse model support the hypothesis that both CD4+ and CD8+ serotype-cross-reactive memory T cells from a primary dengue virus infection alter the immune response during a heterologous secondary dengue virus infection. These data further elucidate potential mechanisms whereby the specific sequence of infection with different dengue virus serotypes influences disease outcomes in humans.
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35

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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36

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

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37

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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38

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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39

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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40

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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41

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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42

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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43

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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44

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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45

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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46

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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47

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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48

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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49

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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50

Lin, Yu-Ying, and 林育瑩. "Dengue Viruses Inhibit Mosquito JAK-STAT Signaling." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/pbqwtx.

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碩士
國防醫學院
微生物及免疫學研究所
101
Dengue fever is a mosquito-borne disease endemic in many countries of the world including Taiwan. This disease is transmitted the dengue virus by Aedes aegypti and Aedes albopictus female mosquito biting to human host. These two mosquito species are prevalent in Taiwan, with Aedes aegypti present only in southern location. Mosquito does not have the adaptive immunity as mammals, only exist the innate immunity to struggle against microbe such as virus. The dengue virus is circulated between the mosquito vector and human being; it might develop some strategy to overcome the innate immunity in the mosquito vector for virus persistency and survival cycle in nature. The innate immune system or mosquito regulated by three main signaling pathways: the Toll-Dorsal/Dif, immune deficiency (IMD)-Rel, and JAK-STAT pathways for defending microbial infection. The Toll pathway is activated by fungi, Gram-positive bacteria, and viruses through pattern recognition receptor (PRRs). The Imd pathway is involves in the protection against Gram-negative. Also, activation of JAK-STAT pathway may lead to defense against viral infection. We had shown that dengue 2 virus infected C6/36 cells and dengue 2 NS3 plasmid transfected C6/36 cells with decreased STAT phosphorylation. The EMSA also showed decreased STAT binding activity in nucleus from dengue NS3 plasmid transfected C6/36 cells. In this study, we assay the JAK-STAT function of these two mosquitoes by infection with DENV-1, DENV-2, DENV-3, and DENV-4 virus in C6/36 (Aedes albopictus mosquito cells) and CCL-125 (Aedes aegypti mosquito cells). The construct 6X ST-luc plasmid contains 6XSTAT (TTCNNNGAA) binding site is for mosquito active STAT binding. The STAT activity of mosquito cells or adult female after four serotype dengue viruses infection were assayed using this 6X ST-luc plasmid and all showed decreased activity. The EMSA also showed decrease STAT binding activity after four serotype dengue virus infection in mosquito cells. All above results demonstrated that four serotype dengue viruses inhibit the JAK/STAT pathway in mosquito cell lines and adult female mosquitoes in vivo.
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