黃熱病毒屬包含登革熱病毒(Dengue virus)、黃熱病毒(Yellow Fever virus)、西尼羅河病毒(West Nile virus)、日本腦炎病毒(Japanese Encephalitis virus)與蜱媒腦炎病毒(Tick-borne Encephalitis virus)會引發許多人類的疾病，其中又以日本腦炎病毒及登革熱病毒常常在東亞及東南亞盛行。黃熱病毒屬的病毒外殼上覆蓋著一層封套蛋白(Envelope protein)，這些封套蛋白被認為是一個主要抗原，在遭受感染的病人體內可以用來產生中和抗體，封套蛋白包含三個區塊，其中第三區塊(domain III)在病毒貼附上細胞和細胞膜融合兩階段扮演很重要的角色，因此有許多研究採用第三區塊來作為藥物開發的標的物，但是到目前為止仍然沒有有效治療的藥物可用。本研究共包含了兩個主題，第一個主題探討的是肝素(heparin)與日本腦炎病毒的交互作用，我們在日本腦炎病毒第三區塊的N端向第二區塊延伸區域發現了一個肝素的結合位置，而且以此位置序列合成的短胜肽也同樣對於肝素具有很強的親和力，說明了日本腦炎病毒與細胞表面的醣類之間的交互作用關係，也對將來藥物設計非常有幫助；第二個主題是利用SELEX技術篩選與第三區塊有高度親和力的核酸適體作為治療登革熱的藥物開發研究，我們建立了一套SELEX技術並以登革熱病毒封套蛋白的第三區塊為篩選標的，結果得到一個結構為四複合體的核酸適體可以辨識第三區塊，而且更進一步發現此核酸適體的結合位置在第三區塊28-32個胺基酸上，這幾個胺基酸在登革熱四個血清型中呈現極高的相似度，使得此核酸適體同時能抑制登革熱四種血清型病毒，本研究發現了第一個利用SELEX產生的可以作為抑制登革熱的核酸適體藥物，更重要的是因為它同時抑制登革熱四種血清型病毒，所以可以避免病毒依賴性增強感染現象(Antibody-Dependent Enhancement of infection)的發生，我們認為它非常有潛力成為治療登革熱的藥物。

Flaviviruses including dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and tick-borne encephalitis virus (TBEV) are associated with human diseases. Particularly, infected cases of JEV and DENV are reported in south-east Asia. The flaviviruses are covered by envelope proteins, which are the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. Among three domains of envelope protein, domain III plays the most important role in viral attachment and fusion. Therefore, previous studies have used domain III as a target for drug development. However, there is still no sufficient treatment available. Our works includes two studies. In the first study, we identified a heparin binding motif located on the N-terminal of JEV E protein domain III is responsible for heparin binding. On the other hand, a synthetic peptide with sequence that is corresponding to this region also demonstrates strong affinity to heparin. Our results provide a basis for further understanding the interactions of flaviviruses and GAGs on the host cell surfaces and would be helpful for drug design. In the second study, we used SELEX technology to generate an aptamer which has the ability to target domain III with parallel G-quadruplex structure. Furthermore, we found that residues Q28, H29, G30 and I32 on a completely identical loop among all four DENV strains are involved in the aptamer binding, which leads to the antiviral activity of selected aptamer against all four DENV strains. Our results provide an aptamer as artificial antibody which solves the problem of antibody-dependent enhancement of infection and thus this aptamer may have the potential to be a therapeutic drug.

Chapter One
Introduction 1
Flavivirus 1
Flavivirus life cycle 3
Envelope protein and fusion 4
Current antiviral strategy 5
Systematic Evolution of Ligands by Exponential Enrichment 6
Specific aim 8
Chapter Two
Identification of a Heparin Binding Peptide from the Japanese Encephalitis Virus Envelope Protein 14
Abstract 14
Introduction 15
Materials and methods 17
Materials 17
Protein preparation and purification 17
Monoclonal Antibody preparation 17
NMR spectroscopy 18
Fluorescence Spectroscopy 18
Enzyme-Linked ImmunoSorbent Assay (ELISA) 19
Results and discussions 20
Identification of heparin binding sites of the JEV envelope protein 20
Mutations of critical residues important for heparin binding 22
Titration with heparin, heparin disaccharides, low molecular weight heparin, and suramin 22
Binding of heparin to a synthetic peptide corresponding to residues 279-297 23
Competitive binding between heparin and the neutralizing antibody mAb E3.3 24
Biological importance 24
Chapter Three
DNA aptamer forms a G-quadruplex can neutralize all four serotypes of dengue viruses 36
Abstract 36
Introduction 37
Materials and methods 40
Preparation and purification of protein 40
Synthesis and preparation of nucleotides 40
In vitro selection of ssDNA aptamers 41
Fluorescence quenching 41
Circular dichroism 42
ELISA 42
NMR spectroscopy 43
Plaque reduction neutralization test 43
Results 45
Preparation of DENV-2 ED3 45
Selection of DNA aptamers target DENV ED3 45
Binding affinities of selected aptamers to DENV-2 ED3 46
Formation of quadruplex structure. 46
In vitro neutralization of all four DENVs by S15 48
Stability of aptamer S15 49
Discussion 50
Chapter Four
Conclusions and prospects 69
Reference……………………………………………………………………………..73

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