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研究生：	詹婷婷 Chan, Ting-Ting
論文名稱：	從人類細胞和繼代細胞培養中純化出的屈公熱病毒顆粒的性質分析 Characterize the properties of chikungunya virus particle derived from ex vivo and in vitro culture system
指導教授：	吳尚蓉 Wu, Shang-Rung
學位類別：	碩士 Master
系所名稱：	醫學院 - 口腔醫學研究所 Institute of Oral Medicine
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	60
中文關鍵詞：	低溫電子顯微鏡、屈公熱病毒、離體環境培養系統、屈公熱病毒囊泡、三維結構重組
外文關鍵詞：	Cryo-electron microscopy, Chikungunya virus, ex vivo culture system, CHIKV vesicle, three-dimensional reconstruction
相關次數：	點閱：84 下載：1
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屈公熱病毒是一種蟲媒病毒，屬於披衣病毒科中的甲病毒屬。其在1953年時於非洲被鑒定並開始散播至亞洲等蚊子肆虐的地區，直到最近又再現並爆發於歐洲以及美洲等五大洲。而目前台灣還沒有本土案例的產生，已知病例皆是由國外旅遊回國後才發現。屈公熱病毒的低溫電子顯微鏡結構已經於2013被發表，其為二十面體病毒，被歸類於 T = 4 準對稱病毒之一。其二十面體上具有12個五聚體環繞於每個五重頂點並帶有30個六聚體環繞於二十面體上雙重軸，而在成熟的屈公熱病毒顆粒表面上共有80個刺突結構，每一個刺突皆由三個E1和E2形成的異源二聚體所組成。在先前的研究中已經得知E1蛋白帶有融合環，而E2蛋白則可能帶有和宿主細胞接觸的接收器和具有在正常環境下能保護融合環的功能。另外，我們也已經得知當屈公熱病毒在感染宿主細胞時其結構是如何變化的。雖然我們已經在結構上對屈公熱病毒有許多的了解，但現在市面上仍然沒有能夠有效的疫苗進行預防，也沒有藥物能夠治療屈公熱。而目前正在發展中的屈公熱疫苗其製備方式皆以體外培養環境為主。我們的合作者，成功大學的彭貴春教授以及其實驗室成員發現登革病毒在體外環境以及病人體內中所呈現的型態有所不同。而由人類初代細胞所培養出的登革病毒和病人體內的病毒呈現一樣的型態。因此我們假設屈公熱病毒在試管內以及體內環境時可能具有不同的型態，以至於試管內所解讀的病毒型態及生化特性無法適用在體內，造成疫苗發展上的困難。
在我們這次的研究中，我們使用減毒過後的屈公熱病毒疫苗株去感染由人類骨髓所取得的初代細胞和非洲綠猴腎上皮細胞。將取得的兩種型態的屈公熱病毒利用低溫電子顯微鏡的技術去確認其型態是否會有差別。由體外環境所產生的屈公熱病毒在經過三維重組後，呈現直徑為60奈米的二十面體單結構，其外表呈現共80個刺突結構。這樣的三維重組結構特徵與2013年所發表的屈公熱病毒結構相似。另外，由人類初代細胞所產生的屈公熱病毒在低溫電子顯微鏡的觀察下呈現與從體外環境所產生的屈公熱病毒完全不同的型態，病毒顆粒被膜所包覆，高度專一抗體免疫金標定實驗證實此膜包裹的粒子是屈公熱病毒，我們在此稱為屈公熱病毒囊泡。由於病毒顆粒外層的膜，使得此離體環境所產生屈公熱病毒的型態呈現一個不規則的型態，因此無法使用適用於純度高且結構均一度高的高解析單粒子三維結構重組技術來得到其三維結構，但從低溫電子顯微鏡得到的影像及免疫金標定實驗我們可以得知，屈公熱病毒和登革病毒一樣由離體環境和體外環境所培養出的病毒會呈現不同的型態。這樣的發現或許可以在研發抗屈公熱藥物和其疫苗的製造提供更多有用的依據。

Chikungunya virus (CHIKV) belongs to Alphavirus genus of Togaviridae family. CHIKV was identified in Africa in 1953 and has recently reemerged, causing explosive outbreak and reaching 5 continents. So far, there is no autochthonous CHIKV infection in Taiwan. CHIKV is icosahedral with T = 4 symmetry arrangement. The viral particle consists of 12 pentamers around each fivefold vertex and 30 hexamers around each icosahedral twofold axis. The outer surface of mature CHIKV particles is composed of 80 spikes, each spike is formed by three copies of an E1–E2 heterodimer. E1 protein contains fusion loop, and E2 proteins carries putative receptor binding site and protects fusion loop before activation. The conformational change of CHIKV fusion has been studied. Although a lot of studies on CHIKV, there are no effective vaccine and medicine which are specific for CHIKV. The currently production of CHIKV vaccine which is developing is in vitro culture system. In 2015, our cooperator, Prof. Perng and his team in National Cheng Kung University, found that dengue virus (DENV) presented different morphologies in vitro and in vivo. And ex vivo DENV which derived from human primary cell shares the same features with in vivo DENV. Hence, we suspected that CHIKV, which is also transmitted by Aede mosquito, may has the same phenomenon.
Here, CHIKV attenuated vaccine strain 181/25 clone was used to infect human primary cells isolated form bone marrow and vero cell line to present the ex vivo and in vitro experiments. Cryo-EM and immunogold labeling were used to identify the morphology. The features of our cryo-EM in vitro CHIKV structure agreed with the features of CHIKV structure published in 2013. On the other hand, cryo-EM images showed that ex vivo CHIKV was wrapped by membrane, called CHIKV vesicle here. Immunogold labeling and affinity grid confirmed that the vesicle-like particle was CHIKV. Even though we couldn’t solve the CHIKV vesicle 3D structure using high resolution single particle reconstruction approach due to the heterogeneity of the ex vivo CHIKV production. The TEM images analyses showed that CHIKV presented different morphologies derived from in vitro and ex vivo culture system. Our finding here may provide the hints for antiviral candidates and effective vaccine development.

Key words: Cryo-electron microscopy, Chikungunya virus, ex vivo culture system, CHIKV vesicle, three-dimensional reconstruction

中文摘要.…………………………………………………………………………………....1
Abstract……………………………………………………………………………………..2
Acknowledgement................................3
Catalogues..........................................4
Figure List.......................................7
Supplementary Figure List.........................8
Table List.......................................9
Abbreviations.....................................10
Introduction.............................12
1 Chikungunya virus............................12
1.1 Chikungunya virus structure.................12
1.2 The life cycle of CHIKV and its conformational change..............................................14
2 CHIKV vaccine development.........................16
3 Transmission electron microscope (TEM) technology..18
3.1 Cryo-electron microscope (Cryo-EM)........19
3.2 Antibody-based affinity grid..............19
4 Rationale...................................20
Materials and Methods......................21
1 In vitro cell cultures.........................21
2 Ex vivo cell handling procedures and cultures.....21
3 CD14 positive cell sorted from PBMCs..............21
4 GM-CSF cultures.................................22
5 CHIKV infection...................................22
6 Virus harvest..................................23
7 CHIKV quantification (plaque assay).............23
8 CHIKV purification.............................24
9 Proteomics assay...............................25
10 Transmission Electron Microscope facility......25
11 Negative-stain EM...............................25
12 Immunogold labeling..........................26
13 Affinity grid................................26
14 Cryo-electron microscope (Cryo-EM) images.......27
15 Three-dimensional reconstruction of CHIKV.........27
Results...................................28
1 The cell types used for in vitro and ex vivo culture system............................................28
1.1 CHIKV could infect vero cells and produced new virus particles....................................28
1.2 The infectivity of CHIKV in the human primary cells (cord blood, bone marrow and peripheral blood mononuclear cells)..........28
2 Characterize CHIKV particles derived from in vitro and ex vivo...................................29
2.1 Observe the morphology of CHIKV by negative-stain EM...........................................29
2.2 Identify CHIKV by immunogold labeling and affinity grid...........................................30
3 Three-dimension reconstruction of CHIKV derived from in vitro and ex vivo................................31
3.1 Cryo-EM images..................................31
3.2 2D analyses of particle images..................32
3.3 Reconstruct the 3D structure of in vitro and ex vivo CHIKV..........................................33
Discussion....................................34
1 Our solved in vitro CHIKV structure agreed with the one published in 2013.................................35
2 Characteristics of in vitro and ex vivo CHIKV....35
3 Whether the CHIKV that present different morphologies in difference culture system effect vaccine production.35
4 The composition of membrane which covered the ex vivo CHIKV particle needs to be explored....................37
5 Whether ex vivo and in vivo CHIKV have same morphology, as DENV does.........................38
6 The production limit of ex vivo CHIKV particles.38
7	Future perspectives.......................38
Conclusion................................40
References.............................41
Figures..............................47
Supplementary Figures.............57
Tables..........................59

                                    

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