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  1. NTU Theses and Dissertations Repository
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請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79945
完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor陳慧文(Hui-Wen Chen)
dc.contributor.authorMeng-Chi Wuen
dc.contributor.author吳孟錡zh_TW
dc.date.accessioned2022-11-23T09:17:53Z-
dc.date.available2021-11-05
dc.date.available2022-11-23T09:17:53Z-
dc.date.copyright2021-11-05
dc.date.issued2021
dc.date.submitted2021-08-12
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79945-
dc.description.abstract鸚鵡玻那病毒感染造成鸚鵡發生高致死性的前胃擴張症,共有八種基因型。其中,最為盛行的是第四型病毒,而第五型病毒則是較罕見的型別,單獨分類在第二群演化支,和水鳥或雀科玻那病毒關係較近,先前研究顯示水鳥或雀科之玻那病毒具有感染哺乳類細胞能力,然而,第五型玻那病毒的病例稀少,其感染性、病原性及宿主與病原之交互作用等目前皆為未知。本實驗室先前自臨床檢體中分別分離到第四型與第五型玻那病毒,而本研究則進一步建立持續性感染玻那病毒的鵪鶉細胞株,用以瞭解病毒感染細胞之生長曲線,並利用RNA定序法初步探究病毒感染後宿主細胞之轉錄體調控。結果顯示,不論是第四型或第五型病毒,皆高度依賴細胞的生長而增殖,而第五型病毒在培養的第五天過後,病毒核酸有非常明顯的增加。轉錄體基因表現結果顯示,兩種型別病毒感染對於宿主細胞之轉錄調控有顯著不同,兩者的差異表現基因數量高達111個;而第五型病毒持續感染細胞與未感染細胞比較之差異表現基因數量有67個,其中的兩個與神經調控相關之基因皆顯著表現增加:VIPR2及LPAR4。另外,將病毒接種至多種禽類及哺乳類來源細胞,持續培養五代或十代,以定量PCR檢驗病毒核酸,並透過免疫化學染色法偵測病毒蛋白質,進而分析對第五型鸚鵡玻那病毒在不同細胞的之複製特性。結果顯示,第五型病毒具有在Vero細胞複製的能力 ,而對於禽類細胞的感受性依序為:QM7 > DF-1和QT6 > DEF,其中DEF只適合初代分離。我們亦將第五型病毒接種於鴨胚胎蛋之尿囊絨毛膜,再檢驗組織內的病毒核酸,結果顯示大部分為陰性,推測此模式不適用於增殖病毒。總結來說,第五型鸚鵡玻那病毒相對於第四型病毒具有不同的生長曲線與基因轉錄體調控,且第五型病毒具有廣泛的宿主細胞感染能力,包含多種禽類細胞和哺乳類Vero細胞,此結果將有利於深入解析鸚鵡玻那病毒對宿主之致病與免疫機制,也將有助於未來動物感染模式之建立。zh_TW
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dc.description.tableofcontents"中文摘要 I Abstract II List of figures VII List of tables IX Chapter 1 Introduction 1 1.1 Structure and characteristics of bornaviruses 1 1.2 Genotypes of bornaviruses 1 1.2.1 Mammalian bornavirus 2 1.2.2 Avian bornavirus (ABV) 2 1.3 Viral life cycle and replication 4 1.4 Host range of bornavirus in vivo and in vitro 5 1.4.1 Mammalian bornavirus 5 1.4.2 Avian bornavirus 6 1.5 Clinical signs and pathogenesis of proventricular dilatation disease (PDD) 7 1.6 Epidemiology and transmission of parrot bornavirus 10 1.7 Diagnostic tools of parrot bornavirus 12 1.7.1 RT-PCR and RT-qPCR 12 1.7.2 Serologic assays 13 1.8 Treatment and control 14 1.9 The objective of this study 17 Chapter 2 Materials and methods 18 2.1 Immortalized cell lines 18 2.2 Preparation of primary duck embryo fibroblasts (DEF) 18 2.3 PaBV-4 or -PaBV-5 persistently-infected cell lines 19 2.4 Phylogenetic analysis 20 2.5 Total RNA extraction and cDNA generation 20 2.6 Growth kinetics of PaBV-4 or PaBV-5 persistently-infected cells 21 2.7 Transcriptomic profiling analysis by RNA-sequencing (RNA-seq) 21 2.8 Preparation of virus stock 23 2.9 Generation of monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against PaBV 24 2.10 Western blotting analysis 25 2.11 Titration of viral stocks 26 2.12 Immunocytochemical (ICC) staining 26 2.13 Nested polymerase chain reaction (nPCR) and agarose gel electrophoresis 27 2.14 Real-time quantitative PCR (RT-qPCR), and quantification by the delta-delta Ct method or standard curve 28 2.15 Replication studies of PaBV-5 in different cells 30 2.16 Inoculation in chorioallantoic membrane (CAM) of duck embryos 30 2.17 Statistical analysis 32 Chapter 3 Results 33 3.1 Phylogenetic analysis 33 3.2 Cellular morphology and viral detection in persistently infected cells 33 3.3 Characterizations of PaBV-4 and PaBV-5 persistently-infected cells 33 3.4 Quality control and sample repeatability after RNA-sequencing 34 3.5 Analysis of differential expression genes (DEGs) 35 3.6 Viral stock titration and protein detection 37 3.7 Growth kinetic of PaBV-5 in QM7 cells 37 3.8 Growth kinetics of PaBV-5 in QT6 cells 38 3.9 Growth kinetics of PaBV-5 in DEF 39 3.10 Growth kinetics of PaBV-5 in DF-1 cells 40 3.11 Growth kinetics of PaBV-5 in Vero cells 41 3.12 Comparisons of viral permissiveness in different cells 42 3.13 PaBV-5 inoculation on CAM of duck embryos 43 Chapter 4 Discussion 45 Chapter 5 Figures and tables 57 Chapter 6 References 102"
dc.language.isoen
dc.subject轉錄體調控zh_TW
dc.subject第五型鸚鵡玻那病毒zh_TW
dc.subject感受性zh_TW
dc.subject複製特性zh_TW
dc.subjectParrot bornavirus type 5en
dc.subjecttranscriptomic profilesen
dc.subjectreplication studiesen
dc.subjectsusceptibilityen
dc.title第五型鸚鵡玻那病毒在不同細胞之複製特性分析與受感染細胞之轉錄體表現探討zh_TW
dc.titleReplication studies of parrot bornavirus type 5 in different cell types and analysis of cellular transcriptomic profiles after infectionen
dc.date.schoolyear109-2
dc.description.degree碩士
dc.contributor.oralexamcommittee王金和(Hsin-Tsai Liu),張麗冠(Chih-Yang Tseng),徐維莉
dc.subject.keyword第五型鸚鵡玻那病毒,感受性,複製特性,轉錄體調控,zh_TW
dc.subject.keywordParrot bornavirus type 5,susceptibility,replication studies,transcriptomic profiles,en
dc.relation.page123
dc.identifier.doi10.6342/NTU202101769
dc.rights.note同意授權(全球公開)
dc.date.accepted2021-08-13
dc.contributor.author-college獸醫專業學院zh_TW
dc.contributor.author-dept獸醫學研究所zh_TW
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