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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/100192完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳乃慧 | zh_TW |
| dc.contributor.advisor | Nai-Huei Wu | en |
| dc.contributor.author | 鄭伊文 | zh_TW |
| dc.contributor.author | Yi-Wen Cheng | en |
| dc.date.accessioned | 2025-09-24T16:48:14Z | - |
| dc.date.available | 2025-09-25 | - |
| dc.date.copyright | 2025-09-24 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-12 | - |
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(2023). 建立禽流感病毒於禽類呼吸道3D細胞培養之感染模式. 臺灣大學獸醫學研究所學位論文. 蔡宜蓓. (2023). 3D腸道類器官禽流感病毒感染模式之建立. 臺灣大學獸醫學研究所學位論文 | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/100192 | - |
| dc.description.abstract | 家禽白血病病毒（Avian Leukosis Virus, ALV）屬於反轉錄病毒科（family Retroviridae），可引發淋巴球性白血病與骨髓球性白血病，雞隻一旦感染，常伴隨嚴重免疫抑制、生產性能下降、腫瘤生成及高死亡率，對全球家禽產業造成重大損失。根據病毒封套蛋白（envelope, env）序列差異，ALV可分為11個亞群（A-K），ALV-A與ALV-J亞群病毒最常見於雞場。ALV依傳播途徑可分為外源性與內源性，兩者間在雞隻體內或雞場環境中可能發生重組，增加了診斷與根除的難度。近年新興的ALV-K被認為可能為外源性與內源性病毒重組之產物，其致病性與其他亞群間的交互作用目前尚未釐清，亦缺乏在台灣地區的流行病學研究。本研究針對近年台灣家禽場所分離到之ALV-A、ALV-J及ALV-K進行親緣關係分析，同時利用RCAS（Replication Competent ALV LTR with a Splice acceptor）載體系統構築帶有台灣ALV-J封套蛋白之重組病毒，進一步探討不同亞群的交互作用。RCAS為禽類反轉錄病毒載體，可攜帶ALV env基因並搭載EGFP或mCherry螢光標記，適用於追蹤無細胞病變效應之ALV感染路徑。本研究比較RCAS病毒於多種永生化細胞株、雞初代細胞及三維（3D）類器官模型中的感染表現，以模擬不同條件下之病毒傳播與交互作用。統計2023至2025年間的家禽腫瘤病例，ALV陽性樣本中以ALV-J最常見，且ALV-J與ALV-K共同感染的現象最頻繁。進一步分析序列發現台灣ALV-J病毒株在近20年間持續產生變異，而ALV-K則相對穩定。透過RCAS病毒感染實驗，發現ALV-A與ALV-J在細胞內呈現不同的傳播模式。此外當與ALV-K共同感染時，ALV-A的感染受到抑制，而ALV-J的感染則顯著增強，顯示不同亞群間存在不一樣的交互作用。我們進一步比較不同支系之ALV-J病毒於單獨感染以及與ALV-K共同感染之差異，結果呈現其感染能力與在共同感染下的反應亦有所不同。本研究闡明台灣ALV之演化趨勢與亞群間的交互作用，並透過RCAS系統提供可量化且可追蹤的ALV感染模型，為後續探討其致病機轉與疫病控制策略奠定基礎。 | zh_TW |
| dc.description.abstract | Avian Leukosis Virus（ALV）, classified into Retroviridae family, is known to induce lymphoid leukosis and myeloid leukosis. Infected chickens often exhibit severe immunosuppression, reduced production performance, tumor formation, and high mortality rates, resulting in significant economic losses for the global poultry industry. Based on the envelope glycoprotein（env）, ALV is classified into 11 subgroups（A-K）, among which ALV-A and ALV-J are the most common subgroup infections in poultry farms. In addition, ALV can be further categorized as exogenous or endogenous depending on the transmission route. Recombination between exogenous and endogenous ALV is frequently observed in poultry farms, which further complicates pathogen detection and eradication. Recently, a novel subgroup, ALV-K, has been identified and is hypothesized to be a recombinant between exogenous and endogenous ALVs. Its pathogenicity and interactions with other ALV subgroups remain poorly understood, and its epidemiological status in Taiwan is largely uncharacterized. In this study, phylogenetic analyses were performed on ALV-A, ALV-J, and ALV-K isolates collected from Taiwanese poultry farms in recent years. To further investigate inter-subgroup interactions, we constructed recombinant viruses using the RCAS system, incorporating envelope proteins from field strains of ALV-J in Taiwan. RCAS（Replication Competent ALV LTR with a Splice acceptor）, an avian retroviral vector, was engineered to carry ALV env genes tagged with EGFP or mCherry, allowing visualization of viral spread in the absence of cytopathic effects. We evaluated RCAS viral infection profiles in multiple immortalized cell lines, primary chicken cells, and three-dimensional（3D）models to simulate viral transmission and interaction under various conditions. Analysis of tumor cases in poultry from 2023 to 2025 revealed that ALV-J was the most frequently detected subgroup, with ALV-J/ALV-K coinfections being particularly common. Sequence analysis showed ongoing genetic variation in Taiwanese ALV-J strains over the past two decades, while ALV-K strains remained relatively stable. Functional assays using RCAS viruses revealed distinct transmission patterns for ALV-A and ALV-J in cells. Notably, in coinfection experiments, ALV-K suppressed ALV-A infection but significantly enhanced ALV-J infectivity, indicating subtype-specific interactions. Further comparisons among ALV-J clades showed differences in infectivity and responses under coinfection with ALV-K. This study elucidates the evolutionary trends of ALVs in Taiwan and highlights the complex interplay among different ALV subgroups. By applying the RCAS system, we established a quantifiable and traceable model for studying ALV infections, providing a foundation for future research on viral pathogenesis and control strategies. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-09-24T16:48:14Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-09-24T16:48:14Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 致謝 I
中文摘要 II Abstract III 目次 IV 圖次 X 表次 XI 縮寫 XII 第一章 緒論 1 第一節 家禽白血病的簡介與歷史背景 1 第二節 病原學 2 1-2.1 病毒結構 2 1-2.1.1 病毒基因體 2 1-2.1.2 病毒蛋白質 3 1-2.2 病毒複製流程 4 1-2.2.1 病毒進入（virus entry） 4 1-2.2.2 反轉錄作用（reverse transcription） 5 1-2.2.3 嵌入宿主基因（integration） 5 1-2.2.4 轉譯、轉錄及蛋白質合成（transcription and translation） 6 1-2.2.5 病毒組裝與釋放（virus assembly and release） 6 1-2.3 病毒分類 6 1-2.3.1 病毒env亞群 6 1-2.3.2 腫瘤類型 7 第三節 流行病學 8 1-3.1 病毒傳播途徑 8 1-3.1.1 外源性病毒（exogenous virus） 8 1-3.1.2 內源性病毒（endogenous virus） 9 1-3.2 宿主與細胞受體 10 1-3.3 臨床症狀 11 1-3.4 不同亞群病毒重組 12 1-3.5 腫瘤性病原共同感染 13 1-3.6 世界流行現況 13 第四節 診斷方法 14 1-4.1 病毒分離 14 1-4.2 分子生物學診斷 15 1-4.3 血清學診斷 15 第五節 預防與控制 16 第六節 RCAS載體系統 17 1-6.1 簡介 17 1-6.2 RCAS載體之宿主範圍 18 1-6.3 RCAS載體序列插入之設計 18 1-6.4 RCAS衍生載體 19 1-6.5 RCAS載體之應用 19 第七節 三維（3D）細胞培養系統 20 1-7.1 簡介 20 1-7.2 類器官（organoid） 20 1-7.2 氣液界面系統（Air-Liquid Interface, ALI） 21 第八節 研究動機 21 第二章 材料與方法 23 第一節 病例收集與檢測 23 2-1.1 病例收集與樣本處理 23 2-1.1.1 臟器樣本處理 23 2-1.1.2 血液樣本處理 23 2-1.2 DNA核酸萃取 24 2-1.3 RNA核酸萃取 24 2-1.4 反轉錄（reverse transcription） 24 2-1.5 聚合酶鏈鎖反應（polymerase chain reaction, PCR） 25 第二節 ALV病毒分離 26 2-2.1 DF-1細胞培養 26 2-2.2 病毒分離與檢測 26 第三節 序列分析與比對 26 2-3.1 病毒基因體定序 27 2-3.1.1 TA cloning 27 2-3.1.1.1 env基因增幅與質體合成 27 2-3.1.1.2 轉型（Transformation） 27 2-3.1.1.3 Colony PCR 28 2-3.2 序列比對與親緣樹狀圖繪製 28 第四節 RCAS重組病毒 28 2-4.1 RCAS(J)21-01-EGFP重組病毒 28 2-4.1.1 RCAS(J)21-01-EGFP重組病毒增殖 29 2-4.1.1.1 解凍細胞 29 2-4.1.1.2 病毒增殖 29 2-4.1.1.3 病毒力價測定 29 2-4.2 RCAS(J)23-03-EGFP重組病毒 30 2-4.2.1 載體（Vector）增幅 30 2-4.2.1.1 質體限制酶酵素切割 30 2-4.2.1.2 Vector PCR 30 2-4.2.2 嵌入基因（Insert）增幅 30 2-4.2.2.1 限制酶酵素切割 30 2-4.2.2.2 AV23-03 env PCR 30 2-4.2.3 以NEBuilder® HiFi DNA Assembly重組質體 31 2-4.2.4 RCAS(J)23-03-EGFP質體增殖與定序 31 2-4.2.5 DF-1轉染（Transfection） 31 2-4.2.6 RCAS(J)23-03-EGFP病毒增殖 32 2-4.2.7 病毒力價測定 32 2-4.3 RCAS(A)-mCherry之重組 32 2-4.3.1 載體（vector）PCR增幅 32 2-4.3.2 嵌入基因（insert）mCherry片段PCR增幅 33 2-4.3.3 以NEBuilder® HiFi DNA Assembly重組質體 33 2-4.3.4 轉型（Transformation）與Colony PCR 33 2-4.3.5 質體萃取 34 2-4.3.6 DF-1轉染（Transfection） 34 2-4.3.7 病毒增殖 34 2-4.3.8 病毒力價測定 34 第五節 細胞培養 34 2-5.1 永生化細胞培養 35 2-5.2 雞初代細胞分離與3D culture培養 35 2-5.2.1 雞胚腎細胞（Chicken Embryo Kidney cell, CEK）分離與培養 36 2-5.2.2 雞氣管與腸道3D culture培養 36 第六節 RCAS病毒與ALV-K共同感染試驗 37 2-6.1 病毒來源與增殖 37 2-6.1.1 病毒來源 37 2-6.1.2 病毒增殖 37 2-6.1.3 病毒力價測定 38 2-6.2 共同感染試驗 38 2-6.2.1 單層細胞病毒感染試驗 38 2-6.2.2 RCAS(J)23-03-EGFP於DF-1細胞之共同感染試驗 38 2-6.2.3 3D culture感染試驗 39 2-6.2.3.1 ALI感染試驗 39 2-6.2.3.2 類器官感染試驗 39 2-6.2.4 免疫螢光分析法（IFA） 40 2-6.2.4.1 單層細胞 40 2-6.2.4.2 ALI 40 2-6.2.4.3 類器官 41 2-6.2.5 病毒力價測定 41 2-6.2.6 Foci面積大小之測定 42 第三章 結果 43 第一節 腫瘤病例檢測與ALV病毒分離 43 第二節 序列分析與比對 43 3-2.1 親緣樹圖繪製 43 3-2.1.1 ALV-A 43 3-2.1.2 ALV-J 44 3-2.1.3 ALV-K 45 3-2.2 env序列比對 45 3-2.2.1 ALV-A 45 3-2.2.2 ALV-J 46 3-2.2.3 ALV-K 47 第三節 RCAS重組病毒 47 3-3.1 RCAS(J)23-03-EGFP重組病毒 47 3-3.2 RCAS(A)-mCherry重組病毒 47 第四節RCAS病毒感染試驗 48 3-4.1 比較RCAS(A)-EGFP與RCAS(J)21-01-EGFP在DF-1中的感染模式 48 3-4.2 RCAS病毒與ALV-K之共同感染試驗 48 3-4.2.1 單層細胞之共同感染試驗 48 3-4.2.2 RCAS(J)21-01-EGFP於3D culture之共同感染試驗 51 3-4.2.2.1腸道類器官 51 3-4.2.2.2 氣管ALI培養 51 3-4.2.3 比較RCAS(J)21-01-EGFP與RCAS(J)23-03-EGFP在DF-1中的感染能力 52 第四章 討論 53 第五章 參考文獻 61 第六章 圖表 72 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 家禽白血病病毒 | zh_TW |
| dc.subject | 家禽疾病 | zh_TW |
| dc.subject | 3D細胞培養系統 | zh_TW |
| dc.subject | RCAS重組病毒 | zh_TW |
| dc.subject | 序列分析 | zh_TW |
| dc.subject | sequence analysis | en |
| dc.subject | Replication Competent ALV LTR with a Splice acceptor（RCAS）recombinant virus | en |
| dc.subject | 3D cell culture | en |
| dc.subject | Avian Leukosis Virus | en |
| dc.subject | poultry diseases | en |
| dc.title | 台灣家禽白血病J亞群病毒的演化及不同亞群共同感染之分析 | zh_TW |
| dc.title | Evolution of Avian Leukosis Virus Subgroup J and Characterization of Co-infection with Different Subgroups in Taiwan | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 王金和;徐維莉;郭瑞琳 | zh_TW |
| dc.contributor.oralexamcommittee | Ching-Ho Wang;Wei-Li Hsu;Rei-Lin Kuo | en |
| dc.subject.keyword | 家禽疾病,家禽白血病病毒,序列分析,RCAS重組病毒,3D細胞培養系統, | zh_TW |
| dc.subject.keyword | poultry diseases,Avian Leukosis Virus,sequence analysis,Replication Competent ALV LTR with a Splice acceptor（RCAS）recombinant virus,3D cell culture, | en |
| dc.relation.page | 131 | - |
| dc.identifier.doi | 10.6342/NTU202503847 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2025-08-14 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 獸醫學系 | - |
| dc.date.embargo-lift | N/A | - |
| 顯示於系所單位： | 獸醫學系 | |
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