臺灣博碩士論文加值系統

　　輪狀病毒 (Rotavirus, RVs)感染主要造成世界上年幼動物及幼童急性病毒性胃腸炎的人畜共通傳染病。輪狀病毒可藉由病毒蛋白衣上第六蛋白質 (Viral protein 6, VP6)之血清學及基因序列分析分成十型 (RVA-RVJ)。RVA為RVs當中最為重要的病原體，因為在人類、各種飼養家畜及野生動物的流行率及致病率皆相當高。而豬流行性下痢病毒 (Porcine epidemic diarrhea virus, PEDV)與RVs皆會於豬隻引起相似的消化道臨床症狀，但這些病原感染無法從肉眼或組織病變中區分其差異。因此，本研究收集從2015年1月初至2016年12月底，各年齡層中臨床上具有下痢症狀的樣本來確定RVA、RVB及RVC三者間的盛行率及混合感染情況。我們採用即時PCR (real-time PCR)來檢測RVA、RVB及RVC的檢出率百分比，同時與PEDV進行比較。結果顯示，PEDV及RVs (RVA/ RVB/ RVC)陽性率分別為46.9%及36.5% (23.2%/ 12%/ 14.9%)。1084個檢體中與PEDV混合感染的RVA、RVB及RVC分別有83例 (7.7%)、56例 (5.2%)及58例 (5.4%)。此外，未與PEDV混合感染的RVs (RVA/ RVB/ RVC)盛行率為 23.7% (15.5%/ 6.8%/ 9.5%)。RVs
檢出率最高的季節為夏季 (6月至8月)，而PEDV檢出率最高的季節在春季 (3月至5月)。近年來的研究大多著重於RVA及PEDV的感染情況，但是目前RVB及RVC在台灣也有較高的感染情況。

Rotaviruses (RVs) infection are zoonosis disease that cause acute viral gastroenteritis in young animals and children worldwide. RVs are classified into ten groups (A-J) based on serological assays and nucleotides sequencing of the viral protein 6 (VP6). RVA is considered the most important pathogen in RVs due to the high prevalence and pathogenicity in both humans and a variety of domestic and wild animals. The porcine epidemic diarrhea virus (PEDV) and RVs cause similar clinical sign in piglets, so the pathogens can't be clearly distinguished from gross or histological lesions. As a result, the samples was collected from all ages with clinical sign of diarrhea from Jan 2015 to Dec 2016 to identified the prevalence and co-infections status between RVA, RVB and RVC. The real-time PCR will be used to estimate the percentage of RVA, RVB and RVC, then compare with PEDV at the same time. The results shows that the positive rates of PEDV and RVs (RVA/ RVB/ RVC) are 46.9% and 36.5% (23.2%/ 12%/ 14.9%). RVA, RVB and
RVC are 83 (7.7%), 56 (5.2%) and 58 (5.4%) co-infection with PEDV in 1084 samples. There are 23.7% (15.5%/ 6.8%/ 9.5%) that are RVs (RVA/ RVB/ RVC) infection without PEDV. RVs and PEDV have highest prevalence in summer and in spring, respectively. Recent study always focused on RVA and PEDV infections, but RVB and RVC shows high infections in swine population from this study in Taiwan.

中文摘要 I
Abstract II
致　謝 IV
目　錄 V
圖表目錄 VIII
第1章 前言 9
第2章 文獻回顧 12
2.1 豬輪狀病毒 12
2.1.1 背景與命名 12
2.1.2 結構與生化特性 12
2.1.3 宿主 13
2.1.4 流行病學調查 14
2.1.5 傳播方式 15
2.1.6 引發之相關疾病 16
2.1.6.1 臨床症狀 16
2.1.6.2 肉眼病變 17
2.1.6.3 組織病理學 18
2.1.7 致病機轉 18
2.1.8 免疫機制 19
2.1.9 診斷方式 21
2.1.9.1 酵素免疫吸附分析法 22
2.1.9.2 細胞培養 22
2.1.9.3 免疫組織化學染色技術 22
2.1.9.4 反轉錄聚合酶鏈鎖反應 23
2.1.10 防疫策略 23
2.1.10.1 控制與預防 23
2.1.10.2 疫苗 25
2.2 豬流行性下痢病毒 27
2.2.1 背景與命名 27
2.2.2 結構與生化特性 27
2.2.3 宿主 28
2.2.4 流行病學調查 28
2.2.5 傳播方式 30
2.2.6 引發之相關疾病 31
2.2.6.1 臨床症狀 31
2.2.6.2 肉眼及顯微病變 31
2.2.7 致病機轉 32
2.2.8 免疫機制 33
2.2.9 診斷方式 33
2.2.9.1 免疫螢光／免疫組織化學技術 33
2.2.9.2 電子顯微鏡 34
2.2.9.3 細胞培養 34
2.2.9.4 酵素免疫吸附分析法 34
2.2.9.5 反轉錄聚合酶鏈鎖反應 34
第3章 材料與方法 36
3.1 實驗設計流程 36
3.2 樣本採集 37
3.2.1 豬隻腸道樣本採集 37
3.2.2 豬隻糞便樣本採集 37
3.3 核酸萃取 37
3.4 反轉錄反應 38
3.5 即時聚合酶鏈鎖反應之建立 38
3.5.1 豬A、C型輪狀病毒樣本之多對引子即時聚合酶鏈鎖反應 38
3.5.2 豬B型輪狀病毒樣本之即時聚合酶鏈鎖反應 39
3.6 瓊脂膠體電泳分析 39
3.6.1 瓊脂膠體制備 39
3.6.2 電泳分析 39
3.7 聚合酶鏈鎖反應產物純化 40
3.8 聚合酶鏈鎖反應產物之選殖 40
3.9 選殖基因確認 41
3.10 質體DNA萃取 42
3.11 定序 42
3.12 核酸序列分析 42
第4章 結果 44
4.1 樣本收集 44
4.2 Real-time PCR陽性檢體篩選 44
4.3 資料統計及分析 44
4.3.1 豬輪狀病毒 (RVs)與豬流行性下痢病毒 (PEDV)之盛行率 44
4.3.2 不同氣溫間差異 46
4.3.3 年齡間差異 47
4.3.4 病原檢出率趨勢 47
第5章 討論 49
第6章 參考文獻 67
作者簡介 86

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