臺灣博碩士論文加值系統

日本腦炎病毒（Japanese encephalitis virus）與Culex flavivirus均屬於黃熱病毒科（Flaviviridae）中黃熱病毒屬（Flavivirus）的病毒，這兩種病毒主要皆由家蚊來傳播，因此可能在養豬場中共同循環。1990年代以來，許多東南亞國家流行的日本腦炎病毒基因型發生轉變，由原本的第三型基因型轉變成以第一型基因型為主要的流行株；而台灣疾病管制局也於2008年首度分離到第一型基因型日本腦炎病毒，但還未知是否亦發生日本腦炎病毒基因型轉變。而Culex flavivirus為只感染蚊子之黃病毒，目前在台灣並無關於Culex flavivirus的相關研究。

為了監控這兩種病毒在台灣豬場中的分子流行病學，本研究於2009年至2010年在臺灣豬場內進行病毒學監控計畫。於三斑家蚊中共分離出37株日本腦炎病毒陽性檢體，經基因序列及演化樹分析發現，所測得之病毒皆屬於第一型基因型Subcluster I及Subcluster II病毒。再經基因指紋及最短距離生成樹(minimum spanning tree)分析發現，台灣的第一型基因型Subcluster I及Subcluster II病毒皆由日本長崎所傳入，且至少傳入三次。此外，在同時分離到第一型基因型Subcluster I及Subcluster II病毒的台中及雲林場，依其分離率進行相對百分比分析後，顯示第一型基因型Subcluster I病毒似乎比Subcluster II病毒具演化優勢。在Culex flavivirus方面，於台中豬場的三斑家蚊中分離到41個Culex flavivirus 陽性檢體；將其中五個Culex flavivirus陽性檢體進行封套蛋白序列分析後，發現與其他國家核苷酸及胺基酸序列的相似度分別為88~94.2% 及96.9~99.5%；再進行親緣演化樹分析後，發現台灣分離到的Culex flavivirus序列與墨西哥及瓜地馬拉的序列較為相近，但屬於不同的分支。最後將台中豬場的病媒蚊分為CxFV陽性及CxFV陰性組，兩個族群中日本腦炎病毒陽性比率分別為7.3%及0.96%，顯示日本腦炎病毒與CxFV可於三斑家蚊上共同感染，然而CxFV對於日本腦炎病毒的傳播是否有影響仍需進一步證實。

Several flaviviruses, including Japanese encephalitis virus (JEV) and Culex flavivirus (CxFV), are transmitted by Culex mosquito, and possible circulating in pig farms in Taiwan. In several Southeast Asia countries, genotype shift of JEV from genotype III to I has been observed since 1990s. Taiwan was one of last countries free from genotype I JEV, but this virus appeared in the end of 2008. CxFV is a novel flavivirus which only infects Culex mosquitoes, but this virus had never been studied in Taiwan.
In order to monitor the molecular epidemiology of both viruses, we conducted virological surveillance among mosquitoes collected from randomly selected pig farms in Taiwan, 2009-2011. A total 37 mosquito pools, consisted of Culex tritaeniorhynchu collected from eight pig farms in six Counties, were JEV RNA positive. Nucleotide sequence followed by phylogenetic tree analysis confirmed all of isolates belonged to genotype I JEV, and fell into two subclusters, I and II. The virus introduction and movement tracked by minimum-spanning tree analysis indicated that the genotype I JEV isolated in Taiwan, including subcluster I and II, were probably origined from Nagasaki, Japan; and introduced to the northern and central region of Taiwan at least three times in the past. According to the relative proportion of subcluster I and subcluster II virus among co-circulating pig farms(including Taichung county and Yunlin county), the subcluster I virus might had evolutional advantage than subcluster II virus. And, total 41 mosquitoes pools collected from Taichung’s pig farm were CxFV RNA positive. E protein gene sequence analysis of 5 CxFV isolates revealed that nucleotide identity and amino acid identity with other countries isolates were 88~94.2% and 96.9~99.5%, respectively. The positive rate of JEV among CxFV-infected and -uninfected mosquito were 7.3% and 0.96%, respectively. The possible influence of CxFV on the transmission of JEV should be studied more deeply in the future.

中文摘要 I
Abstract II
目次 IV
表次目錄 VII
圖次目錄 VIII

第一章 文獻探討 1
第一節 黃病毒之基本介紹 1
第二節 日本腦炎病毒之基本介紹 1
一、流行病學 1
二、病毒學 2
三、傳染途徑及宿主 2
四、臨床症狀 2
五、日本腦炎病毒基因型分型 3
六、日本腦炎病毒基因型之轉變 3
第三節 Culex flavivirus 基本介紹 5
一、新興特異性感染昆蟲之黃病毒 5
二、Culex flavivirus 病毒學 5
三、Culex flavivirus流行病學 6
第四節 Culex flavivirus 對感染其他黃病毒之影響 6
一、蟲媒病毒之重複感染 6
二、西尼羅病毒與Culex flavivirus 6
第二章 研究動機 8
第三章 材料與方法 9
第一節 細胞培養 9
第二節 病媒蚊日本腦炎病毒偵測及Culex flavivirus偵測 9
一、病媒蚊樣本採集 9
二、蚊子均質化 9
三、RNA萃取及反轉錄反應 9
四、日本腦炎病毒Multiplex RT-PCR 10
五、Culex flavivirus RT-PCR 11
六、PCR增幅產物定序 11
七、親源樹狀圖建構 11
八、病毒分離 11
九、統計分析 11
第三節 豬隻血中日本腦炎病毒中和抗體檢測及日本腦炎病毒檢測 12
一、豬隻樣本採集 12
二、血漿及周邊血液單核球細胞分離 12
三、中和抗體偵測 12
四、抗原偵測 13
第四章 結果 14
第一節 日本腦炎病毒Multiplex RT-PCR 14
一、引子設計 14
二、特異度、敏感度及偵測極限 14
三、Multiplex 條件建立 15
第二節 病媒蚊日本腦炎病毒偵測與分析 16
一、病媒蚊樣本描述 16
二、日本腦炎病毒陽性率及最低攜帶病毒率 16
三、病毒分離 17
四、核苷酸及胺基酸序列比對 17
五、日本腦炎病毒親緣樹狀圖分析 17
六、日本腦炎病毒第一型基因型Subcluster I及Subcluster II相對分布率 18
第二節 豬隻日本腦炎病毒中和抗體及抗原偵測 19
一、豬隻血漿中和抗體測定 19
二、豬隻血漿中日本腦炎病毒抗原測定 19
第三節 病媒蚊Culex flavivirus偵測 20
一、Culex flavivirus RT-PCR偵測結果 20
二、病毒分離 20
三、核苷酸及胺基酸序列比對 20
四、親緣樹狀圖分析 20
五、Culex flavivirus於台灣的盛行趨勢 21
第四節 日本腦炎病毒及Culex flavivirus於三斑家蚊中交互關係 21
第五章 討論 22
第一節 日本腦炎病毒基因型之轉變 22
第二節 台灣第一型基因型日本腦炎病毒之來源 23
第三節 第一基因型日本腦炎病毒Subcluster I及Subcluster II 相對分佈情形 23
第四節 台灣的Culex flavivirus 24
第五節黃病毒與Culex flavivirus於家蚊中交互關係 24
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