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研究生:謝耀清
研究生(外文):Yao-Ching Hsieh
論文名稱:牛流行熱病毒之抗原性分析、疫情監控、快速診斷方法與新型雙價疫苗之研發
論文名稱(外文):Study of Antigenic Analysis, Epidemiological Surveillance, Development of Rapid Diagnostic Techniques and A New Bivalent Vaccine of Bovine Ephemeral Fever Virus
指導教授:陳世輝陳世輝引用關係
指導教授(外文):Shih-Hui Chen
學位類別:博士
校院名稱:國立成功大學
系所名稱:生命科學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:95
語文別:中文
論文頁數:138
中文關鍵詞:抗原性分析疫情監控新型雙價疫苗之研發牛流行熱病毒快速診斷方法
外文關鍵詞:Antigenic AnalysisA New Bivalent VaccineBovine Ephemeral Fever VirusDevelopment of Rapid Diagnostic TechniquesEpidemiological Surveillance
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牛流行熱 (bovine ephemeral fever,BEF) 的病原為子彈型病毒科 (Rhabdoviridae) 之牛暫時熱病毒屬 (bovine ephemeral fever virus, BEFV) ,是經由吸血性節肢動物傳染,引起牛隻急性發熱性傳染病,造成牛隻死亡或淘汰、流產、產乳量下降、治療費用極高及乳汁的廢棄等鉅大損失。本省氣候高溫多濕,對蚊蟲媒介的根除並不容易,隨著抗體的消長,週期性的爆發是可以預期的。過去40年來,本省曾爆發8次BEF疫情,本病若能早期診斷、早期治療,則預後良好,但是目前BEF市售疫苗仍選用1984年分離株製備,急待改進,而台灣地區本病之流行狀況,病毒變異標準、傳統血清學及快速分子診斷技術也都尚未建立,故本研究即以台南縣家畜疾病防治所保存之1984及1996年病毒株以及1999至2004年在台南縣轄內牛場分離之病毒株為材料,進行下列實驗,包括 (1)病毒分離及確認:本試驗分別於1999、2001及2004年檢測疑似感染病牛之血液檢體及解剖5頭感染BEF牛隻,經BHK-21細胞分離、反轉錄酶聚合酶鏈鎖反應 (RT-PCR) 、核酸序列定序、電子顯微鏡檢查及動物接種確認為BEFV; (2)不同分離株抗原性分析:分析17株病毒G醣蛋白全長基因序列,結果顯示2004年分離之病毒株與1996至2001年分離病毒株核酸序列差異約1-1.2%,而與1984年疫苗株差異度約2.1-2.5%,經樹狀親源分析發現1984年與1996至2004年分離株可被區分為2群; (3)建立分子診斷技術:本研究針對病毒醣蛋白G基因設計特異性引子及核酸探針,建立RT-PCR、TaqMan即時定量聚合酶鏈鎖反應 (TaqMan-based real time PCR) 及生物活性增幅雜合反應(bioactive amplification with probing, BAP)等快速診斷方法,結果顯示BAP反應敏感度可偵測1 copy病毒。經檢測及比較34個臨床檢體之敏感度,以BAP反應檢出率72.73%最高,可達到早期偵測之效果; (4)研發酵素免疫結合吸附法(ELISA)套組:因血清中和抗體試驗操作過程費時又費力,無法大量篩檢樣品,本研究以純化BEF病毒來研發ELISA檢測套組,並經抗原抗體定量試驗與4次中和抗體相關性重複性試驗,結果顯示此套組之OD值與中和抗體相關性佳,相關係數達0.80 (R值); (5)牛群免疫接種疫苗成效監控:以血清中和抗體試驗在2002-2004年進行牛隻血清抗體監測,結果顯示每年牛隻經疫苗補強後,血清中和抗體力價約維持4-5個月後開始消退,中和抗體力價呈逐年下降(P<0.0025) ,以2004年7月份中和抗體力價消退最為顯著,在8月份隨即又爆發BEF; (6)不活化種毒更新疫苗之效力評估:本研究同時選用1984及1999年之分離株混和之,不活化後以傳統氫氧化鋁膠當佐劑或改用水包油包水 (w/o/w) 佐劑製成雙價混合油質疫苗,分別免疫接種小鼠與牛隻,結果顯示不論免疫小鼠、天竺鼠、仔牛或懷孕母牛,均無任何不良反應,安全性佳;免疫牛隻方面,(w/o/w)雙價疫苗接種8週後血清中和抗體力價揚升至256倍以上,並持續6個月之久,且抗體力價揚升高於氫氧化鋁膠疫苗32倍,當可參考為推廣之新疫苗。希望上述研究成果可供BEFV分離、鑑定、快速診斷、流行病學監控及疫苗改選等之參考。
Bovine ephemeral fever (BEF), an arthropod-borne disease of cattle, is caused by the bovine ephemerovirus of the Rhabdoviridae family. It can trigger an acute febrile infection and an increase rate of the death or cull of cattle, abortion, decline of milk production, high cost of treatment, or abdication of milk. The weather in Taiwan is hot and humid; therefore, it is not easy to eliminate mosquitoes. As the BEF viral antibody declines is expectable the regular outbreak of BEF. In the past 40 years, Taiwan has had eight of BEF epizodes. If the BEF can be diagnosed in early stage, it can be fully cured. The seed virus of current BEF vaccine is an 1984 isolate and has been used over 20 years. Therefore, it’s efficacy may have declined. In addition, the standard technique, vaccination program, epidemiological surveillance, and rapid diagnosis procedure on BEF have not been established in Taiwan. In this study we used the viral strains which are isolated from 1984 to 2004 and kept in the Tainan Hsien Livestock Disease Control Center (THLDCC) for a series of experiments as follows: (1)Virus isolation and identification: Examining the samples of blood collected from allegedly BEFV infected cows in 1999, 2001 and 2004, and dissecting two infected cows. By the use of inoculation into BHK-21 cell line, the RT-PCR assay, nucleotide sequencing, electron microscopic examination and mouse inoculation, we confirm that the virus is BEFV; (2)Antigenic analysis of different isolates: The study analyzed the sequences of G-encoding gene of 17 BEFV isolates and showed that the sequence difference between 2004 and 1996-2001 isolates was 1-1.2%. On the other hand, the difference between 2004 and 1984 isolates was 2.1-2.5%. In the phylogenetic analysis, 1984 and 1996-2004 isolates have been classified into 2 different groups; (3)Establishment of molecular diagnostic technique: We designed a pair of primers and nucleic acid probe based on G-glycoprotein gene of BEFV to establish RT-PCR, TaqMan-based real time PCR and bioactive amplification with probing assay (BAP). The sensitivity of BAP is about one copy of virus. In the thirty-four clinical cases detected by RT-PCR, TaqMan-based realtime PCR, and BAP, BAP technology shows higher sensitivity than other methods for detecting early stage of BEFV infection; (4)Development of ELIAS kit: Because the process of serum neutralization(SN) test is time-consuming and laborious, we developed an ELISA kit by using purified BEF virus to detect the antibody. We preceded SN test and ELISA together. The result showed that the correlation index between the ELISA kit and SN test is 0.8; (5)The antibody monitoring of cattle herd: The study uses the SN test to detect BEF antibody in blood samples collected from 2002 to 2004, and the result indicates that after the second injection of BEF vaccine, the BEF antibody titer level maintains 4-5 months. After that, the antibody titer declines every year. The decline of the antibody titer is most obvious in July, 2004, followed by the outbreak of BEF in August, 2004 in Tainan; (6)The efficacy evaluation of new inactive vaccine: The combination of 1984 and 1999 BEFV isolates was prepared, inactivated, then emulsified in the form of water-in-oil-in-water emulsion (w/o/w). The mice, guinea pigs, calves and pregnant cattle were immunized with this new vaccine, and the conventional vaccine preparation using Al(OH3) as adjuvant was also inoculated into separate animals for comparison. Eight weeks after the vaccination on cattle, SN titer is increased over 256-fold and remained at this level for 6 months. The conventional vaccine showed 32-fold lower in SN titer. We believe that the above strategies and results will be helpful in improving the BEFV isolation, identification, rapid diagnosis, epidemiological study, and new vaccine development.
項目 頁次
中文摘要 I
英文摘要 III
致謝 VI
目錄 VII
縮寫目錄 XII
表目錄 XIII
圖目錄 XV
第一章、緒論 1
第二章、文獻探討 4
第一節 牛流行熱的歷史背景 4
第二節 病毒之特性 9
第三節 流行病學 16
第四節 經濟重要性 19
第五節 台灣牛流行熱疫情及疫苗使用現況 20
第六節 台灣牛流行熱監控及病毒變異 22
第七節 分子生物學快速診斷技術開發 23
第八節 血清學檢驗技術:ELISA試劑套組開發 27
第三章、材料與方法 30
第一節 牛流行熱病毒分離及確認 30
(一)病史及前置作業 30
(二)細胞培養 30
(三)病毒分離與增殖 30
(四)病毒純化 31
(五)病毒力價(TCID50)測定 31
(六)間接螢光染色法 31
(七)哺乳小鼠腦內接種 32
(八)電子顯微鏡檢查 32
(九)反轉錄聚合酶鏈鎖反應 32
(十)電泳分析與核酸序列定序 33
第二節 牛流行熱病毒分離株抗原性及親緣關係分析 33
(一)含病毒病材RNA萃取及RT-PCR 33
(二)DNA純化與核酸序列定序分析 33
(三)親緣關係樹狀圖分析 34
(四)野外病毒分離株之交叉中和試驗 34
(五)不同年代病毒分離株抗原性之預測值分析 35
第三節 牛流行熱分子診斷技術之建立 35
(一) TaqMan探針即時定量聚合酶鏈鎖反應 35
(1)BEF病毒核酸序列分析及引子/探針設計 35
(2)TaqMan探針即時定量PCR參考標準曲線建立 36
(3)TaqMan探針即時定量PCR特異性試驗 39
(4)以TCID50定量病毒及以TaqMan探針即時定量PCR檢測病毒含量之相關 39
(二) 生物活性增幅雜合反應 40
(1)BEF病毒核酸序列分析及引子/探針設計 40
(2)樣品處理之方法 40
(3)反轉錄聚合酶鏈鎖反應最佳化測試 41
(4)轉殖 41
(5)單管反轉錄聚合酶鏈鎖反應 41
(6)生物活性增幅雜合反應之研究及最佳化測試 42
第四節 血清學ELISA試劑套組之研發 42
(一)病毒純化 42
(二)病毒濃度測定 42
(三)抗原盤病毒塗鍍測試 43
(四)ELISA套組與中和抗體力價之相關性試驗 43
(五) ELISA套組敏感度及特異性測試 43
第五節 牛流行熱免疫成效監控 44
2002-2004年監控計畫 44
第六節 牛流行熱不活化疫苗效力及免疫適期試驗45
(一)不同市售疫苗效力評估 45
(二)一次與二次免疫效力分析 45
(三)不同免疫適期比較試驗 45
(四)新型複合雙價油質疫苗製作及測試 45
第四章、結果 48
第一節 牛流行熱病毒分離及確認 48
(一)牛流行熱臨床症狀、肉眼與組織病變 48
(二)病毒分離、細胞病變與間接螢光抗體染色 48
(三)哺乳小鼠腦內接種與電子顯微鏡檢查 49
(四)反轉錄聚合酶鏈鎖反應 49
第二節 牛流行熱病毒分離株抗原性及親源關係分析50
(一)DNA純化與核酸序列分析 50
(二)親源關係樹狀圖 50
(三)不同年代病毒分離株交叉中和試驗 51
(四)不同年代病毒分離株抗原性之預測值分析 52
第三節 牛流行熱分子診斷技術之建立 52
(一) TaqMan探針即時定量聚合酶鏈鎖反應 52
(1)TaqMan探針即時定量PCR參考標準曲線建立 52
(2)標準曲線重複性評估試驗 53
(3)以TaqMan探針即時定量聚合酶鏈鎖反應偵測臨床病例54
(4)TaqMan探針即時定量PCR特異性試驗 54
(二) 生物活性增幅雜合反應 54
(1)生物活性增幅雜合反應敏感度之測試 54
(2)生物活性增幅雜合反應特異性之測試 55
(3)以生物活性增幅雜合反應偵測臨床病例 55
第四節 血清學ELISA試劑套組之研發 55
(一)抗原盤病毒塗鍍測試結果 55
(二)ELISA套組與中和抗體力價之相關性試驗 56
(三)ELISA套組敏感度及特異性測試 56
第五節 牛流行熱免疫成效監控 57
(一)2002年抗體監測結果 57
(二)2003年抗體監測結果 57
(三)2004年抗體監測結果 58
第六節 牛流行熱不活化疫苗效力及免疫適期試驗 58
(一)不同市售疫苗效力評估 58
(二)一次與二次免疫試驗 60
(三)不同免疫適期比較試驗 60
(四)雙價油質疫苗效力評估 61
第五章、討論 63
第一節 牛流行熱病毒分離及確認 63
第二節 牛流行熱病毒分離株抗原性及親源關係分析65
第三節 牛流行熱分子診斷技術之建立 67
第四節 血清學ELISA試劑套組之研發 69
第五節 牛流行熱免疫成效監控 71
第六節 牛流行熱不活化疫苗效力及免疫適期試驗73
第六章、結論 77
參考文獻 80
表 94
圖 108
作者簡歷及發表著作 132
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