王金和臺灣大學：獸醫學研究所鄭宛芯Cheng, Wan-HsinWan-HsinCheng2007-11-282018-07-092007-11-282018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/59967家禽網狀內皮症病毒 (avian reticuloendotheliosis virus, REV)，在台灣家禽疾病中較不被重視，但現場感染雞隻可發現雞隻出現生長不良、羽毛生長異常、胸腺及華氏囊萎縮等症狀發生，但是尚無有效的預防控制方法，所以目前種雞場最重要的是區分感染及未感染的雞隻，以避免病毒傳播的可能；並檢測現場使用之疫苗是否有REV污染，以釐清現今台灣所使用的疫苗在病毒傳播上所扮演的角色。因此本研究以ELISA方式調查台灣種雞及土雞REV抗體陽性率，同時由血液中抽取DNA檢測病毒核酸，並由臨床送檢病例的血液樣本與腫瘤乳劑中進行病毒分離，此外也從動物用藥品檢定分所取得禽痘疫苗與馬力克疫苗以進行REV污染檢測。在抗體陽性率結果顯示在種雞場之抗體陽性率為83％，土雞場之抗體陽性率為8％，而病毒核酸檢測結果在土雞的感染率為29％，由結果可知台灣雞隻REV感染情形相當普遍。病毒分離方面由現場病例中分離到兩株病毒，編號分別為3122/03與3295/04。而疫苗檢測結果方面，則從18種禽痘病毒疫苗中分離出一株且此分離株具有細胞病變 (cytopathic effect, CPE) 現象；在馬立克病毒疫苗方面，17種疫苗都未分離到REV病毒。由於目前商用ELISA套組檢測方式以偵測抗體為主，所以本研究欲想發展偵測抗原之ELISA，以便現場診斷之大量運用，所以本研究則利用REV分離株3122的部分envelope基因選殖出來，進行重組蛋白3122env的表現並針對選殖部分的胺基酸片段發展單株抗體，結果共獲得3株單株抗體mAb1、mAb2及mAb3，進一步利用單株抗體來偵測REV抗原，結果可順利偵測到重組蛋白與REV病毒，顯示這些單株抗體具有應用在偵測抗原型ELISA之潛力。綜和本實驗之結果，由血清學、病毒核酸檢測及病毒分離方面，皆充分顯示出台灣REV感染情形相當普遍，此外亦從疫苗株分離到REV，更代表此疾病在雞群中之潛在危險性。Avian reticuloendotheliosis virus (REV) infection is common, but not ubiquitous. The REV-infected chickens showed stunted, pale, and abnormal feather development. The principal lesions include runting, and atrophy of the thymus and bursa of Fabricius. No procedures have been applied in commercial practice for the control of RE, so the most important thing in the field is to distinguish the infected and uninfected chickens. In this study, we used commercial ELISA kits to detect REV-specific antibodies in color-breed chickens, breeder chickens, and broilers; meanwhile we detected the viral DNA in blood by polymerase chain reaction (PCR). Clinical materials, including whole blood, plasma, tumor tissues, and litter, were collected for virus isolation. We collected fowl pox virus (FPV) vaccines and Marek’s disease (MDV) vaccines provided by branch institute of animal drugs inspection animal health research institute for detection of REV contamination, and tried to isolate REV from these vaccines. The result of prevalence investigation showed 83% antibody positive rate in breeder chickens and 8% in color-breed chickens. The result of detection of REV in native color hybrid blood samples by PCR showed 29% positive rate, which meant that the infection of REV was popular in Taiwan. Two REVs, named 3122/03 and 3295/04, were isolated from clinical materials. Among eighteen FPV vaccines we collected, eleven of them showed REV LTR sequences integration and one REV was isolated from these vaccines. This isolate had cytopathic effect in DF1 cell line. No REV was isolated from seventeen MDV vaccines. We cloned partial envelope gene for the expression of recombinant protein used for the production of monoclonal antibody to develop antigen-capture ELISA. The results showed that we had selected three monoclonal antibodies which could recognize the recombinant protein and REVs. These monoclonal antibodies have potential for developing of antigen-capture ELISA. Based on the results of serology, REV DNA detection, and virus isolation, the REV infection in Taiwan is common. Specially, the isolation of REV from FPV vaccines shows a potential danger in the field.目錄 目錄----------------------------------------------------------------------------------------------Ⅰ 表次----------------------------------------------------------------------------------------------Ⅶ 圖次----------------------------------------------------------------------------------------------Ⅷ 附錄----------------------------------------------------------------------------------------------Ⅹ 中文摘要-------------------------------------------------------------------------------------ⅩⅠ 英文摘要-------------------------------------------------------------------------------------ⅩⅡ 第一章 緒言-------------------------------------------------------------------------------------1 第二章 文獻探討-------------------------------------------------------------------------------3 2.1 歷史背景------------------------------------------------------------------------------------3 2.2 家禽網狀內皮症病毒介紹---------------------------------------------------------------3 2.3 病毒分類------------------------------------------------------------------------------------3 2.4 病毒型態與特性---------------------------------------------------------------------------4 2.5 致癌基因------------------------------------------------------------------------------------4 2.6 家禽網狀內皮症病毒基因結構與病毒蛋白------------------------------------------5 2.7 家禽網狀內皮症病毒之宿主特異性---------------------------------------------------5 2.7.1 病毒複製之宿主特異性----------------------------------------------------------------5 2.7.2 自然與實驗感染宿主-------------------------------------------------------------------6 2.8 病毒傳播------------------------------------------------------------------------------------6 2.8.1 水平傳播----------------------------------------------------------------------------------6 2.8.2 垂直傳播----------------------------------------------------------------------------------7 2.8.3 污染的生物性材料----------------------------------------------------------------------8 2.9 潛伏期---------------------------------------------------------------------------------------8 2.10 臨床症狀-----------------------------------------------------------------------------------9 2.11免疫抑制-----------------------------------------------------------------------------------10 2.12 病理變化----------------------------------------------------------------------------------10 2.13 血清學-------------------------------------------------------------------------------------10 2.14 家禽網狀內皮症病毒之實驗室診斷-------------------------------------------------11 2.15 區別診斷----------------------------------------------------------------------------------11 2.16 家禽網狀內皮症病毒污染活毒疫苗及插入FPV野外分離株基因中的情形12 2.17 家禽網狀內皮症病毒污染MDV活毒疫苗的情形--------------------------------14 2.18 家禽網狀內皮症病毒之免疫與治療-------------------------------------------------15 2.19 家禽網狀內皮症病毒之預防與控制-------------------------------------------------15 第三章 材料與方法---------------------------------------------------------------------------16 第一節 血清學調查---------------------------------------------------------------------------16 3.1.1血漿樣本採集----------------------------------------------------------------------------16 3.1.1.1 採樣方式-------------------------------------------------------------------------------16 3.1.1.2 採樣來源-------------------------------------------------------------------------------16 3.1.2 血清學檢測------------------------------------------------------------------------------16 3.1.2.1 抗體檢測-------------------------------------------------------------------------------16 3.1.2.2 結果判定-------------------------------------------------------------------------------17 第二節 屠宰場有色雞群血液樣本之REV病毒核酸檢測-----------------------------17 3.2.1 血液樣本採集---------------------------------------------------------------------------17 3.2.1.1 採樣方式-------------------------------------------------------------------------------17 3.2.1.2 採樣來源-------------------------------------------------------------------------------17 3.2.2 血液之DNA萃取-----------------------------------------------------------------------17 3.2.3 聚合酶鏈鎖反應------------------------------------------------------------------------18 第三節 病毒分離------------------------------------------------------------------------------19 3.3.1 病材採樣---------------------------------------------------------------------------------19 3.3.1.1 血液樣本之採樣----------------------------------------------------------------------19 3.3.1.2 腫瘤組織之採樣----------------------------------------------------------------------19 3.3.2 細胞培養---------------------------------------------------------------------------------20 3.3.3 冷凍與解凍細胞------------------------------------------------------------------------20 3.3.4 病毒分離---------------------------------------------------------------------------------21 3.3.5 病毒偵測---------------------------------------------------------------------------------21 3.3.5.1 血液之DNA萃取--------------------------------------------------------------------21 3.3.5.2 細胞之DNA萃取--------------------------------------------------------------------21 3.3.5.3 聚合酵素鏈反應 --------------------------------------------------------------------22 3.3.5.4 病毒RNA之萃取---------------------------------------------------------------------22 3.3.5.5 反轉錄聚合酵素鏈反應 -----------------------------------------------------------22 3.3.6 聚合酶鏈反應產物之選殖 (cloning)-----------------------------------------------23 3.3.6.1 聚合酶鏈反應產物之純化----------------------------------------------------------23 3.3.6.2 聚合酵素鏈反應產物之選殖-------------------------------------------------------24 3.3.7 免疫螢光分析 (Immunofluorescence assay, IFA)---------------------------------24 3.3.8 病毒力價測定---------------------------------------------------------------------------25 3.3.9 病毒中和試驗 (Virus neutralization test)-------------------------------------------26 3.3.10 病毒之濃縮與純化-------------------------------------------------------------------26 3.3.11 電子顯微鏡之病毒型態-------------------------------------------------------------27 3.3.12 蛋白質之定量-------------------------------------------------------------------------27 3.3.13 病毒核酸序列的定序及分析-------------------------------------------------------27 3.3.13.1 核酸序列的定序--------------------------------------------------------------------27 3.3.13.2 序列之分析--------------------------------------------------------------------------28 第四節 疫苗中REV污染檢測及自疫苗中病毒分離-----------------------------------28 3.4.1疫苗來源----------------------------------------------------------------------------------28 3.4.2禽痘疫苗之病毒分離-------------------------------------------------------------------28 3.4.3禽痘疫苗之DNA萃取-----------------------------------------------------------------29 3.4.4馬立克疫苗之病毒分離----------------------------------------------------------------29 3.4.5 馬立克疫苗之DNA萃取--------------------------------------------------------------29 3.4.6聚合酵素鏈反應檢測-------------------------------------------------------------------29 3.4.7病毒RNA之萃取------------------------------------------------------------------------30 3.4.8反轉錄聚合酵素鏈反應----------------------------------------------------------------30 3.4.9巢式聚合酵素鏈反應 (Nest-PCR)---------------------------------------------------31 第五節 單株抗體------------------------------------------------------------------------------31 3.5.1 單株抗體之製備------------------------------------------------------------------------31 3.5.1.1封套基因選殖--------------------------------------------------------------------------31 3.5.1.2 建構表現載體-------------------------------------------------------------------------32 3.5.1.3 免疫計畫-------------------------------------------------------------------------------32 3.5.1.4 單株抗體篩選-------------------------------------------------------------------------33 3.5.1.5 表現質體之保存----------------------------------------------------------------------33 3.5.2 重組蛋白之表現------------------------------------------------------------------------34 3.5.2.1重組蛋白之確認-----------------------------------------------------------------------34 3.5.3單株抗體之確認與應用----------------------------------------------------------------35 3.5.3.1單株抗體之確認-----------------------------------------------------------------------35 3.5.3.1.1 免疫螢光分析 (Immunofluorescence assay, IFA) ----------------------------35 3.5.3.1.2 SDS PAGE及西方墨點法 (Western blotting）-----------------------------35 3.5.3.1.3 免疫墨點法 (Immunodot blot assay)-------------------------------------------36 3.5.3.1.4 病毒中和試驗 (Virus neutralization test) -------------------------------------36 第四章 結果------------------------------------------------------------------------------------37 第一節 血清學調查--------------------------------------------------37 4.1.1 台灣種雞群之血清學調查------------------------------------------------------------37 4.1.2 台灣有色雞群之血清學調查---------------------------------------------------------37 第二節 REV抗原檢測------------------------------------------------------------------------37 4.2.1屠宰場有色雞群血液樣本之REV病毒核酸檢測---------------------------------37 第三節 病毒分離與病毒偵測---------------------------------------------------------------38 4.3.1 病材採樣---------------------------------------------------------------------------------38 4.3.1.1 病例介紹－2003年至2004年現場病例之家禽網狀內皮症病毒分離情形----------------------------------------------------------------------------------------------------38 4.3.2 病毒來源---------------------------------------------------------------------------------39 4.3.3 聚合酵素鏈鎖反應---------------------------------------------------------------------40 4.3.4 反轉錄聚合酵素鏈鎖反應------------------------------------------------------------40 4.3.5 聚合酵素鏈反應產物之選殖 (cloning)--------------------------------------------40 4.3.6 病毒力價測定---------------------------------------------------------------------------40 4.3.7 病毒中和試驗 -------------------------------------------------------------------------40 4.3.8 病毒之濃縮與純化---------------------------------------------------------------------41 4.3.9 免疫螢光分析---------------------------------------------------------------------------41 4.3.10 電子顯微鏡之病毒型態-------------------------------------------------------------41 4.3.11 蛋白質之定量--------------------------------------------------------------------------41 4.3.12 序列分析-------------------------------------------------------------------------------42 4.3.13免疫墨點法-----------------------------------------------------------------------------42 4.3.14 SDS PAGE及西方墨點法------------------------------------------------------------42 第四節 疫苗檢測------------------------------------------------------------------------------42 4.4.1 禽痘疫苗檢測---------------------------------------------------------------------------42 4.4.2 馬立克疫苗檢測------------------------------------------------------------------------43 4.4.3 序列分析---------------------------------------------------------------------------------43 第五節 單株抗體------------------------------------------------------------------------------43 4.5.1 重組蛋白之表現------------------------------------------------------------------------43 4.5.1.1 重組蛋白之確認----------------------------------------------------------------------43 4.5.2 單株抗體之確認------------------------------------------------------------------------43 4.5.2.1免疫螢光分析 ------------------------------------------------------------------------44 4.5.2.2 免疫墨點法 --------------------------------------------------------------------------44 4.5.2.3 SDS PAGE及西方墨點法 ---------------------------------------------------------44 4.5.2.4 病毒中和試驗 ----------------------------------------------------------------------44 第五章 討論與結論---------------------------------------------------------------------------46 第六章 參考文獻------------------------------------------------------------------------------53 表次 Table 1: Serological investigation of reticuloendotheliosis antibody positive rate from different breeds of chickens during 2003 to 2005---------------------------------------64 Table 2: Positive rates of polymerase chain reaction for reticuloendotheliosis virus in broiler breeder and native color hybrid blood samples collected during 2004----- 64 Table 3: Reticuloendotheliosis virus has being isolated from these clinical cases and antibody positive rates of different flocks at Yilan in November, 2004--------------65 Table 4: Antibody positive rates of different flocks in 3273 chicken farms at Miaoli in August, 2004---------------------------------------------------------------------------------65 Table 5: Reticuloendotheliosis virus has being isolated from these clinical cases and antibody positive rates of different flocks in 3295-3299 chicken farms at Changhua in September, 2004--------------------------------------------------------------------------66 Table 6: Detection of reticuloendotheliosis virus (REV) integrated in fowl pox vaccines by polymerase chain reaction and virus isolation ---------------------------67 Table 7: Detection of reticuloendotheliosis virus (REV) integrated in Marek’s disease vaccines by polymerase chain reaction and virus isolation ---------------------------68 Table 8: Titer of 3 isolates of reticuloendotheliosis virus propagated in chicken fibroblast as detected by polymerase chain reaction and immunofluorescence assay -------------------------------------------------------------------------------------------------69 Table 9: Western blotting analysis of concentrated antigens (isolate 3122/03, 3295/04, 3134/03) and of recombinant protein (3122/03 env protein) recognized with anti-reticuloendotheliosis virus antiserum (Charles River), mAb1, mAb2, and mAb3------------------------------------------------------------------------------------------69 Table 10: Detection of reticuloendotheliosis virus (REV) against different antibodies -------------------------------------------------------------------------------------------------70圖次 Figure 1: Schematic diagram of a retrovirus virion indicating various structures and proteins----------------------------------------------------------------------------------------71 Figure 2: The structure of the 9.0 Kb genome of reticuloendotheliosis virus A strain (REV-A; helper virus)----------------------------------------------------------------------72 Figure 3: The structure of the 5.7 Kb genome of reticuloendotheliosis virus T strain (REV-T; defective virus)-------------------------------------------------------------------72 Figure 4: Schematic representation of reticuloendotheliosis virus (REV) provirus integrated into the genomes of fowl poxvirus (FPV) field and vaccine strain viruses. (A) as were as of the long (B) and short (C) REV LTR remnants present in the FPV genome are shown---------------------------------------------------------------------------73 Figure 5: Maps of the env5/env6、env1/env2、rel8/rel4 primers for polymerase chain reaction ---------------------------------------------------------------------------------------73 Figure 6: Seroprevalence of reticuloendotheliosis virus at different ages and different breed-------------------------------------------------------------------------------------------74 Figure 7: Reverse transcriptase polymerase chain reaction analysis of the reticuloendotheliosis virus (REV)---------------------------------------------------------75 Figure 8: Detection of isolate 3122/03 from clinical signs in case 3121 to 3126 by immunofluorescence assay ----------------------------------------------------------------76 Figure 9: Purification of the isolate 3122/03 of reticuloendotheliosis virus (REV) for mmunodot blot assay, and western blot --------------------------------------------------77 Figure10: Negative stain electron micrograph of reticuloendotheliosis virus isolate 3295/04----------------------------------------------------------------------------------------78 Figure 11: Virus titration (TCID50) of REV isolates by PCR-- --------------------------79 Figure 12: DF1 cells infected with isolate 3134/03 of reticuloendotheliosis virus----80 Figure 13: Detection of isolate 3134/03 from fowl poxvirus vaccine by immunofluorescence assay---------------------------------------------------------------- 81 Figure 14: (A) SDS-PAGE analysis of different purification virus and expressed protein (B) Western blotting profile of isolate reticuloendotheliosis virus obtained following incubation of the nitrocellulose membrane with polyclonal antibody (Charles River)-------------------------------------------------------------------------------82 Figure 15: TA cloning of reticuloendotheliosis virus (REV) envelope gene-----------83 Figure 16: The envelope gene sequence of isolate 3122/03------------------------------84 Figure 17: The envelope gene sequence of isolate 3295/05------------------------------85 Figure 18: The partial envelope gene sequence of isolate 3134/03----------------------86 Figure 19: Percentage identity and divergence of the nucleotide sequences (nt. 22 – 1455) of the envelope gene of reticuloendotheliosis virus (REV) isolates 3122/03, 3134/03, 3292/ 05, and published sequences -------------------------------------------87 Figure 20: Phylogenetic tree of the nucleotide sequence of the envelope gene of reticuloendotheliosis virus (REV) isolates 3122/03, 3134/03, 3295/05, and published sequences-------------------------------------------------------------------------88 Figure 21: SDS-PAGE analysis of E. coli-expressed protein. E. coli BL21 (LysE) cells with pET 3122/03 env.----------------------------------------------------------------------89 Figure 22: Immunodot blot assay by using monoclonal antibodies 1, 2 and 3 and polyclonal antibodies (Charles River)----------------------------------------------------90 Figure 23: Western blotting assay using monoclonal antibodies 1, 2, 3, and polyclonal antibodies (Charles River)------------------------------------------------------------------91 Figure 24: Polymerase chain reaction analysis in the Marek’s disease (MD) vaccine92 Figure 25: Polymerase chain reaction analysis of the fowl poxvirus vaccine ---------93 Figure 26: Fluorescent micrograph of isolate 3295/04 of reticuloendotheliosis virus infected DF1 reacted with monoclonal antibody 2 (magnification 400x)-----------94附錄 Appendix 1. 95% 的機率至少可以檢出一個陽性個體所需的樣本數---------------95 Appendix 2: The prevalence of reticuloendotheliosis antibody in layer breeder chickens in 2004.----------------------------------------------------------------------------96 Appendix 3: The prevalence of reticuloendotheliosis antibody in broiler breeder chickens---------------------------------------------------------------------------------------97 Appendix 4: The prevalence of reticuloendotheliosis antibody in colored breeder chickens.--------------------------------------------------------------------------------------98 Appendix 5: Serological investigation of reticuloendotheliosis virus in native color hybrid chicken sera collected from the different slaughter house.-----------------------99 Appendix 6: IDEXX data--------------------------------------------------------------------100 Appendix 7: Detection of LTR-region of REV in colored chicken from a slaughter house by PCR.------------------------------------------------------------------------------101 Appendix 8: Positive values (S/P value) of REV antibody positive serum samples from clinical cases 3121 to 3126.--------------------------------------------------------102 Appendix 9. Solution formula used in this study-----------------------------------------103 Appendix 10. 中英對照縮寫------------------------------------------1052128047 bytesapplication/pdfen-US家禽網狀內皮症病毒封套基因avian reticuloendotheliosis virusenvelope gene[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59967/1/ntu-94-R92629019-1.pdf