蔡向榮臺灣大學：獸醫學研究所張能輝Chang, Neng-HuiNeng-HuiChang2007-11-282018-07-092007-11-282018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/59956本研究為探討台灣地區家鴨禽流感病毒及家鴨在禽流感病毒流行傳播鏈中與跨物種感染上可能扮演的角色，於2002-2007年對家禽市場及禽鳥養殖場的家禽進行病毒分離及血清學監測，共分離出6株H4N6亞型流感病毒，且無其他亞型。分析16株流感病毒株自3個來源獲得，包括6株H4N6亞型流感病毒及連同其他分讓自家畜衛生試驗所及台大公衛學院流行病學研究所的10株1998至2006年H4亞型基因序列 (共計16株)，分別進行其所有的8段基因核酸定序後，經過序列比較分析得知，這些來自不同鳥種及不同分離年代之H4亞型流感病毒，個別病毒株之間的各別基因相異度達0.4-14.5％，顯示臺灣地區H4N6亞型流感病毒之差異性，且家鴨病毒源自境外移入的野鴨。以HA基因論，各個病毒株之蛋白酵素切割位周邊鹼性氨基酸皆僅有1個，表示均為弱毒株或無毒株。就NA基因言，其中的A/Duck/Changhua/0648/06 (H4N6) 於氨基酸序列52至71區段中缺損8個氨基酸，對病毒特性的影響程度則有待進一步分析研究。另進行各別基因之種系親緣關係分析後，得知16株H4亞型區分在4個小支群，並且台灣地區雖然至少曾有4株野鴨H4N6亞型流感病毒於不同年代侵入家鴨群，但可能皆未再於家鴨中持續傳播；而所有的H4N6亞型流感病毒株皆屬歐亞種系。至於以2006年家鴨H4N6亞型流感病毒 (A/DK/Changhua/5911/2006) 為抗原對台灣地區2002至2007年期間採集的禽鳥血清檢體進行血球凝集抑制抗體調查後，可知禽場陽性率為 7.4％ (6/81場)，其中雞隻陽性率 0.38 ％ (7/1826隻)、鵪鶉 0％ (0/96隻)、鴨 0％ (0/857隻)、鵝 0％ (0/266隻)，顯示H4 亞型流感病毒感染台灣家禽群的情況並不普遍。本研究成果表明臺灣地區家鴨及野鴨H4N6亞型流感病毒皆源自歐亞種系，且可由野鴨跨物種感染台灣家鴨群，但未能在家鴨中廣泛穩定傳播，亦未有進而跨物種傳播給其他種類家禽的現象發生。To investigate avian influenza viruses in domestic ducks in Taiwan and possible roles of domestic ducks in transmission chain and cross-species infection of avian influenza, virological and serological surveillance have been conducted on live bird markets and avian farms during 2002-2007. Six viruses of H4N6 subtype were isolated and identified from 3 sources whereas none of the other HA subtypes were found. In total, 16 viral isolates included those 6 H4N6 viruses and the other 10 H4 viruses isolated from migratory and domestic ducks during the period of 1998 to 2006, which kindly provided from the Animal Health Research Institute, Council of Agriculture and the Graduate Institute of Epidemiology College of Public Health, National Taiwan University, were subject to genome sequencing. After sequence comparisons, the divergence of 0.4-14.5% was observed among all 8 individual corresponding genes of H4 viruses from different species and isolation dates. The results revealed further that H4N6 subtype viruses in Taiwanese poultry are genetically diverse and migratory duck might be the one of sources of H4N6 viruses isolated from domestic ducks. HA sequence comparisons showed that only one basic amino acid residues was found around proteolytic cleavage site in HA. It suggested that all 16 H4 isolates were low or non- pathogenic. The deletion of 8 amino acid residues over a region of aa 52-72 in deduced NA sequence was found in A/Duck/Changhua/0648/06 (H4N6), compared to the remaining Taiwanese H4N6 viruses as well. The impact of truncated NA on virus is unclear and awaited for further analyses. Phylogenetic relationships based on all 8 individual corresponding genes among the 16 H4 viruses and reference viruses have indicated that all Taiwanese H4 isolates were distributed into 4 small clusters in Eurasian lineages. The domestic and migratory duck H4 viruses isolated from the same year were grouped together, distingnished separated from the other isolates from different years. It implies that at least 4 invasions of H4N6 viruses from migratory ducks to domestic ones were independently occurred in different years in Taiwan and migratory duck viruses might have not perpetuated and disseminated in the infected domestic duck population. To investigate the prevalence of H4 virus in Taiwanese poultry, serum from several species of domestic poultry collected during 2002-2007 were subjected to hemagglutination-inhibition assays using A/DK/Changhua/5911/2006 (H4N6). Low seroprevalences were observed as the following: 0.38% in chicken (7/1826) over 7.4% of chicken flocks (6/81), 0% in quail (0/96), 0% in duck (0/857), 0% in goose (0/266), respectively. These results showed that H4 subtype virus is not prevalent in poultry in Taiwan. Conclusively, even though Taiwanese H4N6 viruses are quite diverse with each others, all are in Eurasian lineages regardless of their species origins. Furthermore, the finding from this investigation implies that H4N6 viruses might transmit from migratory birds to domestic poultry in Taiwan but might not be disseminated stably and efficiently in new hosts after cross-species infections.目錄 誌謝------------------------------------------------------------------------------------------------I 中文摘要-----------------------------------------------------------------------------------------II 英文摘要----------------------------------------------------------------------------------------Ⅲ 目錄----------------------------------------------------------------------------------------------Ⅴ 圖目錄-------------------------------------------------------------------------------------------Ⅸ 表目錄----------------------------------------------------------------------------------------ⅩⅡ 附錄--------------------------------------------------------------------------------------------XⅢ 第一章 緒言-------------------------------------------------------------------------------------1 第二章 文獻回顧-------------------------------------------------------------------------------2 2-1 家禽流行性感冒病毒歷史背景---------------------------------------------------------2 2-1.1 歷史背景----------------------------------------------------------------------------------2 2-2 流行性感冒病毒特性 2-2.1病毒分類及構造--------------------------------------------------------------------------4 2-2.2流行性感冒病毒蛋白質結構及功能--------------------------------------------------5 2-2.2.1 血球凝集素-----------------------------------------------------------------------5 2-2.2.2 神經胺酸酶-----------------------------------------------------------------------6 2-2.2.3 核蛋白-----------------------------------------------------------------------------6 2-2.2.4 基質蛋白--------------------------------------------------------------------------6 2-2.2.5 非結構蛋白-----------------------------------------------------------------------7 2-2.2.6 聚合酶蛋白-----------------------------------------------------------------------8 2-2.3 流行性感冒病毒的複製----------------------------------------------------------------9 2-2.4 流行性感冒病毒化學特性------------------------------------------------------------10 2-3 流行性感冒病毒之感染宿主範圍與跨物種傳播-----------------------------------11 2-3.1 宿主範圍---------------------------------------------------------------------------------11 2-3.2 A型流行性感冒病毒的自然保毒宿主----------------------------------------------13 2-3.3 中間宿主扮演跨物種傳播的角色---------------------------------------------------14 2-3.4 家禽流行性感冒病毒H4亞型之流行病學----------------------------------------16 2-4 流行性感冒病毒抗原變異性、病原性及致病病害---------------------------------17 2-4.1 流行性感冒病毒抗原變異性---------------------------------------------------------17 2-4.2 流行性感冒病毒病原性---------------------------------------------------------------18 2-4.3 流行性感冒病毒傳播途徑及病徵---------------------------------------------------19 2-5 家禽流行性感冒病毒之實驗室診斷--------------------------------------------------20 2-5.1 病原分離---------------------------------------------------------------------------------20 2-5.1.1 雞胚胎蛋接種------------------------------------------------------------------21 2-5-1-2 細胞培養------------------------------------------------------------------------21 2-5.2 檢測病毒抗原---------------------------------------------------------------------------21 2-5.2.1 平板凝集法---------------------------------------------------------------------21 2-5.2.2 反轉錄聚合酶鏈反應---------------------------------------------------------22 2-5.2.3 RealcTime Reverse Transcriptase Polymerase Chain Reaction-----------22 2-5.3 血清學檢測抗體-----------------------------------------------------------------------22 2-5.3.1 瓊膠免疫擴散法---------------------------------------------------------------23 2-5.3.2 酵素結合免疫吸附法---------------------------------------------------------23 2-5.3.3 血球凝集抑制試驗------------------------------------------------------------23 2-5.3.4 神經胺酸抑抑制試驗---------------------------------------------------------24 第三章 材料與方法---------------------------------------------------------------------------25 3-1 H4亞型血清學調查----------------------------------------------------------------------25 3-1.1 樣本來源---------------------------------------------------------------------------------25 3-1.1.1 雞---------------------------------------------------------------------------------25 3-1.1.2 鵪鶉------------------------------------------------------------------------------25 3-1.1.3 鴨---------------------------------------------------------------------------------25 3-1.1.4 鵝---------------------------------------------------------------------------------25 3-1.1.5 豬---------------------------------------------------------------------------------25 3-1.2 採樣方法--------------------------------------------------------------------------------25 3-1.3. 血清樣本抗體檢測--------------------------------------------------------------------25 3-1.3.1 製備紅血球溶液---------------------------------------------------------------25 3-1.3.2 血清處理------------------------------------------------------------------------26 3-1.3.3 紅血球凝集試驗---------------------------------------------------------------26 3-1.3.4 H4亞型血球凝集抑制試驗---------------------------------------------------27 3-2. 家禽流行性感冒病毒之病毒分離及核酸序列分析-------------------------------27 3-2.1 病毒分離之檢體來源------------------------------------------------------------------27 3-2.2 病毒核酸序列分析之檢體來源------------------------------------------------------27 3-2.3 檢體採樣方法---------------------------------------------------------------------------27 3-2.4 檢體的運輸、儲存與處理------------------------------------------------------------28 3-2.5 病毒增殖---------------------------------------------------------------------------------28 3-2.6 病毒RNA之萃取-----------------------------------------------------------------------29 3-2.7 反轉錄反應------------------------------------------------------------------------------30 3-2.8 聚合酶鏈反應---------------------------------------------------------------------------30 3-2.9 增幅PB2、PB1、PA、HA、NP、NA、MP、NS八段基因全長------------31 3-2.10 瓊膠醣凝膠電泳----------------------------------------------------------------------33 3-2.11聚合酶鏈反應產物之純化-----------------------------------------------------------33 3-2.12 聚合酶鏈反應產物之選殖----------------------------------------------------------34 3-2.13 Colony PCR-----------------------------------------------------------------------------34 3-2.14 核酸序列之定序----------------------------------------------------------------------35 3-2.15 病毒核酸序列之分析--------------------------------------------------------------- 36 第四章 結果------------------------------------------------------------------------------------37 4-1 H4亞型血清學調查----------------------------------------------------------------------37 4-1.1 雞------------------------------------------------------------------------------------------37 4-1.2 日本鵪鶉---------------------------------------------------------------------------------37 4-1.3 鴨------------------------------------------------------------------------------------------37 4-1.4 鵝------------------------------------------------------------------------------------------37 4-1.5 豬------------------------------------------------------------------------------------------37 4-2 以反轉錄聚合酶鏈反應增幅八段基因全長之結果--------------------------------38 4-2.1 增幅H4基因全長----------------------------------------------------------------------38 4-2.2 增幅N6基因全長----------------------------------------------------------------------38 4-2.3增幅Matrix基因全長------------------------------------------------------------------38 4-2.4增幅PA、PB1、PB2基因全長-------------------------------------------------------39 4-2.5增幅NP及NS基因全長---------------------------------------------------------------39 4-3 家禽流行性感冒病毒核酸序列之分析-----------------------------------------------40 4-3.1 H4亞型核酸序列分析及胺基酸序列分析-----------------------------------------40 4-3.2 N6亞型核酸序列分析及胺基酸序列分析-----------------------------------------41 4-3.3 Matrix gene亞型核酸序列分析及胺基酸序列分析------------------------------42 4-3.4 Polymerase (PA) 亞型核酸序列分析及胺基酸序列分析------------------------42 4-3.5 Polymerase (PB1) 亞型核酸序列分析及胺基酸序列分析----------------------43 4-3.6 Polymerase (PB2) 亞型核酸序列分析及胺基酸序列分析----------------------44 4-3.7NP亞型核酸序列分析及胺基酸序列分析------------------------------------------45 4-3.8 NS亞型核酸序列分析及胺基酸序列分析-----------------------------------------46 第五章 討論------------------------------------------------------------------------------------47 5-1 H4亞型血清學調查----------------------------------------------------------------------47 5-2 H4N6病毒核酸序列分析及胺基酸序列分析----------------------------------------50 第六章 參考文獻------------------------------------------------------------------------------79 附錄----------------------------------------------------------------------------------------------90 圖錄 Figure 1. Structure of an influenza A virus. http://www.biografix.de --------------------4 Figure 2. Krug, RM, Lamb, RA Orthomyxoviridae: The Viruses and Their Replication 2001 Fields Virology. 4th edition, editors: Knipe DM, Howley PM,. Philadelphia: Lippincott Williams & Wilkins. ISBN: 0-7817-1832-5 Orthomyxoviridae: The Viruses and Their Replication.----------------------10 Figure 3. The H4-1 of AIV were ampified from H4-8f and H4-777r by RT-PCR.---53 Figure 4. The H4-2 of AIV were ampified from H4-759f and H4-1704r by RT-PCR. ----------------------------------------------------------------------------------------53 Figure 5. The N6-1 of AIV were ampified from N6-1f and N6-2r by RT-PCR.------54 Figure 6. The N6-2 of AIV were ampified from N6-2f and N6-2r by RT-PCR.------54 Figure 7. The PA1 of AIV were ampified from PA1-1f and PA1-1r by RT-PCR.---55 Figure 8. The PA2 of AIV were ampified from PA2-1f and PA2-1r by RT-PCR.----55 Figure 9. The PB1-1 of AIV were ampified from PB1-1f and PB1-1r by RT-PCR.--56 Figure 10. The PB1-2 of AIV were ampified from PB1-2f and PB1-2r by RT-PCR. ---------------------------------------------------------------------------------------56 Figure 11. The PB2-1 of AIV were ampified from PB2-1f and PB2-1r by RT-PCR. ----------------------------------------------------------------------------------------57 Figure 12. The PB2-2 of AIV were ampified from PB2-2f and PB2-2r by RT-PCR. --------------------------------------------------------------------------------------- 57 Figure 13. The NP-1 and NP-2 of AIV were ampified from NP-1f、NP-1r and NP-2f、NP-2r by RT-PCR.-------------------------------------------------------58 Figure 14. The NS of AIV were ampified from NS-1f、NS-1r by RT-PCR.----------58 Figure 15. The MP of AIV were ampified from Bm-M-1 and Bm-M-1027r by RT-PCR.---------------------------------------------------------------------------59 Figure 16. The showing hemagglutination (HA) with chicken red blood cells.-------59 Figure 17. A. Percentage identity and divergence of the nucleotide sequences of HA genes..-----------------------------------------------------------------------------60 Figure 17. B. Percentage identity and divergence of amino acid sequences of H4 HA genes.----------------------------------------------------------------------------60 Figure 18. Phylogenetic tree based on the nucleotide sequences of the H4 HA genes by MEGA version 3.0. ------------------------------------------------61 Figure 19. A. Nucleotide sequences identity (%) of N6 NA genes.---------------------62 Figure 19. B. Amino acid sequences identity (%) of N6 NA genes. --------------------62 Figure 20. Phylogenetic tree based on the nucleotide sequences of the N6 NA genes by MEGA version 3.0.-------------------------------------------------63 Figure 21. A. Nucleotide sequences identity (%) of the MP genes of H4N6.----------64 Figure 21. B. Amino acid identity (%) of the MP genes of H4N6.----------------------64 Figure 22. Phylogenetic tree based on the nucleotide sequences of the MP genes by MEGA version3.0--------------------------------------------------------------65 Figure 23. A. Nucleotide sequences identity (%) of the PA genes of H4N6.-----------66 Figure 23. B. Amino acid identity (%) of the PA genes of H4N6.-----------------------66 Figure 24. Phylogenetic tree based on the nucleotide sequences of the PA genes by MEGA version 3.0.------------------------------------------------------------67 Figure 25. A. Nucleotide sequences identity (%) of the PB1 genes of H4N6.---------68 Figure 25. B. Amino acid sequences identity (%) of the PB1 genes of H4N6.--------68 Figure 26. Phylogenetic tree based on the complete nucleotide sequences of the PB1 genes by MEGA version 3.0.-------------------------------------------69 Figure 27. A. Nucleotide sequences identity (%) of the PB2 genes of H4N6.---------70 Figure 27. B. Amino acid sequences identity (%) of the PB2 genes of H4N6.--------70 Figure 28. Phylogenetic tree based on the complete nucleotide sequences of the PB2 genes by MEGA version 3.0.-------------------------------------------71 Figure 29. A. Nucleotide sequences identity (%) of the NP genes of H4N6.----------72 Figure 29. B. Amino acid sequences identity (%) of the NP genes of H4N6.---------72 Figure 30. Phylogenetic tree based on the nucleotide sequences of the NP genes by MEGA version 3.0.------------------------------------------------------------73 Figure 31. A. Nucleotide sequences identity (%) of the NS genes of H4N6.----------74 Figure 31. B. Amino acid sequences identity (%) of the NS genes of H4N6.---------74 Figure 32. Phylogenetic tree based on the nucleotide sequences of the NS genes by MEGA version 3.0.------------------------------------------------------------75 表目錄 Table 1. Serological results showing HI seroprevalence in chicken in Taiwan.-------76 Table 2. Comparison of amino acid sequence of HA cleavage site of H4 form Taiwan. ------------------------------------------------------------------------------------------76 Table 3. Alignment of nucleotide sequences encoding the region of delection of the NA stalk of Taiwanese H4N6 viruses.---------------------------------------------77 Table 4. Alignment of amino acid sequences of the NA stalk of H4N6 viruses isolated from Taiwan.--------------------------------------------------------------------------78 Table 5. Range of nucleotide and amino acid percent identity among all reported influenza isolates form Taiwan domestic duck and wild duck.----------------79 Table 6. Comparison of A/DK/Changhua/0648/2006 and other influenza isolates of nucleotide and amino acid percent identity form Taiwan domestic duck and wild duck.-----------------------------------------------------------------------------80 附錄 附錄1. The serum samples of chickens in different regions of the Taiwan from May, 2006 to February, 2007.-------------------------------------------------------------90 附錄2. The serum samples of Japan quail in Changhua County from August, 2006. ------------------------------------------------------------------------------------------92 附錄3. The serum samples of duck in the central Taiwan from May, 2002 to December, 2002 and 2004-2006.---------------------------------------------------93 附錄4. The serum samples of meat-type goose in Changhua , central Taiwan from 2005-2006.----------------------------------------------------------------------------94 附錄5. All the samples were collected from poultry farms or pig farms in Changhua County from July, 2006 to September, 2006.------------------------------------95 附錄6. Summary of viral isolates .----------------------------------------------------------96 附錄7. Alignment of deduced amino acid sequences of H4 hemagglutinin.-----------97 附錄8. Alignment of deduced amino acid sequences of the NA stalk of H4N6.-----101 附錄9. Alignment of deduced amino acid sequences of the MP genes of H4N6.----104 附錄10. Alignment of deduced amino acid sequences of the PA genes of H4N6.---106 附錄11. Alignment of deduced amino acid sequences of the PB1 genes of H4N6.--109 附錄12. Alignment of deduced amino acid sequences of the PB2 genes of H4N6.--112 附錄13. Alignment of deduced amino acid sequences of the NP genes of H4N6.---115 附錄14. Alignment of deduced amino acid sequences of the NS genes of H4N6.---118en-US家禽流行性感冒病毒H4N6亞型跨越物種傳播血清學調查avian influenzaH4N6cross-species transmissionprevalencephylogeny.[SDGs]SDG3thesis