台灣地區跨越宿主傳播的人流行性感冒病毒偵測與分子及血清流行病學探究

• Main Authors: 廖宜真, Liao, Yi-Jen
• Other Authors: 金傳春, 臺灣大學：流行病學研究所
• Format: Thesis
• Language: Chinese; English
• Published: 2004
• Subjects: 跨越宿主傳播; 分子與血清流行病學偵測; A型流行性感冒病毒; Molecular and Serological Surveillance; Interspecies Transmission; Influenza A Viruses; Main Island; Avian flu
• Online Access: http://ntur.lib.ntu.edu.tw/handle/246246/56223; http://ntur.lib.ntu.edu.tw/bitstream/246246/56223/1/ntu-93-R91842010-1.pdf

1997年至今香港、中國大陸、荷蘭、越南與泰國等地陸續發生新型禽流感病毒(H5N1、H9N2、H7N7)跨越宿主直接感染人及死亡病例，流行病學調查發現多數個案均有動物接觸史，血清流行病學研究也證實家禽工作者和醫護工作人員為感染H5N1的高危險群。台灣及金門接近香港與中國大陸，兩岸人民往來密切，加上有豬、禽的飼養與侯鳥遷徙過冬的生態條件，本研究於2002年12月至2004年3月間在台北、桃園、宜蘭、彰化、台南、屏東及金門六縣市建立「整合性」的人流感病毒偵測點，擬自分子及血清流行病學兩層次分別探討住家附近有飼養動物、發病前曾接觸動物或疫區旅遊史之患者、家畜獸醫相關工作者以及偵測到新型動物流感病毒(如H1N2、H3N1、H5、H7)時，此高危險人群是否較易感染動物流感病毒H5、H6及H7；另一方面以金門作為台灣地區偵測大陸流感病毒的前哨站，並探討流感病毒的基因組成變異與演化上之關係。 2002至2004年的兩個流感流行季中，共分離到31株人流感病毒。由HA的基因組成與演化樹分析，發現自2002年底始所分離到的A型流感病毒株均屬類福建株[A/Fujian/411/02-like(H3N2)]，所幸八段基因全落在「人流感病毒」的支群，也沒有與禽類相同的唾液酸受體結合位(sialic acid receptor binding sites)之胺基酸；此外一名住家附近有飼養雞且父母親為畜牧工作者(ID＃618)、一名住家附近有雞、豬、鴿的飼養場(ID＃712)和另一名有養鳥且住家附近有養雞的患者(ID＃704)；HA基因各有Asn31Asp、Ser45Asn和Gly479Glu三處的突變，發現其與豬/禽流感病毒的氨基酸一致，且僅有一名發病前曾接觸過豬隻的29歲患者(ID＃639)其NA基因Arg210Met的突變，是與禽類N2亞型流感病毒相同，出現。其他內部基因的氨基酸突變卻沒有與動物流感病毒的一致關係。 比較兩流行季間的流感病毒演化情形，發現共有九處的氨基酸置換有統計的顯著差異，包括HA基因靠近抗體結合位的Gln156His (7/13 vs. 17/17, p=0.0009)、Ser193Asn (1/13 vs. 9/17, p=0.048)與其他兩處Ser189Asn (0/13 vs. 4/17, p=0.027)、Asn547Asp (0/10 vs. 6/13, p=0.006)；NA基因的抗體結合位Glu199Lys (0/9 vs. 6/17, p=0.025)與其他三處Val216Gly、Thr265Ile、Ile307Val (0/11 vs. 4/17, p=0.049)。HA基因Ser227Pro的置換是由金門的流感病毒株在第一個流行季先發生變異，進而在第二個流行季中成為台灣本島流感病毒主要的氨基酸，另外以第二個流行季中金門與台灣病毒株比對，發現HA1的三處胺基酸在金門已先有所突變：[Leu157Ser、Tyr159Phe及Val309Ile (0/12 vs. 2/3, p=0.01)]，或可作為未來前瞻追蹤2005年台灣本島流感病毒株是否有類似的氨基酸趨變，以及早做防疫準備。 2003年2~3月針對59位動物防治所員工、90位家禽工作者、46位彰化與金門豬農以及24位金門安養院老人，進行共219人的血清流行病學偵測，結果並未檢測出有抗台灣禽流感病毒[A/Duck/Taipei/ A30/2002(H5N2)、A/Duck/Tainan/A45/2003(H7N7)與A/Chicken/Taiwan/ 3153/2004(H6N1)]三種亞型之中和抗體存在。 經此分子與血清流行病學兩研究結果，尚未發現台灣地區有人感染H5、H6與H7三型動物流感病毒，但是人流感病毒仍有流行季間以及台灣、金門兩地的地理之變異，尤其是住家附近有動物飼養者的流感病毒有特殊胺基酸差異。未來及早建立完善的人/禽/豬流感病毒「整合性」偵測系統與流感病毒基因庫，對於我國防範新型流感病毒的入侵與研究不同宿主的流感病毒在生態上的微觀與巨觀演化趨變必有助益。 The emergence of novel influenza viruses poses a pandemic public health threat. Since 1997, new avian influenza A viruses such as H5N1, H9N2 and H7N7 influenza viruses emerged in Hong Kong, China, Netherlands and Hong Kong, China, Vietnam and Thailand, resulting in human fatal cases and most of these cases had contact histories of avian. Seroepidemiological studies reveal that poultry workers and health care workers are the high-risk populations. Since Taiwan and Kinmen have high densities of swine/domestic avian farms, habitats of migrating birds and tremendous numbers of travelers going to mainland China and Hong Kong, integrated human influenza virological surveillance systems had been established in six areas (Taipei, Yilan, Taoyuan, Changhua, Tainan, Pingting County and Kinmen islet) from Dec. 2002 to Mar. 2004. The specific aims of this study are: (1) to investigate the potential avian-to-human transmission by evaluating molecular changes in human influenza A viruses in those high risk populations who live near the poultry or farms with animal flu epidemics or novel influenza viruses isolated (H1N2, H3N1, H5 and H7), and who have contacts with swine or avian or travel histories to China/Hong Kong before the onset of illness, (2) to evaluate their serological responses to H5, H6 and H7 avian influenza viruses, and (3) to monitor micro-evolutionary changes of those human influenza isolates obtained from Taiwan main island versus Kinmen islet. All of 31 influenza A viruses in total isolated from two flu seasons from Dec. 2002 to Mar. 2004 were A/Fujian/411/02-like (H3N2) by phylogenetic and amino acid analysis of HA gene and the sialic receptor binding sites of HA did not have any avian flu specific amino acid signatures either. All other 7 genes also belong to the lineage of human influenza viruses. Three patients, including: (l) ID#618 lived near the chicken farm and her parents are poultry workers, (2) ID#712 lived close to the chicken, pig and pigeon farms, and (3) ID#704 raised birds and lived near the chicken farms, had specific mutation sites at Asn31Asp, Ser45Asn and Gly479Glu in the HA1, respectively and their amino acids at these three sites were similar to avian and swine flu H3 viruses. In NA gene, Arg210Met, which was found in one patient ID#639 who had contact with pigs before illness, was also identical to avian N2 subtype. No such relationships in amino acids between human flu isolates and animal flu viruses were observed among the rest of 6 internal genes. In comparing human influenza isolates obtained between two flu seasons, 9 amino acids substitution in HA and NA genes were found significantly different, including: (1) two sites near the antibody combining sites of HA [Gln156His (7/13 vs. 17/17, p=0.0009) and Ser193Asn (1/13 vs. 9/17, p=0.048)], (2) another two sits of HA [Ser189Asn (0/13 vs. 4/17, p=0.027) and Asn547Asp (0/10 vs. 6/13, p=0.006)], (3) one of the antibody epitopes of NA [Glu199Lys (0/9 vs. 6/17, p=0.025)], (4) other 3 mutated residues of NA [Val216Gly, Thr265Ile and Ile307Val (0/11 vs. 4/17, p=0.049)], and (5) Ser227Pro in HA gene was initially observed only in Kinmen isolates in 2002~2003 flu season, but such a Pro then became as a dominant amino acid in Taiwan’s 2003~2004 flu isolates. Additionally, three mutated amino acids in HA1, including Leu157Ser, Tyr159Phe and Val309Ile, which were found in 2/3 Kinmen isolates during 2003~2004 (0/12 vs. 2/3, p=0.01), can be served as a basis to monitor possible changes of 2005’s flu isolates in Taiwan island. Seroepidemiological study on 219 high risk populations, including 59 animal health research institute workers, 90 poultry workers, 46 swine workers, and 24 elderly in Kinmen found that all of them were seronegative against three subtypes of Taiwan’s avian flu viruses [A/Duck/Taipei/A30/02 (H5N2), A/Duck/Tainan/A45/03 (H7N7) and A/Chicken/Taiwan/3153/04 (H6N1)] by micro-neutralization test. In conclusion, our molecular and serological epidemiology studies have not had any evidences to support avian to human transmission of novel influenza viruses in Taiwan area. Furthermore, human influenza viruses H3N2 had temporal changes in HA and NA genes between two seasons and spatial differences in Taiwan vs. Kinmen. Four flu patients living near animal farms showed specific amino acid signatures of HA and NA identical to animal influenza A viruses. Future efforts will monitor the microevolution and macroevolution of influenza viruses in human, avian and swine obtained in the same and different areas over years for better understandings in the mechanisms that emerging novel human flu viruses might have public health significance. 目 錄 致謝 i 中文摘要 iii 英文摘要 vi 目錄 ix 表目錄 xiii 圖目錄 xiv 第一章 緒言 1 第二章 文獻探討 2 第一節 A型流行性感冒病毒特徵 2 一、病毒結構與分類 2 二、病毒複製 4 三、抗原變異 5 四、生態與流行病學相關的重要宿主 6 第二節 流行性感冒病毒「跨越宿主傳播」之歷史回顧 11 一、二十世紀的三次世界大流行 12 二、近年禽流感病毒感染人群事件 14 (一) H5N1亞型 14 (二) H9N2亞型 16 (三) H7N7亞型 17 三、人類流行性感冒病毒高危險群之血清流行病學探究 18 (一) 偵測禽流感病毒抗體的方法 19 (二) 家禽工作者 19 (三) 醫護人員 20 第三節 人類流行性感冒病毒H3N2亞型之演化 20 一、 病毒的遺傳演化 21 二、 氨基酸置換的探討 21 第四節 台灣地區A型流行性感冒病毒現況 22 一、人流行性感冒 23 二、豬流行性感冒 24 三、禽流行性感冒 25 第三章 研究目的與假說 29 第四章 材料與方法 31 第一節 研究地區 31 第二節 研究對象 31 第三節 問卷設計 33 第四節 實驗診斷 33 第五節 結果分析與統計方法 45 第五章 結果 .47 第一節 2002-2004年台灣地區流行性感冒病毒感染者之描述 流行病學分析 47 一、流行趨勢 47 二、人口學變項 48 第二節 流行性感冒病毒跨越宿主傳播之探討 49 一、接種SPF雞蛋的結果 49 二、演化樹分析 50 三、胺基酸分析 52 第三節 流感病毒在2002年到2004年間的演化 55 一、HA基因 55 二、NA基因 56 三、由2004年的金門流感病毒株來預測2005年的台灣流感 病毒之基因變異 56 第四節 高危險群之血清流行病學探究 56 一、 人口學與職業暴露史資料 57 二、 禽流感病毒血清抗體偵測 59 第六章 討論 60 第一節 動物流感病毒跨越宿主傳播的因素與佐證 61 第二節 本研究至今並無發現人類感染動物流感病毒之佐證 65 第三節 接觸動物患者的流感病毒分子層次之影響 68 第四節 研究限制 69 第五節 未來研究方向 70 第六節 建議事項 71 參考文獻 74 表 81 圖 95 附錄 106 本研究發表於各研討會之摘要 113