Characterization, Variation and Wild Bird Surveillance of Avian Influenza Viruses in Taiwan

本研究分為3部分，第1部分為野鳥禽流感病毒監測。為了監測高病原性家禽流行性感冒在野鳥帶毒情形，自1998年9月起在台灣進行了10年的野生鳥類禽流感帶毒監測。1998~2007年十年期間共監測29,287個樣本數，樣本來自台灣北、中、南、東及離島(金門和澎湖)各地區的溼地之野鴨、鷸鴴及其他鳥類。所有樣本以雞胚胎接種方法分離病毒，以血球凝集及反轉錄聚合酶鏈鎖反應方法鑑定禽流感病毒，分離出H5及H7亞型病毒則進行無特定病原雞隻之靜脈接種病原性指數 (IVPI) 及以血球凝集蛋白切割位胺基酸序列分析方法來鑑定病毒之病原性。結果所有監測樣本之毒帶毒盛行率為0.81% (237/ 29,287)，鳥種帶毒盛行率以野鴨1.1% (229/20,812) 為最高。年季節帶毒盛行率以9~12月1.06% (186/17,493) 為全年最高峰，該季為野鴨自北方遷徙來台渡冬之候鳥季前期，1~4月之季節樣本帶毒盛行率為0.51%，為野鴨棲息後陸續北返之候鳥季後期，5-8月非候鳥季，沒有分離到任何禽流感病毒。所有分離到的禽流感病毒株分屬於37種不同HA/NA亞型組合，最大亞型群為H4N6 (n=86)，其次為H3N8 (n=20)及H10N3 (n=16)。選擇28株H4病毒株進行HA基因之HA1核酸序列分析，其演化樹呈現分離株年份與演化有相關性。所有分離之H5和H7亞型病毒株之病原性分析結果皆為低病原性禽流感病毒株。本研究證實，臺灣候鳥低病原性禽流感帶毒普遍且亞型種類多，但未曾監測到H5N1 或其他高病原性禽流感病毒株，顯示自1998以來的十年期間，藉由候鳥帶毒傳播H5N1病毒入台的風險性低。第2部分進行2008年臺灣雞隻H5N2禽流感病毒之分離與特性分析。分析株於2008年10月分離自高雄某一養雞場內臨床外觀健康蛋雞的一個共泄腔拭子檢體，經分離鑑定為低病原性H5N2禽流感病毒株。分析發現病毒的血球凝集蛋白切割位具有4個鹼性氨基酸，該切位具有高病原性禽流感病毒的特徵性。分離株的靜脈接種病原指數為0.89，顯示該病毒的毒力正往高病原性演化的途中。監測分離株所在蛋雞場週邊半徑3公里146場2,916件檢體，監測結果病毒皆為陰性。該分離株的八段基因分析結果證實此病毒與2003年台灣株一樣為重組病毒株，其HA與NA基因片段屬於美洲基因群，其他基因片段屬於歐亞基因群。 為瞭解H5N2低病原性禽流感病毒毒力演化趨勢，第3部分以臺灣2003年12月首例分離之H5N2低病原性禽流感分離株v1209進行試驗，設計以14日齡雞胚胎連續繼代感染50代來探討病原性變化情形。結果在繼代至第10代時，試驗株在沒有胰蛋白酶的培養條件之下，對雞胚胎纖維芽細胞感染後形成病毒斑的現象由陰性轉變為陽性，且在日齡雛雞腦內感染試驗下，對小雞的之致病指數(ICPI)由原株的0.19，增強至1.64，證實病毒繼代至第10代對細胞及雛雞腦部感染的毒力明顯升高。檢測繼代株之雞隻靜脈接種致病指數(IVPI)，發現第40代及第50代的IVPI值分別為1.45及1.92，皆高於高病原性的判斷值(1.2)，計算感染造成的死亡率分別為70%及100%，只有第50代繼代株的感染死亡率高於世界動物衛生組織訂的HPAI判斷值。分析繼代株之血球凝集蛋白切割位胺基酸序列，發現在第20代及第30代的切割位鹼性胺基酸由原株的3個(-REKR-)增加為5個(-RRKKR-)，但分析第40代及第50代的切割位鹼性胺基酸則與原株相同。本試驗結果顯示影響H5N2禽流感病毒的病原性不完全是由HA0切位鹼性胺基酸數目的多寡所決定。This study is divided into three parts; the first is monitoring of AIV in migratory birds. The study has been performed in Taiwan since 1998. From 1998 to 2007, 29,287 samples were collected from wild ducks, shorebirds, and other wild birds in the four wetlands around Taiwan and two outside islets, Penghu and Kinmen. Virus isolation was performed for all collected samples by inoculating chicken embryos, and the AIV in the allantoic fluid was identified by reverse transcription polymerase chain reaction and hemagglutination. The pathogenicity of isolated AIV H5 and H7 subtypes was determined by intravenous pathogenicity index (IVPI) test in specific-pathogen-free chickens and hemagglutinin (HA) cleaveage sequencing. The AIV prevalence from those samples was 0.81% (237/29,287). The peak prevalence reached 1.06% (186/17,493) from September to December, in which migrating ducks came from the north; the prevalence during January-April was 0.51; however, no virus was isolated during May-August. The partial HA genes of 28 H4 AIVs were sequenced and analyzed. The phlylogenetic tree showed that a correlation existed between isolation years and evolutional distances. All of the H5 or H7 AIVs isolated were determined to be low pathogenic AIVs by IVPI test and the HA cleavage sequences.The second part is isolation and characterization of H5N2 influenza virus from a chicken in Taiwan in 2008. During the surveillance of avian influenza, an H5N2 influenza A virus was isolated from a cloacal swab sample of apparently healthy chicken in Taiwan in October 2008. It was found that the heamagglutinin (HA) of the virus had a pair of dibasic amino acid residues at the cleavage site, which is a marker of highly pathogenic avian influenza virus. Intravenous pathogenicity index of the isolate was 0.89, indicating that the virus was on the way of acquisition of high pathogenicity in chicken. Virus isolation was negative in 2,916 birds of 146 farms in the circle of 3-km radius around the farm where the virus was isolated. Genetic analysis of the eight segments of the isolate indicate that the isolated virus was a reassortant whose HA and neuraminidase gene segments belonged to the American lineage and the others to Eurasian lineage.The third part is in order to understand the virulence tendency of H5N2 low pathogenic avian influenza virus, the virus strain of v1209 isolated in Taiwan 2003 was used for this study. After serial passages in fourteen-day-old chicken embryonic eggs, the pathogenicity of different passages were compared with the original strain before passage. The plaque formed at the 10th passage but not at 5th passage. The intracerebral pathogenicity index (ICPI) in the 10th passage was 1.64, higher than that of the 5th passage of 0.71. The intravenous pathogenicity index (IVPI) of the 40th passage was 1.45, while the original strain was 0.0. All the tests indicated that the pathogenicity of v1209 strain was higher in high passage than in low passage after serial passages in 14-day-old chicken embryo. However, it is noteworthy that, the number of basic amino acid in haemagglutinin cleavage site in this test was not correlated to pathogenic change. The amino acid sequence of cleavage site was changed from -REKR- in original to -RRKKR-at the 20th passage and the 30th passage, and then changed back to -REKR- at the 40th and the 50th passages. It indicates that the number of basic amino acid in haemagglutinin cleavage site is not related completely with pathogenicity