台灣家禽流行性感冒病毒H4亞型血清學調查 與病毒核酸序列分析

Abstract

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.