2005-2006年台灣活禽市場家禽流行性感冒病毒的分子流行病學

Abstract

1997年香港發生人類的高致病性H5N1禽流感疫情，突顯人禽交界面在流行病學的重要性。其中以活禽市場扮演的角色格外重要。此外，分子流行病學發現，由2005-2006年中國青海湖H5N1高致病性禽流感病毒株和其後在歐洲及非洲爆發的禽流感疫情之H5N1病毒株極為類似，間接證實侯鳥可能是此次在歐、非洲疫情散播病毒的兇手。台灣位處侯鳥遷徙路徑之中，也由於水禽是禽流感病毒的天然宿主敦促台灣不容忽視有得到外來禽流感病毒株的機會。有鑒於此，本研究於2005年9月至2006年10月，選擇人禽交界的北部一大活禽市場，監測其是否有低致病性H5型禽流感病毒的活動，並仔細分析所分離得的低致病性H5型禽流感病毒株的分子特性、隨時間的分子演化及其在公共衛生的防疫意義。 做法是以鴨糞便所分離的26支鴨類低致病性H5亞型禽流感病毒(包含禽流感病毒混合型別)作為研究重點。因此本研究兩大目標：（一）探討此26支低致病性H5亞型禽流感病毒株的分子流行病學及生物特性，及（二）探察混合多型的檢體中，其禽流感病毒株之分子特性是否和單一型別之病毒株有所差異。方法上，使用狗腎細胞[Madin Darby canine kidney (MDCK) cells]及病毒斑純化(plaque purification)，來分離混合多型的病毒株。擇選來自單一病毒斑的病毒株，再以反轉錄-連鎖聚合酶反應(reverse transcriptase- polymerase chain reaction, RT-PCR)得到11株低致病性H5亞型禽流感病毒株的八段之各段完整序列，並分析其與亞洲已知的高與低致病性H5分離株的核酸序列親緣樹關係、病毒致病力、和抗藥性相關的胺基酸位置及比對分析病毒和宿主細胞的結合位置的胺基酸。 禽流感病毒八段基因的核酸親緣樹分析，結果發現所有26支鴨類低致病性H5亞型禽流感病毒均非源於2003年流行於台灣的雞類低致病性H5N2禽流感病毒株(HA相似度為76.2%-79.4%；NA相似度為84.3%-85.5%)，且約有15.4% (4/26)H5病毒株的M基因可能和1999年台灣本土雞類的低致病性H6N1禽流感病毒株[A/chicken/ Taiwan/165/99 (H6N1)]共同演化(相似度為96.5%-99.3%)。又有11.6%(3/26)的H5病毒株之NS基因是較相似於2000年日本北海道鴨類H5N3病毒株[A/duck/Hokkaido/447/ 2000 (H5N3)](相似度為94.8%-99.8%)。不僅如此，研究中更發現在2006年10月(候鳥來台之際)，自台南鴨所分離到的一株H5病毒[A/duck/Taiwan/DV647/2006 (H5Ny)]，是和研究中其他的H5病毒株於NA之外的其他7段基因片段均較不相似(相似度為70.1%-93.2%)，顯示此病毒株可能從境外移入。 探察2005年9月至2006年10月26支鴨類低致病性禽流感病毒株之胺基酸，發現四大重要結果。(一)11株H5N2八段基因中，以HA基因的相似度為最小 (88.6%-98.9%)。而N2的基因的相似度為最大(94%-100%)。（二）於HA蛋白切割位的胺基酸序列顯示所有的分離株均為低致病性禽流感病毒(---R/)，但是在A/duck/Taiwan/DV647/2006 (H5Ny)有多於一個的鹼性胺基酸(REKR/)。檢視狗腎細胞是否會造成病毒與細胞結合位(receptor binding site, RBS)的變異時，發現單一型別的H5病毒及混合他型別的H5病毒株兩者於HA蛋白胺基酸表現模式(patterns)相一致，顯示狗腎細胞傳代一代尚未改變結合位的胺基酸。且HA蛋白RBS有10/14(71.4%)個胺基酸位置是和1997年自新加坡鴨類低致病性H5N3病毒株[A/duck/Singapore/Q-F119-3/1997(H5N3)]相同。另本研究的低致病性H5禽流感病毒株HA胺基酸序列和目前所知高致病性H5禽流感病毒株的結構已有很大的差異，未來台灣這些低致病性H5禽流感病毒的HA蛋白結構仍須多方突破。（三）於其他和致病能力相關的胺基酸(PB1-F2, PB2-627, NS1-92, NA Stalk)均沒有高致病能力，且依舊保有低致病性禽流感病毒特徵，又並沒有在與干擾素相關的NS1-92上產生變異。（四）均未產生對兩類抗病毒藥物-剋流感與金剛胺的於NA與M蛋白抗藥性相關胺基酸位置之改變。以上再再顯示本研究中的低致病性H5型禽流感病毒株的HA蛋白可能為台灣所特有，且多月的持續存在隱示其地方性流行之潛力。 綜觀此研究，台灣2005~06年自活禽市場健康鴨得的26支低致病性H5亞型禽流感病毒尚無健康威脅。同時發現許多台灣前所未聞的低致病性H5亞型禽流感病毒的特性，但是仍有許多未知尚待解答，包括：究竟不同的細胞培養是否會改變這些病毒的分子特性而形成病毒異變株群(quasi-species)，以及HA蛋白醣化作用對於抗體和病毒結合的影響。更重要的是，比較低致病性H5型禽流感病毒基因和胺基酸序列是否會隨著時間趨變以及此變化是否和高致病性H5亞型禽流感病毒有何異同處，又能否由低致病性H5禽流感病毒的分子演化脈絡以探察其轉變為高致病性H5禽流感病毒之可預測性及其分子機轉，將更有助於公共衛生的提早防治成效。

Interface areas between avian species and humans including live-bird markets (LBMs) had been documented their epidemiological importance since the highly pathogenic avian influenza (HPAI) virus H5N1 was isolated from human cases visited LBM in Hong Kong in 1997. The molecular epidemiology studies proved that the high similarity between the HPAI H5N1 isolates from Qinhai Lake in China and those from the subsequent spread to Europe and Africa during 2005-2006, suggesting the role of migratory birds. Taiwan, is located amid the flying route of migratory birds, thus elevates the possibility of imported avian influenza viruses. To avoid public health threat, we monitored the molecular changes of the 26 low pathogenic avian influenza (LPAI) H5 viruses isolated from healthy ducks through a virological surveillance system established in a large LBM in Northern Taiwan during Sept. 2005- Oct. 2006 and investigated their public health implications. The aims of the study are to understand the molecular epidemiology and biological characteristics of the 26 LPAI H5 viruses and to examine any molecular differences between single H5 subtype and mixed subtype AI viruses. To separate the mixed viruses, we used one generation plaque purification on [Madin Darby canine kidney (MDCK) cells] and employed reverse transcriptase-polymerase chain reaction (RT-PCR) from the single plaque to get the full-length sequences, particularly each of the eight segments of all the 11 H5N2 viruses. Phylogenic analysis of our LPAI H5 isolates compared with different HPAI and LPAI H5 viruses in Asia and characterization of amino acids (AAs) involved in receptor binding sites, pathogenesis related sites, and anti-viral drug resistant sites were further investigated. Phylogenic analysis showed that all the 26 duck LPAI H5 viruses were strikingly different from the 2003 Taiwan’s chicken LPAI H5N2 viruses (HA similarity:76.2%-79.4%; N2 similarity:84.3%-85.5%). Additionally, 15.4% (4/26) of the M gene of the studied duck LPAI H5 viruses might have the co-evolution with the 1999 Taiwan’s chicken H6N1 virus [A/chicken/Taiwan/165/99 (H6N1)] (similarity: 96.5%-99.3%). In NS gene, 11.6% (3/26) of our duck LPAI H5 viruses were grouped much closer to A/duck/Hokkaido/447/2000(H5N3) (similarity:94.8%-99.8%). Interestingly, A/duck/Taiwan/DV647/2006 (H5Ny) isolated from Tainan in October of 2006 when migrating birds flew into Taiwan, had its seven segments other than NA gene were very different from our other H5 isolates (similarity:70.1%-93.2%) implied that it might be an imported H5 virus. Examining the AAs of the 26 duck LPAI H5 viruses revealed four major findings. First, the HA gene showed the lowest similarity (88.6%-98.9%) while the N2 gene showed the greatest one (94%-100%) among the eight segments of the 11 H5N2 viruses. Second, all of them were LPAI viruses demonstrated by the AAs at the cleavage site of HA protein (---R/), however, A/duck/Taiwan/DV647/2006 (H5Ny) virus contained more than one basic aa “REKR/” at such a cleavage site. In evaluating the possible changes of the receptor binding site (RBS) of HA due to passage of the single plaque-derived AIV (PP-AIV) on unnatural host cells, we found that the patterns of AAs of single vs multiple subtyped LPAI H5 duck viruses were quite similar implying that one passage of the PP-AIV on MDCK cells was insufficient to change RBS. In addition, 71.4% (10/14) of these RBS had the same sites as LPAI A/duck/ Singapore/ Q-F119-3/1997 (H5N3) virus. Furthermore, the sequences of the antigenic epitopes of HA protein of our LPAI H5 isolates were very much different from those currently available HPAI H5 viruses; thus we can’t map the antigenic sites of our LPAI H5 viruses to the known structure of HPAI H5 viruses. Third, all the known AAs at the sties related to viral pathogenicity (PB1-F2, PB2-627, NS1-92, NA Stalk) maintained all the molecular traits of LPAI and no mutation occurred at NS1-92 for IFN. Lastly, the analysis of antiviral important AAs of NA and M2 proteins of our LPAI H5 isolates found that all of them were sensitive to the neuraminidase inhibitor, Tamiflu and adamantane derivatives, respectively. All above findings further supported that our LPAI H5 viruses are very unique to Taiwan and persistence of these LPAI H5 viruses over several months may imply the potential endemicity of these LPAI H5 viruses in certain parts of Taiwan. In summary, Taiwan’s 2005-2006 LPAI H5 viruses isolated from healthy ducks of one LBM had no public threat to humans and provided us the first time to learn biologic characteristics of LPAI H5 viruses. However, many questions still remained. Whether may the quasi-species of H5 be different in using less suitable cell lines to culture? How does the glycosylation of viral HA proteins affect receptor binding sites and their subsequent interactions with antibodies? Most importantly, would changes of LPAI H5 viral AAs and nucleotides have special patterns while time goes by? If it does, are their patterns helpful to understand the transition from LPAI to HPAI so that the trends towards to HPAI become more predictable and avoidable? We believe that thorough understanding the underlying mechanisms involved in stepwise micro-changes at each of the eight segments from LPAI to HPAI will definitely help to prevent future public health threat of pandemic influenza.