2005-2006年臺灣活禽市場分離鴨流感H5N2病毒特性研究

Abstract

人類感染禽流行性感冒 (簡稱流感) 病毒 (avian influenza virus, AIV)，常因為病毒具有跨宿主傳播的潛力，致世人恐於全球性大流行 (pandemic) 的健康威脅。水禽是流行性感冒病毒的主要自然宿主，曾提供往昔全球性大流行病毒株的前驅基因片段。有鑑於家鴨對於流感病毒演化的重要性，活禽市場例行監測所得的流感病毒可有助明瞭病毒演化及宿主趨向性 (host range) 的資訊。近年來，臺灣出現數次雞H5N2病毒疫情。並且，之前研究描述22株自活禽市場鴨群分離的H5N2病毒，八段病毒基因片段中具有不同的演化來源，這些病毒的基礎病毒學特性卻未曾被研究。因此，本研究的目標為：(1) 探討這些具有不同基因組合 (gene constellation) H5N2病毒其複製效率的差異；(2) 比較在哺乳類及禽類細胞中，具有不同表現型 (如；病毒溶斑大小) 的病毒間其生長特性的差異；及 (3) 探討影響病毒表現型差異的分子機轉。 做法是挑選2005至2006年間、代表不同基因組合 [在病毒鹼性聚合酶2 (PB2) 和神經氨酸酶 (NA) 蛋白的核酸基因序列差異 >5%] 的四株鴨流感病毒株 (DV30, DV192, DV413 , DV518) 做為研究對象。首先將此四株病毒感染犬腎細胞MDCK和雞胚纖維細胞DF1，藉由對病毒間質基因的病毒核醣核酸 (M-vRNA) 定量方法，測量被感染細胞其培養液中的核酸產量，觀察病毒的生長狀態。接著嘗試自病毒對於MDCK細胞的溶斑 (plaque) 大小特徵，區分其在哺乳類細胞的表現型；而純化具不同表現型的病毒株，使用50% 組織細胞感染劑量 (TCID50) 再次評估它們對於MDCK及DF1兩不同宿主細胞的動態生長特性。所有的病毒株均會進行基因定序，比較其胺基酸組成。最後，以人類肺癌細胞A549及前述兩細胞測量對於這些病毒株的細胞接合 (cell binding) 的結果，以得知病毒血球凝集素蛋白 (HA) 差異對於細胞接合的影響。並以微基因體實驗 (minigenome assay) 探討不同病毒株的聚合酶活性，研究是否因病毒聚合酶體 (polymerase complex) 胺基酸位點的差異影響病毒生長。 結果顯示此四株鴨H5N2病毒株的生長，在細胞培養液中的核醣核酸表現量相似。然而，DV518病毒株在犬腎細胞MDCK和雞胚纖維細胞DF1的感染，可以觀察到高於其他病毒株的核酸複製數 (copy number)。因DV518和DV413此兩病毒株僅具有三個胺基酸位點的差異，接著探討這些差異是否為影響病毒生長的決定因素。 為進一步了解DV413及DV518病毒株，自DV518病毒株感染的MDCK細胞產生的不同型態病毒溶斑中，純化大、小溶斑兩種不同表現型的病毒株 (518 S 及 518 L)。結果發現DV413較兩株518病毒株在MDCK細胞上產生較小的病毒溶斑。大溶斑的518 L株能在MDCK細胞達到最高的病毒力價，但兩株純化自DV518的病毒對DF1細胞產生相近的病毒量，而DV413則明顯較另兩株病毒為低。於無特定病源 (specific pathogen-free) 雞胚蛋的病毒感染實驗中，三株病毒株表現與DF1細胞有相同的趨勢。 以不同來源的細胞 (A549, MDCK, DF1) 進行細胞接合實驗，結果並沒有發現明顯的受器特異性 (receptor specificity) 特徵。但兩株自DV518純化且均具有HA蛋白170D位點變異的518 S與518 L病毒株，較DV413病毒株在MDCK細胞對於病毒核醣核酸的測量都有比較強的貼附 (核酸複製數的比較p<0.05)。此外，518 L較518 S 具有PB2 蛋白E73D，以及酸性聚合酶 (PA) 蛋白P224S的變異，僅在感染人類腎臟細胞293T後，產生明顯高於518 S的冷光訊號。此研究顯示病毒胺基酸可能存在有特定決定因子參與病毒其在哺乳類細胞複製，指出可能有宿主特異的潛在因子交互作用。 總結，本研究嘗試探討此三株自田野分離之鴨流感H5N2病毒胺基酸特定位點改變與病毒表現型的關係。實驗的結果和過去研究的佐助，顯示保存在鴨子的異質病毒族群中，具有能在哺乳類細胞中相對適應且複製更好的分子決定因素。未來有待藉由反基因技術 (reverse genetic)，確認這些特定胺基酸位點對於禽流感病毒進入細胞、病毒複製，以及其與宿主間交互作用的獨立影響。

Human infection with avian influenza viruses (AIV) has always raised global health concern of emerging novel inter-species transmissible virus that might have potential to cause pandemic. Waterfowls, as the predominant nature reservoir of AIV, harbor precursor genetic lineages contributing to viral evolution on past pandemic strains. Routine virological surveillance at live-bird markets (LBMs) can acquire essential information on viral evolution and host range through AIV isolates. In recent years, several outbreaks of H5N2 in chickens occurred in Taiwan. Additionally, previous study identified 22 duck H5N2 viruses from a LBM with different gene origins in eight segments, but their virological properties remained unclear. Therefore, the specific aims of this study were: (1) to investigate the replication efficiencies of those H5N2 isolates with differ ent gene constellations, (2) to compare the growth properties of H5N2 isolates with different viral phenotypes in both avian and mammalian cells, and (3) to elucidate the possible molecular factors contributing to such phenotypic variations. The four duck H5N2 influenza viruses, DV30, DV192, DV413 and DV518, isolated during 2005-2006 representing distinct gene constellations were selected for their phylogenetic differences in PB2 and NA genes (>5% nucleotide identities). The Madin-Darby Canine Kidney cell (MDCK) and chicken embryo fibroblast cell (DF1) were firstly infected with these viruses to monitor their replication by measuring the levels of viral RNA using real-time polymerase chain reaction (qPCR) targeting viral M gene. Second, viral phenotypes were characterized by the size of plaques in MDCK cells infected with various strains. Besides, the strains possessing different plaque morphologies were further plaque-purified, and 50% tissue culture infectious dose (TCID50) method was performed to evaluate the growth kinetics of viruses in MDCK and DF1 cells. Third, all of the viral strains plaque-purified were sequenced to compare the differences in amino acids. And subsequently, cell binding test with human lung carcinoma cell (A549), MDCK and DF1 cells were investigated. In addition, minigenome assays in 293T and DF1 cells were conducted to clarify possible roles of amino acid changes in viral polymerase complex proteins conferring differences in viral growths. Results from the growth properties of the four duck H5N2 viruses showed similar replication in M gene of vRNA; however, DV518 had relatively higher levels of the same transcripts in both MDCK and DF1 cell lines among all the four isolates. DV413, which had only three amino acids different from DV518, presented significant lower virus yields than DV518, implying possible underlying determinants of viral growth. To further investigate the residues between DV413 and DV518, two strains of DV518 were plaque-purified in MDCK cells, and their plaque morphologies together with that of DV413 in MDCK were characterized. Plaques generated by DV413 were smaller than both 518 L, which had large plaque size, and 518 S. Higher virus titers were observed in MDCK cells infected with 518 L strain, than those infected with either 518 S or DV413. However, both of these two strains of 518 showed similar growth kinetics in DF1 cells, whereas DV413 displayed significant lower replication efficiency. Furthermore, viral yield after in ovo inoculation in SPF embryonated chicken eggs also correlated with the results in DF1 cells.

The results of binding assay to assess the receptor specificity of these H5N2 viruses presented no apparent pattern in mammalian and avian cells. However, DV518 isolates, possessing 170D in HA, showed enhanced binding to MDCK cells based on higher levels of attached viral gene (both 518 S v.s. DV413 and 518 L v.s. DV413, p<0.05). In addition, the 518 L, possessing the amino acid substitutions of E72D in PB2 and P224S in PA, demonstrated significant higher polymerase activities by luciferase signals in minigenome assay only in 293T cells but not in DF1 cells than those infected with 518 S virus. These results support that possible molecular determinants may be involved in differences in viral replication in mammalian cells, implying a potential marker associated with host-specific factors. In summary, this study tried to elucidate the relationship between amino acid changes and phenotypic variations among the three Taiwan duck influenza H5N2 viruses isolated in the field. Our data and supports from previous studies provides important insights on molecular determinants that may also be present in the heterogeneous populations of duck low pathogenic avian influenza viruses, which would be capable to adapt and to replicate better in mammalian cells. Future studies need to use reverse genetics to verify the role of each point mutation identified from this study affecting viral entry, replication, and interaction with host.