探討2009年H1N1流感病毒株之基因體變異對病毒本身感染能力之影響

Abstract

流感病毒藉由飛沫傳播，經常造成急性嚴重的呼吸道及肺部疾病，尤其是A型流感病毒，其曾經造成數次世界性大流行如西元1918年西班牙流感及西元2009年新型豬流感。西元2009年H1N1新型豬流感為三重組病毒，其基因來源包含人、豬以及禽流感病毒，最初於美國及墨西哥爆發，而後流行至世界並造成約135萬人感染，28萬人死亡。在2009~2016年間，病毒仍有造成數波之流行並持續演化，其分支由最初之clade 1演進至clade11.4。實驗室最初利用病毒溶斑滴定法(Plaque assay)測定H1N1標準株A/California/07/2009之病毒價數時，發現此病毒株之溶斑尺寸大小不一，可明顯區分為大型(L)與小型(S)溶斑。而根據先前研究指出，病毒之致病性及基因之變異可能會影響其溶斑尺寸之大小，顯示在此病毒株中可能含有兩群不同之病毒，因此本篇論文想分析大小溶斑病毒之基因變異並希望了解這些突變位點是否會持續存在病毒株中並影響病毒之演化。 我們將不同尺寸之病毒溶斑純化(plaque purification)並培養數代，所得之病毒株稱為L clone及S clone。為了了解兩株病毒之感染與生長差異，將L及S clone感染MDCK細胞後，進行病毒結合細胞試驗、病毒進入細胞試驗、病毒之核酸複製能力及病毒複製生長能力，發現L clone病毒複製能力較高，但在病毒結合及進入細胞試驗的量皆少於S clone；而在分析病毒核酸複製能力之實驗時，S clone的vRNA、mRNA及cRNA生成量皆較低，顯示L clone具有較佳的複製效率。接著，分析病毒的八段基因序列後發現HA基因上的D114N、D144E及D239G較有可能改變血凝集素與唾液酸之結合力；而在NA基因上則發現了T226A之改變；另外，在NP基因上亦觀察到兩個突變位點：D53E及D101G，這些突變位點多位於蛋白質功能區，可能會影響病毒蛋白之功能。本研究發現於病毒HA、NP與NA基因的位點會影響病毒感染複製能力並導致溶斑型態改變。

Influenza viruses cause spread by droplets and could cause acute and severe respiratory and lung diseases, especially influenza A virus, which is known to cause several worldwide pandemics,which an 1918 pandemic flu and 2009 pandemic flu. The pandemic H1N1 virus of 2009 (2009 H1N1) was a triple-reassortant virus, which was migrated from with human , avian and swine flu. Initial 2009 pandemic outbreak was observed in the Unite State and Mexico, and then the viruses spread rapidly around the world. 2009 H1N1 has caused about 1.35 million infections and 280 thousand deaths in 2009. From 2009 to 2016, 2009 H1N1 still caused several waves of epidemics and continued to evolve. Its branch evolved from the original clade 1 to clade 11.4. Our laboratory has identified two virus variants with different plaque sizes, designated as L and S clones, from the standard strain A/California/07/2009 (Cal07). Since previous studies have demonstrated that different plaque sizes of influenza A virus might implicate different virulence and pathogenicity of viruses, we aimed to characterize the phenotypes of 2009 H1N1 L and S clones and to use reverse genetics to determine the amino acid substitutions which might contribute to the phenotypic differences. First, L and S clones were individually passaged and purified by plaque purification assay. The derived L and S clones were used for subsequent virus characterization and full genome construction. The virus growth kinetics was first determined in MDCK cells by collecting the cultured supernatants at 0, 3, 6, 12, 24, 48 and 72 hours post infection (hpi). The L clone exhibited faster replication kinetics, especially at 12 hpi. To determine whether the delayed growth kinetics is due to differential efficiency of viral RNA replication, the levels of vRNA, mRNA and cRNA were respectively determined. Compared to the S clone, a 2 fold increase of viral mRNA and cRNA and a 1000 fold increase of vRNA of the L clone was observed by real-time RT-PCR. These results indicate that the L clone is more efficient in viral replication than the S clone. Next, the eight genome fragments were individually PCR-amplified and sequenced. Among several mutations on HA gene, D114N, D144E and G239D are likely to change the binding ability to HA and sialic acid, T226A of NA and two variants (D53E and D101G) of NP were also identified to be unique for the L or the S clones. These mutation sites are located in protein functional regions, which may affect the function of viral proteins. Our study found that mutations on HA, NA and NP gene may affect the replication and plaque size of 2009 H1N1.