腸病毒71型屬於小RNA病毒，含一條長約7.4 kb的正股RNA。自1969年於美國加州首次分離，台灣於1998年爆發大流行，造成78名死亡病例，自此腸病毒71型約3至5年就會出現流行，流行基因型在C型與B型之間變化，主要是因為病毒抗原性的改變而造成流行。而2008年及2012年欲發現重複出現的基因亞型，B5基因型造成大流行，但此二年B5基因型之病毒抗原性上並無太大差異，因此本研究進一步探討除了抗原性外，2008年與2012年腸病毒71型B5基因型的胺基酸變異是否造成病毒特性改變。首先，我們發現在兩年病毒間具有18個胺基酸變異點，其中有3個位在結構蛋白部分，這部分變異並沒有影響病毒抗原性；而其他變異位點皆位在非結構蛋白部分。為了研究這些突變位點造成的病毒特性變異，我們先利用臨床病毒株進行病毒生長速率分析，將病毒感染細胞後，在不同時間點收取病毒液，並利用病毒溶斑試驗進行病毒濃度測定。結果發現2012年病毒的生長速率較2008年快。同時也進行溫度耐受性測試，顯示2008年臨床病毒株比2012年較具有溫度耐受性。而從文獻中得知，3D蛋白部分可影響病毒複製與溫度耐受性，因此我們再進一步利用感染性克隆平台，將2008年與2012年病毒的3D蛋白基因部分與腸病毒71型骨架N7008TW99 (EV71 backbone) 的3D部分進行置換，構成重組病毒，再利用重組病毒進行病毒特性之研究。先利用生長速率分析，發現在病毒快速複製時期，帶有2012年3D部分的重組病毒具有較高的病毒濃度；由於病毒RNA是由3D聚合酶合成而來，因此，接下來利用Real-time PCR方法定量病毒RNA量。結果發現2012年病毒RNA量上也有較高之量。而在溫度耐受性實驗中，帶有2008年3D的重組病毒則和2012年相同，均變成嚴重溫度敏感性病毒，因此可得知不僅只有3D區域能調控病毒對於溫度的耐受性，尚有其他區域會影響此病毒特性。從上述結果中得知，非結構蛋白中3D區域變異會影響病毒RNA的複製與病毒顆粒的生成。另外，由2008年與2012年3D區域胺基酸序列分析發現，其中在第383個位點和第396個位點有胺基酸的差異，因此推測主要調控的位點可能是這兩個位點。同時，利用外源性胍適應性試驗(Guanidine resistance assay)測試病毒株的保真性(fidelity)，可以看到在第383與396位點具有相同胺基酸序列的3D置換病毒：r3D654TW12與r3D1745TW08，對胍具有敏感性，且在胍耐受比率上比其他兩株明顯偏低，表示這兩株重組病毒的保真性相較其他較高，這樣的結果告訴我們第383與396個位點對病毒複製具有一定調控能力。另外，由本研究中挑選的四株病毒3D區域胺基酸序列分析發現，其中在第383個位點和第396個位點有胺基酸的差異，因此推測主要調控的位點可能是這兩個位點。接著，我再利用單點突變技術(site-directed mutagenesis)與感染性克隆系統做出具有單一位點突變的病毒株，同樣利用多步生長曲線觀察病毒複製速度，發現這七個位點對於病毒複製有一定的影響；然而在外源性胍耐受性試驗的結果，推測可能這兩個位點需要同時存在變異才能對於病毒保真性有影響。同時我也利用適應性試驗(Fitness assay)觀察，帶有單一位點突變的病毒與未帶有突變病毒在宿主細胞中複製速度是否有差異，而結果仍然無法完全確定何者為主要調控因子。最後，經過蛋白質結構對照，可以發現此二位點皆位在3D聚合酶的thumb位置，而相關研究中指出thumb位置和聚合酶與引子的結合有相關，因此可以推測這兩個位點影響病毒複製是藉由與VPg結合的變化而造成病毒複製速度改變，而使病毒毒力改變。本研究的結果將有助於了解3D複製酶基因變異對腸病毒71型病毒複製的影響。

Entervirus 71 is a major pathogen causing hand-foot-and-mouth disease or herpangina, but occasionally causes central nervous system demages. From 1998 to 2013, EV71 caused outbreaks every 3-5 years in Taiwan because of antigenic changes, but in both 2008 and 2012 years were caused by genotype B5. To examine why the same genotype EV71 could cause two large outbreaks, we examined the isolates from National Cheng Kung University Hospital to compare the full-length sequences. There were 16 amino substitutions in the non-structural region between isolates from 2008 and 2012. We compared two B5 viruses of each from 2008 and 2012, and found 2012 EV71 had higher growth kinetics and 2008 were temperature resistant. For further investigation, we constructed 2008 and 2012 3D reverse genetic (rg) virus by using N7008TW99 backbone and six single mutation viruses in 3D region. 2012 3D rg viruses had higher replication rate than 2008 3D rg viruses which was similar to native viruses. We found r3D654TW12 and r3D1745TW08 had higher fidelity by guanidine resistance assay. In sequence comparison, we found sites 383 and 396 in 3D region might play important role for EV71 replication. Furthermore, six single mutant viruses showed higher replication rate than rM202TW12 backbone virus, and low fidelity. In fitness assay, we found no difference between 2008 and 2012 substitutions. These findings suggested that the position at 383 and 396 in 3D region may be important for viral polymerase activity and replication. The results will help to understand the mechanism of EV71 replication and virulence.

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