近十年來，腸病毒71型被認為是一個全球性的新興病毒。它是一種大小約為30奈米，屬於微小核糖核酸病毒科(Picornaviridae family)裡腸道病毒屬(enterovirus genus)的單股RNA病毒。腸病毒71的蛋白殼是一個由VP1、VP2、VP3以及VP4結構蛋白所組成的二十面體。腸病毒71是造成手足口疾病(hand-foot-and-mouth disease, HFMD)大爆發的元凶，可能會造成嚴重的中樞神經反應表現，像是遲緩性癱瘓、脊髓灰質炎、急性腦脊髓炎、急性腦幹腦炎和無菌性腦膜炎等等。這些病症也使得腸病毒71型有著高死亡率以及癒後長期性的神經後遺症，所以目前關於腸病毒71型的研究依然是大家所持續進行的研究。根據國家衛生研究院周彥宏老師實驗室在先前的生化實驗與動物實驗中，發現RD (Rhabdomyosarcoma)細胞產出之病毒相較於Vero細胞產出的病毒在噬菌斑測定與小鼠病徵反應上有著顯著的差異；另外根據定序的結果我們也發現到這兩個細胞產出的病毒在VP1蛋白的區域有胺基酸變異產生。因此我們想以低溫穿透式電子顯微鏡結合單粒子三維結構重組技術去解析這兩種來自不同細胞株的腸病毒71有何表徵上的差異。由我們解析出的結構結果顯示，它們兩者在5-對稱軸處有著明顯的差異，RD產出的病毒在5-對稱軸是個開口結構，然而從Vero產生的病毒是個突起結構．為了大大地提升樣品的數量以增加結構解析度，我們在銅網的碳膜鋪上蛋白Ａ(protein A)及與病毒專一性高的抗體來抓取更多的病毒顆粒。結果顯示此免疫親和銅網 (antibody-based affinity grid) 可以成功地抓取更多的病毒顆粒．但解析出來的結構解析度不如預期，可能的原因是所使用的抗體造成形態上的變化，以致樣品異值性高，解析度無法提高。未來若要成功使用免疫親和銅網來增加樣品數，專一性高，但卻不會造成形態上變化的抗體是首選，此外，使用高加速電壓及高階影像照相系統都是增加結構解析度的關鍵。最後，這個研究的發現對於腸病毒腸71型的研究提供了另一個思路。

The statistical report showed that Enterovirus 71 (EV71) was identified as an emerging virus worldwide in recent decade years. Enterovirus 71 is a ~30 nm single-stranded RNA virus from the enterovirus genus in the Picornaviridae family. Enterovirus capsid is an icosahedron, which is made up by VP1, VP2, VP3 and VP4 structural proteins. The infection of EV71 usually causes outbreaks of hand-foot-and-mouth disease (HFMD). EV71 can lead to severe neurological manifestations, such as flaccid paralysis, poliomyelitis, acute encephalomyelitis, acute brainstem encephalitis and aseptic meningitis. Those maladies bring on high mortality rates or long-term neurological sequelae in survivors. Therefore, the studies about EV71 is still popular. The previous studies from our cooperative laboratory –Dr. Chow in NHRI in Taiwan showed that the viruses produced by Rhabdomyosarcoma (RD) cell has different plaque result and symptom reaction in mice comparing to ones produced in Vero. Sequencing result showed that there were several amino acid differences in VP1 region. Here, we would like to characterize the RD-derived EV71 (EV-R) and Vero-derived EV71 (EV-V) particles by cryo transmission electron microscopy (cryo-TEM) and three-dimensional reconstruction. The cryo-TEM structure showed that the structural differences were located at 5-fold axis, there was an opening structure in the 5-fold axis of EV-R structure while a bump structure in EV-V. In order to elevate the concentration and purity of specimen, antibody-based affinity grid approach was used. Affinity grid indeed successful improved the number of particles, however the structural resolution was nor improved. The possible reason is that the antibody used in our affinity grid caused the conformational changes of the virus. To successfully implement the affinity grid for structural improvement, choosing the right antibody which will not cause the conformational change will be critical. Besides, using TEM with higher accelerating voltage and Direct Detection Device (DDD) will make high resolution structures possible. These findings of the mutant EV71 here may provide a new direction for study EV71 research.

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