腸病毒71型 (Enterovirus 71；EV71) 在分類上是屬於小RNA病毒科 (Picornaviridae)，不含外套膜的正股RNA病毒，它可造成手足口症，嚴重者可併發神經系統病變，如：無菌性腦膜炎、腦幹腦炎等，或肺部發炎浸潤，甚至造成死亡。而在過去文獻中指出，當細胞受到病毒感染時，細胞死亡扮演著一個調節發炎反應的角色。此外，活化的T淋巴球可分泌出一些細胞激素，例如：Fas/ Fas ligand (FasL), TNFR/TNF-α，藉由引起細胞凋亡的訊息傳遞路徑而造成細胞或器官的傷害；而此研究第一部分利用腸病毒71型感染周邊血液單核球細胞，探討其所分泌出來的sFasL或TNF-α在肺臟或神經細胞死亡所扮演之角色。而實驗結果發現，腸病毒71型感染單核球細胞後72小時及96小時間sFasL及TNF-α濃度都會持續增加，但是sFasL所分泌的濃度比起TNF-α而言並不足以去引起神經細胞 (SK-N-SH cell) 死亡。此外，利用了TNF-α之中和抗體發現，神經細胞死亡率有減少的現象。另外，以NF-κB抑制劑作用於周邊血液單核球細胞後顯示其會抑制腸病毒71型感染細胞分泌細胞激素TNF-α。此結果顯示腸病毒71型感染周邊血液單核球所分泌之TNF-α是經由NF-κB之訊息傳遞路徑，此可能進而造成細胞或器官之傷害。

　　而第二部分進行的是腸病毒71型基因重組 (Genetic recombination)之研究，此現象經常發生在正股RNA病毒，之前的研究學者拿non-polio enteroviruses (NPEV) 分析發現到在自然傳染途徑中血清型 (serotype)間的重組是經常發生的現象且重組也在腸病毒演化過程佔了一個重要的角色。本研究想去探討腸病毒71型演化史中，基因重組所扮演之角色及其與病毒致病性之關聯。我們分析腸病毒71型基因體中的部分片段，包含5'-UTR, VP4VP2, VP1, 2B和3D區域。我們分析了1998年之前及之後的30株腸病毒71型臨床株，並利用PHYLIP 程式的Parsimony, Maximum likelihood及Neighbor-joining method等三種方法來繪製演化樹 (phylogenetic tree)。最後以Neighbor-joining method來呈現結果。由演化樹的結果得知30株檢體中共有四株檢體 (N0003-TW-05, S0584-TW-04, N3340-TW-02及236-TW-86) 可能發生了突變或基因重組，其中兩株為1986年所分離病毒株 (236-TW-86及252-TW-86)，其在5'-UTR區域由演化樹結果發現236-TW-86是屬於腸病毒71型C基因型然而在252-TW-86中5'-UTR區域則是屬於腸病毒71型B基因型，但是在其他區域均屬於腸病毒71型基因型B，此結果可能顯示1998年大流行前後病毒株最大的差異可能是位於5'-UTR之位置；此外，亦發現三株重組病毒株，首先發現的是一株2002年病毒株 (N3340-TW-02) 其發現基因重組的位置在2B及3D間，在2B區域屬於腸病毒71型基因型B，其餘區域則屬於腸病毒71型基因型C。而其他兩株2004及2005年病毒株 (N0003-TW-05, S0584-TW-04) 亦是相同情形。更進一步，我們想要知道發生重組的位置是位於何處? 所以將N3340-TW-02的全長序列定序出來，利用SimPlot程式分析，發現發生重組的位置可能是在3000~3500及5500~6000核苷酸之間。

　　此外，藉由病毒溶斑法 (plaque assay) 、生長曲線 (one-step growth curve) 及39.5℃溫度耐受性 (temperature-resistant) 等方法來分析重組病毒 (recombinated EV71) 之生物特性，結果發現重組病毒均屬於溫度耐受性；在生長曲線分析中，發現N3340-TW-02其複製 (replication) 速度比起其他未重組病毒還快。以上結果顯示基因重組可能改變了病毒之生物特性，與病毒致病性之關聯則待進一步探討。

　Enterovirus 71 (EV71), a non-enveloped, positive strand RNA virus belongs to Picornaviridae family. EV71 is a frequent cause of epidemics of hand-foot-and-mouth disease (HFMD) in young children, and can cause severe brainstem encephalitis (BE) and pulmonary edema (PE) with high fatality rates. Cell death plays a central role in modulation of the inflammatory response during viral infection. In addition, activated T lymphocytes can release cytokines which result in apoptotic signaling pathways such as the Fas/ Fas ligand (FasL), TNFR/TNF-αlead to of cell death. The first part of study was designed to investigate of the cell death induced by the release FasL or TNF-αfrom the peripheral blood polymorphonuclear cells (PBMC) upon EV71 infection. The results showed that the concentrations of sFasL and TNF-α increased continouslly during 72 and 96 hours postinfection in EV71-infected PBMC by ELISA. However, the concentration of sFasL is not enough to cause cell death when compared with concentrations of TNF-α. In addition, adding neutralization antibody against TNF-α showed the reduction of cell death. Furthermore, inhibition of TNF-αproductionwas observed when inhibitor of NF-κB was pretreated with PBMC. The showing indicated that EV71-induced TNF-α production via NF-κB pathway which may resulted in cell death and tissue damage.

　In the second part, genetic recombination of EV71 was examined. genetic recombination is a common feature among positive-strand RNA viruses. Analysis of non-polio enteroviruses (NPEV) prototype strains has suggested that interserotypic recombination is a frequent event during natural transmission and that it may play a significant role in enterovirus evolution and virulence. To examine the role of genetic recombination in the evolution of the EV71, the partial sequences of EV71, including the 5'-UTR, VP4VP2, VP1, 2B and 3D regions, of EV71 were examined. Thirty EV71 clinical isolates before and after 1998 epidemic were compared with the homologous sequences from all other enterovirus by phylogenetic analysis using PHYLIP Neighbor-joining method.

　Four isolates (N0003-TW-05, S0584-TW-04, N3340-TW-02 and 236-TW-86) among 30 clinical isolates were identified to have the evidence of genetic mutation or recombination. Among VP4VP2, VP1, 2B and 3D sequences of two isolates from 1986 (236-TW-86 and 252-TW-86), they all belonged to genotype B. However, the 5'-UTR region of isolates 236-TW-86 belong to EV71 genotype C, indicating genetic recombination between genotype B and C in 5'-UTR region. In addition, three isolates from 2002 (N3340-TW-02), 2004 (S0584-TW-04) and 2005 (N0003-TW-05), respectively, were identified to have genetic recombination between 2B and 3D region. Sequence of 2B fragment of these isolates belonged to genotype B. However, the sequence of 5'-UTR, VP4VP2, VP1 and 3D belonged to genotype C. Using SimPlot program to check full-length sequence, the localization of recombination was identified between 3000~3500 and 5500~6000 in N3340-TW-02 isolate.

　Moreover, the virological properties of recombinated EV71 was examined, including plaque assay, one-step growth curve and temperature-resistant assay. In 39.5℃ temperature-resistant assay, the data showed that they all belong to temperature-resistant. Furthermore, N3340-TW-02 showed faster growth than non-recombinated EV71. Taken together, these results indicated that genetic recombination phenomenon may have alter their virological and virulence properties.

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