台灣克沙奇病毒之分子流行病學與中樞神經感染之分子影像分析診斷

Coxsackievirus B in Taiwan：Molecular Epidemiology and Magnetic Resonance Imaging infection in Central Nervous System

李侑真(Yu-Chen Li)

指導教授 ： 彭健芳

高雄醫學大學/健康科學院/醫學檢驗生物技術學研究所/碩士(2009年)

摘要

科沙奇病毒B群（Coxsackievirus B, CB）屬於小RNA病毒屬，基因型為人類腸病毒B型，是一種沒有套膜直徑30nm的病毒，二十面體的結構蛋白中含有一正股RNA，包含了六個血清型CB1到CB6 。主要是經由飛沫、糞口及接觸等途徑感染，所造成的疾病包括：呼吸道症狀、手足口病（HFMD）、胰臟炎、胰島素依賴型糖尿病（IDDM）、心肌炎、肌炎、中樞神經系統感染、無菌性腦膜炎、腦膜炎、猛爆性肝炎、類似敗血症休克甚至死亡。文獻指出尤其是新生兒感染科沙奇病毒B群通常包含中樞神經系統感染和引起嚴重心肌炎或猛爆性肝炎。
CB3感染出現於亞洲（台灣、日本、中國、韓國），美洲（美國），歐洲（德國、法國），尤其最近於2005年在台灣發生大流行。分析1992~2005年高雄醫學大學附設醫院病毒室所分離出腸病毒臨床病毒株，結果得知在這20年間克沙奇病毒B3（CB3）曾造成1996（26.4％）、1999（42.1％）、2000（6.4％）及2005（88.2％）年造成四次大流行。1999年台灣疾病管制局成立病毒性感染症合約實驗室，其後幾年仍持續發生不同型的腸病毒流行。因此快速準確的鑑定與分型對腸病毒流行之掌握是刻不容緩的事。此外，多種有效的腸病毒單株抗體之開發對早期診斷是很重要的。
因此本研究共分兩部分，第一部分希望藉由調查台灣CB3分子流行病學，以了解國內C B3病毒株的基因型其流行動向；第二部分利用實驗室另外發展一株NPEV（non-polio enterovirus）單株抗體PY-CY（經免疫螢光染色證實可偵測多種腸病毒，包含腸病毒71型（EV71）、伊科病毒30型（E30）、克沙奇病毒B1~B6）結合超順磁氧化鐵奈米粒子Superparamagnetic iron oxide nanoparticles (SPIONs) 用於MRI檢查試劑，並配合病理切片觀察感染小鼠的中樞神經影像變化，作為評估此非侵襲性檢驗試劑在腸病毒感染早期診斷的效果。
第一部分自1992~2005年間隨機挑選高雄榮總與高雄醫學大學附設醫院病毒室所分離出之CB3臨床病毒株共有29株。先經免疫螢光染色確認，再定序利用5’VP1引子222，292經由PCR得到之290 bp 病毒VP1核苷酸產物片段。在基因庫搜尋出其他83株世界CB3分離株序列共112株，利用Kimura 2-parameter算出序列距離再以MEGA 4.0分析序列基因並以bootstrap 驗算1000次得演化樹狀圖。參考Pairwise comparison數值取18％基因變異程度加以分群將CB3病毒株分為I~V群。第I、II、III群為美洲和歐洲；第IV、V群為亞洲；而台灣株分佈於第IV和V群內。
第二部分動物實驗方法使用實驗動物Balb/c 4-6周齡母鼠，隨機分為各條件實驗組與正常對照組，均在相同條件下飼養。實驗組每隻腹腔注射400TCID 50 CB4病毒0.4 mL，正常對照組每隻腹腔注射生理鹽水0.4 mL。分別在感染後1、2、3、4周做MRI，在未施打CLIO-EDBE-PY-CY （20 m mol/kg）前先一次Prescreen,接下來施打0.1mL CLIO-EDBE-PY-CY（20 m mol/kg）後0、10、20、30、60分鐘時掃描，完畢後安樂死取腦部做病理切片。在結果方面，對照組未看到腦部的任何變化，實驗組中CB4感染小鼠一周時注射CLIO-EDBE-PY-CY，腦部MRI無任何變化；CB4感染小鼠後第二周時注射CLIO-EDBE-PY-CY，由於腸病毒感染腦部造成腦部血腦障壁（BBB）損傷，因此MRI顯影有看到CLIO-EDBE- PY-CY通過血腦障壁（BBB）進入腦部結合在病毒感染位置；CB4感染小鼠後第三周時注射CLIO-EDBE-PY-CY，腦部MRI顯影更為明顯；然而在CB4感染小鼠4周時注射CLIO-EDBE-PY-CY，腦部MRI並無任何變化，但是我們一到四周的腦部病理切片上都觀察不到任何淋巴球侵潤。

關鍵字

克沙奇病毒 ； 分子流行病學

並列摘要

The Coxsackievirus B (CB) are members of the Human enterovirus B (HEV B) species in the Enterovirus genus within the Picornaviridae family .CB are small non-enveloped virus (30 nm), their single-stranded plus-sense RNA genome is packaged in an icosahedral capsid protein. Transmission occurs through the faecal-oral or respiratory routes. The diseases range from respiratory symptom, hand-foot and mouth disease (HFMD), pancreatitis, insulin-dependent diabetes mellitus (IDDM), central nervous system infection, myocarditis, myositis, aseptic meningitis, meningitis, fulminant hepatitis, sepsis-like picture or death. Neonatal Coxsackievirus infections usually involve the central nervous system (CNS) and may cause serious complications of myocarditis or fulminant hepartitis.
Recently, CB3 activities have been reported in Asia (including: Taiwan, Japan and China, Korea), America (US) and Europe (Germany and France).
According to the Centers for Disease Control and Prevention (CDC) ?{Taiwan Surveillance System installed since 1999, CB3 became predominant 2005 outbreak. CB3 became predominant in 1996 (26.4％)、1999 (42.1％)、2000 (6.4％) and 2005 (88.2％)outbreaks. Enterovirus isolates in this study were obtained from 1992 to 2005. The data of Kaohsing Medical University Hospital showed that several outbreaks occurred in the past two decades.
It is important to understand the trend of epidemiology of enteroviruses for the control measures of our government. Besides, a powerful panel of enterovirus mAbs is critical for the early diagnosis.
To address thses issues, firstly, we revealed molecular the epidemiology of CB3 in the Secondly; we further developed a noninvasive imaging system based on the expression of a monoclonal antibody (mAb) on the cell surface to trap a clinical imaging probe, extent and persistence of Enterovirus in living animals. In this project, a Superparamagnetic iron oxide nanoparticles(SPIO) has approved by the FDA and utilized for imaging in patients will be chosen as imaging probes for detection. The monoclonal antibody (PY-CY) can detect most enterovirus, including Enterovirus 71 (EV71), Echovirus 30 (E30), and Coxsackievirus B1-6 by immunofluorescence assay.
The aim of our study was to test the Superparamagnetic iron oxide nanoparticles as a contrast agent capable of targeting HEV infection in the CNS by MRI.
Part I：To analyze the genotype of CB3 virus in Taiwan, 29 strains in this study were isolated from Kaohsiung Veterans General Hospital and Kaohsiung Medical University Hospital during 1992-2005. After confirmed by the immunity fluorescence technique, a fragment of 290 bp in 5' VP1 were amplified by primer set 222 and 292. Phylogenetic analysis was constracted together with 83 worldwide reference sequence in GenBank. Five genotype (assigned I~V group) were dicipted by by Molecular Evolutionary Genetics Analysis, Version 4.0 (MEGA 4) software. The data reveled that GI, GII and GIII were dominant in America and Europe, while GIV and GV were prevalent in Asia and Taiwan.
Part II：We used 4-week-old BALB/c mice were either sham inoculated (control mice) or were inoculated intraperitoneally with a single dose of the 04/2522 strain of HEV (400X TCID50, 400 μL). Balb/c mice were scanned by MRI at 1, 2, 3, 4 weeks post-infection. CLIO-EDBE-PY-CY (20 mmol/kg) was intravenously injected to the HEV infected mice or control mice. Whole brain images were acquired by a 3.0 T MR Scanner (Sigma; GE Medical Systems, Milwaukee, WI) using pulse sequence TR/TE ratio of 3000/90. In the MRI image, the contrast group and the CLIO were clear. Strong MRI signal was detected in the brains of HEV infected mice using CLIO-EDBE-PY-CY contrast agent 3 weeks post-infection. On the brain pathological section all the out-of-sight any lumphocyte.

並列關鍵字

Coxsackievirus ； Molecular Epidemiology

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