台灣克沙奇病毒B2分子流行病學研究

Molecular epidemiology of Coxsackievirus B2 in Taiwan

黃怡蒨(Yi-Chien Huang)

指導教授 ： 彭健芳

高雄醫學大學/健康科學院/生物醫學檢驗學研究所/碩士(2013年)

摘要

台灣因地處亞熱帶，全年都能有腸病毒病例出現，其中克沙奇B 型病毒會造成心肌炎、腦膜炎和嬰幼兒嚴重感染，尤其是克沙奇B2 (Coxsackievirus B2；CV-B2) 引發急性肝炎及致命腦膜腦炎。CV-B2 在台灣1999 和2006 年造成兩次流行。病毒外殼蛋白VP1 為序列與血清型有非常密切的關聯，3D 為RNA 聚合酶具高度保守的核酸序列；因此VP1序列目前被廣泛運用來做為種系分析， 3D序列可用於鑑定病毒是否有重組的依據。本研究使用84株 (324nt) 之CV-B2病毒 (分離自1947-2011年) 並以CV-B1、B3-B6 原型株為外群 (Out group) 總共89株病毒，分析其VP1 與3D 核酸序列，藉此了解CV-B2 流行病學、時空分佈及其分子特徵。以jMODELTEST 使用赤池準則選出最適合的突變取代模式為GTR+I+G ；使用Neighbor-Joining (N-J)、Maximum-Likelihood (M-L)、MrBayes與Monte Carlo Markov Chain(MCMC)四種程式分析序列。結果顯示VP1可分成三群分別GI、GII及GIII，GI 為1988-1989 年間台灣及美國病毒株，GII分佈於歐美、西非與中國 (1979-2010) ，GIII分布於中國、南韓與台灣 (1987-2011) ，台灣1989年病毒株屬於GI，1991年後皆為GIII。3D區分成C-I~C-VI，C-I~C-III相似於VP1的GI-GIII，除了AY37310 (VP1分布於GII，3D分布於C-I) 、AY373102與AY373104 (VP1分布於GII，3D自成一群) 。根據VP1 序列分析每年CV-B2 的VP1與3D進化速率分別為4.35× 10-3 S / S /Y與4.81×10-3 S / S /Y；並驗算得到B2的TMRCA 出現在1917 年。台灣CV-B2的病患年齡範圍主要在1-5歲 (58.8%) ，男女比例為1.75:1 (男性為63.6%，女性為36.3%) 。綜合結果顯示CV-B2之分群與地域分佈相關，台灣病毒株1990年以屬於GI，1990後轉變成GIII；根據3D與VP1樹型與氨基酸變異點可發現CV-B2在1976-1977與外群有重組現象；值得注意的為本實驗GIII群中臺灣臨床分離株序列與南韓的水源與臨床之分離株類似，因此環境與致病病毒之間的關連是值得密切注與進一步探討的。

關鍵字

系統發育分析 ； 克沙奇B2 ； TMRCA

並列摘要

Geographily, Taiwan is located in a subtropical zone, where Enterovirus clinical cases appear all around the year. Among the enterovirus infectious diseases, Coxsackievirus B2 (CV-B2) serotype causes myocarditis, meningitis, and severe infections in infants. In particular, CV-B2 contributes to acute hepatitis and fatal meningitis. Two large-scale outbreaks of CV-B2 have been occurred in Taiwan in 1999 and 2006, respectively. The viral capsid VP1 region is the target site associated with sequencing and serotyping for Enterovirus. The three-dimensional (3 D) region of RNA polymerase contains the consensus sequence.This study was to investigate the relationship between volutionary dynamics and genetic history of CV-B2. Global VP1 sequence in 84 strains of CV-B2 (324 nt) and 3D region of 34 strains of CV-B2 (456 nt) collected for 6 decades in 1947-2011 were obtained and subjected to comprehensive evolutionary analysis using a suite of phylogenetic and population genetic methods. In order to estimate the divergence times, the closely related ancestors of CV-B1and B3-B6 were used as out group.The jModelTest is a valuable tool to carry out statistical selection of nucleotide substitution according to the Akaike information criterion (AIC) to obtain the optimal model selection of GTR+I+G method. The methods of phylogenetic analysis are including the Neighbor-Joining (N-J), Maximum-Likelihood (M-L), MrBayes and Monte Carlo Markov Chain (MCMC).The VP1 sequence can be divided into three genogroups of GI-GIII. The GI group occurred in 1947-1989 distributed between Taiwan and America.In contrast, GII group occurred in 1979-2010 was distributed in America, Europe, West- Africa and China. The GIII group emerged in Taiwan, China and Southern Korea in 1987-2011. The 3D region can be divided into four clusters of C-I~C-IV, and cluster C-I ~ C-III is similar to that the VP1 sequence of group GI-GIII in all CV-B2 strains tested, except of AY37310 (VP1 distributing in the group GII, 3D distributing in the cluster C-I), AY373102 and AY373104 (VP1 distributed in group GII, and 3D becoming an new out group). According to the VP1 sequence analysis of the annual CV-B2, VP1 and 3D evolution rate were 4.35 × 10-3 Substitution / Site / Year and 4.81 ×10-3 Substitution / Site / Year , respectively. Therefore, the time to most recent common ancestor (TMRCA) of CV-B2 appeared in 1917. In Taiwan, the age of patient of CV-B2 infection was ranged in 1-5 years old (58.8%). The sex ratio of male to female was 1.75:1 (63.6% in men, 36.3% in women). Our data showed that original CV-B2 belonged to group GI (in 1990 or earier) and then gradually shifted into group GIII (1999-2011) in Taiwan. Based on phylogenetic analysis and point mutation in amino acid of 3D and VP1, CV-B2 became stable steadily and has recombination associating with the out groups in 1976-1977. Of note, the group GIII occurring in our clinical virus strain were similar to that of water environmental sources and clinical strains isolated in southern Korea. Accordingly, the association between environmental sources and pathogenic virus is valuable to notify and to investigate further.

並列關鍵字

phylogenetic analysis ； Coxsackievirus B2 ； TMRCA

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