- 學位論文
克沙奇A24變異株之族群動態演化分析
Phylodynamic analysis of Coxsackievirus CA24v
摘要
急性出血性結膜炎(AHC)是一種接觸的高傳染性疾病，特徵是突發性的疼痛腫脹、畏光及結膜下出血。克沙奇病毒A24變異株（CA24v）是造成AHC致病之一。我們過去的分子流行病學報告將病毒株分基因型I到IV (GI-GIV），並指出病毒基因群演化是按照時間順序而演化發展，每一大流行階段都由新興的病毒株導致。為了瞭解近年來臺灣CA24v爆發族群動態演化（phylodynamics）。隨機挑選1985-2010年臨床分離的臺灣CA24v 18株，序列分析其VP1基因片段（804bp）和3D基因片段( 636bp ) ，將定序結果與基因庫41世界分離株包含外群(原型CA24和Poliovirus - P1、P2、P3)作多序列比對，接著進行了系統發育和族群動態演化分析。族群動態演化樹是使用鄰接法(Neighbor joining, NJ)、最大似然法(Maximum likelihood, ML)和蒙地卡羅馬可夫法 (Markov Chain Monte Carlo, MCMC) 進行構建，NJ和ML樹使用MEGA5.0軟體，MCMC方法使用MrBayes及Bayesian Evolutionary Analysis Sampling Trees (BEAST)軟體。族群動態演化分析的結果與我們過去的報告結果是一致的，所有的基因群都有高支持度（>75），各病毒基因群具有年代特徵，即不同基因群是來自不同年代分離。CA24v的原型株獨立成單一分群，本實驗沒有檢測出GII基因型序列（在1975年至1976年分離）。GIII群大部份分離至亞洲 (1985到1986年)和1987年為南美洲分離株。GIV群共分為4個 (0, 2-4) 基因亞群，GIV-C0為多明尼加哥和巴西株，GIV-C1可能因序列不夠長未被納入本研究進行分析，本研究新定序的1991年所有病毒株和一株分離於2008年的病毒株都屬於 GIV-C2，2007年印度株和2007到2008年中國株聚集為GIV-C3，2010年臺灣株和日本株聚集為一個新的亞群GIV-C4；本亞群分離年代橫跨11年及多個地理區域(如：中國，臺灣，韓國，日本和印度（亞洲），巴西和瓜德羅普島（南美洲），突尼斯和加蓬（非洲），西班牙（歐洲）; 和澳大利亞（大洋洲），其核苷酸和胺基酸相似度97%/100%。本篇研究進一步證明了我們過去的報告顯示每一波大流行即新出現一個基因群或基因亞群，這盛行的基因群將導致2-3 年的大流行，然後換下一個基因群導致下一波流行。由於病毒的演化持續進行，為了瞭解CA24v病毒株在臺灣的發展過程，值得進一步重建時空分佈過程及分子特徵分析。
並列摘要
Acute hemorrhagic conjunctivitis (AHC) is a highly contagious disease characterized by sudden subconjunctival hemorrhage resulting in pain, swelling, and photophobia. Coxsackievirus A24 variant (CA24v) is one pathogenic cause of AHC. Our previous reports depicted chronological trends in epidemics involving Genotypes 1-IV (GI-GIV). To understand epidemiological trends in recent CA24v outbreaks in Taiwan, a VP1-based phylogenetic analysis was performed to explore the evolution of CA24v and its epidemiological relationships. Eighteen strains of CA24v strains isolated in Taiwan during 1985-2010 were randomly selected for analysis. A VP1 gene fragment (804bp) and gene fragment (636bp) were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. Multiple sequence alignments were performed in another 41 sequences isolated worldwide. One additional prototype CA24 strain and Poliovirus-1, 2, 3 were also sampled as outgroup. The phylogenetic tree was constructed by performing neighbor-joining and maximum likelihood methods with MEGA5.0 software. The phylogenetic analysis results were consistent with previous reports. All viral strains clustered by isolation year had high bootstrap values (>75). Due to the insufficient number of VP1 virus sequences isolated before 1985, the prototype strain of CA24v was divided into a separate branch and designated Genotype I. No GII sequences (isolated during 1975-1976) were observed. Strains isolated in 1985-1986 were clustered into GIII. Interestingly, all strains isolated in 1991 and one strain isolated in 2008 were clustered into GIV-C2’. The 2010 strains were clustered into a new subcluster GIV-C4.This study further supports our previous report of clear chronological trends revealed by the CA24v dendrogram, i.e., one outbreak for each emerging subgenotype. Most CA24v outbreaks resulted from a newly emerging subcluster that circulated for 2-3 years before being replaced by the next emerging subcluster. For example, the new genetic subtype GIV-C4 caused the 2000-2010 outbreaks. In contrast, the 1991 isolates in this study were clustered into subgenotype GIV-C2, which was characterized by a wide range of isolation years (1984-2006) and a broad geographic region (e.g., China, Taiwan, Korea, Japan and India (Asia); Brazil and Guadeloupe (South America); Tunisia and Gabon (Africa); Spain (Europe); and Australia (Oceania)). Because of the wide geographic distribution of GIV-C2, this subset needs further molecular characterization and further study of its temporal and spatial distribution.