探討嚴重急性呼吸道症候群冠狀病毒於宿主體內之基因序列變異

Abstract

2002年11月在中國廣東省出現一種具有高度傳染性和高致死率的新興感染症，旋即造成全球性的大流行，WHO將其命名為嚴重急性呼吸道症候群（SARS），鑑定後確認病原體是一種新型的冠狀病毒，即SARS冠狀病毒（SARS-CoV）。SARS冠狀病毒為RNA病毒，而RNA病毒的特性之一為具有類種（quasispecies）的特性。這是因為RNA病毒用來複製基因體的反轉錄酶或RNA 複製酶缺乏校對的功能。雖然文獻上已有許多SARS冠狀病毒基因序列之報告，但是絕大多數都是比較不同SARS冠狀病毒株之間的差異。對於SARS冠狀病毒在單一個體中序列變異之程度及其類種的情形尚不明瞭。 本研究第一部分在於探討2003年台灣地區SARS病人漱口水檢體中SARS冠狀病毒之變異程度，分析包括 S（spike）基因、N（nucleocapsid）基因、E（envelope）基因及ORF1b基因部分之序列。利用RT-PCR, PCR和clonal sequencing的方式分析每個檢體中各基因不同克隆的核苷酸序列，並利用電腦軟體計算平均歧異度（mean diversity）及兩兩比對之差異比例（p-distance）以代表各基因之變異程度；對胺基酸序列亦做相同之計算。此外亦計算有表型核酸取代之比例（proportion of nonsynonymous differences，dN）和無表型核酸取代之比例（proportion of synonymous differences，dS）。實驗結果顯示SARS冠狀病毒在病人體內的確以類種的形式存在。另外本研究所得之平均歧異度和先前研究HIV-1及HCV感染之急性期在人體內之變異程度相當，也和登革病毒在人體內之變異程度差不多。同時我們亦考慮了clonal sequencing過程中可能產生的錯誤率，此錯誤率低於我們所觀察到的平均歧異度。因此本研究的結果應該可以反映病人體內的SARS冠狀病毒基因之變異，並不能全部歸因於實驗誤差。從本研究大多數基因dN/dS的結果顯示，SARS-CoV在宿主內的序列變異承受某種負向選擇壓力。由SARS冠狀病毒各基因序列之分析也發現因為核苷酸突變或缺失或增加而產生終止密碼子的情形，顯示有缺陷性病毒的存在。 第二部分則是分析兩位病人在不同時間點之漱口水檢體，探討SARS冠狀病毒在人體內變異程度隨時間之變化，包括分析S基因、N基因、E基因及ORF1b基因部分序列。利用RT-PCR, PCR, clonal sequencing和電腦軟體進行分析，結果顯示SARS冠狀病毒在體內各基因部分片段之變異程度隨感染之進行而有變化，大多數基因的變異程度會隨著時間呈現下降或維持穩定的趨勢，這可能與病毒在體內之逐漸清除有關。由本研究之實驗結果顯示SARS冠狀病毒在體內是以類種的方式存在，各基因均有一些變異；這些變異雖然不大，但可能仍是未來發展疫苗或各種抗病毒藥物的時候所必須考量的因素。

In November 2002, a new infectious disease with relative high transmissibility and mortality rate emerged in Guangdong, China. Later the disease caused a worldwide outbreak. WHO named the novel disease as severe acute respiratory syndrome (SARS). A novel coronavirus, the SARS-associated coronavirus (SARS-CoV), was identified as the etiological agent of SARS. SARS-CoV is a RNA virus, which is known to exist as quasispecies.This is due to the nonproofreading nature of reverse transcriptase and RNA polymerase of RNA virus. While sequence variation of different SARS-CoV isolates have been reported by several groups, little is known about the extent of sequence variation of SARS-CoV in individual patient. In the first part of this study, we investigated the extent of intrahost sequence variation of SARS-CoV in throat wash samples from confirmed SARS patients during the 2003 outbreak in Taiwan. We used RT-PCR, PCR and clonal sequencing of fragments of the spike (S) gene, envelope gene (E), nucleocapsid gene (N) and ORF1b gene. We then calculated the mean diversity and p-distance to determine the extent of nucleotide and amino acid sequence variation. We also calculated the proportion of nonsynonymous differences (dN), the proportion of synonymous differences (dS), and dN/dS ratio. We found that SARS coronaviruses exist as quasispecies in vivo. The extent of intrahost sequence variation of SARS-CoV is in the same range as those reported for dengue virus and acute stage of HIV-1 and HCV infection. We also did our own control to determine the error rates due to PCR and clonal sequencing, which were found to be lower than the observed mean diversity in this study. Thus the sequence variation observed in this study can not be totally attributed to in vitro artifacts and may reflect the actual sequence variation of SARS-CoV in vivo. The dN/dS ratios of most genes in this study suggest that intrahost sequence variation of SARS-CoV is under certain negative selection pressure.We also found stop codons resulting from deletion or insertion, suggesting the presence of defective viruses. In the second part of this study, we analyzed sequential throat wash samples from two patients. To study whether the extent of sequence variations changes with disease progression, we used a similar strategy including RT-PCR, PCR and clonal sequencing to examine sequence variation of part of S, E, N and ORF1b genes over time. There is a trend of decrease or stabilization in the extent of sequence variation in most genes examined.This may be due to viral clearance in vivo. Our findings of the quasispecies of SARS-CoV in vivo, though the extent of sequence variation is low, suggest that future drug and vaccine development for SARS-CoV should also take this into consideration.