丙型肝炎病毒(HCV)株的全基因组序列测定: 完成HCV 基因型6 全套17 个亚型基因组的测序
文献类型：学位论文
| 作者 | 李春华 |
| 学位类别 | 博士 |
| 答辩日期 | 2007-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 季维智 |
| 关键词 | 丙型肝炎病毒 基因型 基因序列 地方性传播的 夫妻间传播 静脉吸毒 |
| 其他题名 | Whole genomes of hepatitis C virus (HCV): completing a full panel of genomes for HCV genotype 6 |
| 学位专业 | 动物学 |
| 中文摘要 | 丙型肝炎病毒（Hepatitis C virus, HCV）的全基因组序列测定，曾经由于许多方面的条件限制而难于完成。但是，其对于研究HCV分子病毒学、流行病学、进化和致病性却至关重要，特别是在临床应用中，不同序列的基因型决定α-干扰素治疗的不同效果。在本研究完成之前，HCV基因型6仅有6个亚型有其全基因组序列。因此，本研究的主要目的在于，测定HCV变异株代表基因型6其余的11个亚型和新亚型的全基因组序列，并深入分析。 本研究从样品分别来自于中国、泰国，和在美国及加拿大生活的东南亚国家移民的HCV感染者。因为样品有限，改良传统的PCR方法，摸索出“桥”和“岛”DNA全序列扩增法，从每例样品100μl血清或从100μl血清中获得的cDNA中测定了13个HCV全基因组核苷酸序列。 以来源于Genbank的已知基因型6的七个全长序列为参考对所测定的13个亚型全序列进行共同分析显示，这些全基因组核苷酸的两两比较相似率变化范围为71.9%--82.7%，著地， 这四对序列间的相同率高于标准定义的HCV基因亚型之间的范围值75%-80%。为了进一步理解和证实这些亚型间的遗传相似性，本研究还测定了代表这4对亚型的病毒原型株的全基因序列，结果显示了相同的核苷酸水平上的变异范围，这为HCV基因亚型的分类提供了新的认识，亦强调了全长序列对于分类的重要性。 从系统发育方面的分析证实，本研究所测得的13个分离株都属于基因型6。在系统发育树上，每个病毒株代表一个独立的枝。并形成了高度分化的HCV基因型6分枝，从而清楚显示，各亚型的独立分布。本研究至此完成了基因型6中17个亚型的全序列测定，而km41和gz52557因缺乏其临床上和流行病学上的多个感染病例的证实，而继续保留其亚型未命名状态。结合来源于Los Alamos HCV database的基因型6的已知部分序列的变异株进一步分析，发现各相近亚型变异株均来自东南亚或东南亚国家移民，这提示了这些HCV的相同感染源。 为了探讨HCV夫妻间传播的可能性，本研究还测定了来自于泰国的两位感染HCV的献血员及其感染HCV的配偶。这4个基因序列C-0044 和C-0046之间核苷酸相同率为98.1%，而C-0185和 C-0192之间为97.8%。文献研究感染HCV的夫妇间的部分亚基因序列的相同率为96.3%至100%，本研究结果与此范围相符，并第一次用全基因组序列提示了HCV在夫妻间传播的可能性。 本研究还测定了基因型6的另一个变异株的全基因组序列：HK6554，香港的某患者，与上文中的GX004一起，均为静脉吸毒者，并共同感染了HCV和HIV-1。分析结果还表明了一种趋势，即是在中国南方，基因亚型6e有从以前的地方性传播方式转为现有的流行性传播方式。这种转变可能由于静脉吸毒感染HCV的人群的网络传播而加快。 综上，本研究用传统PCR、简并引物结合链特异引物的方法有效地测定了共21个病毒株的全基因序列。该方法也可用于其它分子流行病学的研究，特别在测定珍贵的病毒序列然而样品量又受限时。本研究所测定的全基因组序列代表HCV中最古老、分化最多、地方性传播、又可能动物源性的基因型6的全套17个亚型。这有助于进一步理解HCV基因亚型的分类意义、更准确评价HCV的进化和起源，亦有助于发现HCV新的变异株和提高临床诊断、治疗，为将来HCV的流行及公众健康的预测、预防和疫苗的制备奠定了坚实的分子遗传学基础。 |
| 英文摘要 | Entirely sequencing the hepatitis C virus (HCV) genome has been a critical object of study but often difficult to attain for many reasons. However, this is a prerequisite for completely understanding of HCV molecular virology, epidemiology, evolution, and pathogenesis. Particularly, this is a major determinant associated with the outcome of -interferon therapy. When the recent HCV nomenclature was updated, only six subtypes of genotype 6 had their entire genome sequences clarified, while the other 11 subtypes remained to be fully described. The first purpose of this study was to entirely sequence isolates representing these 11 subtypes and two other variants that may qualify for two new subtypes. Six reference sequences representing subtypes of genotype 6 were retrieved from Genbank and co-analyzed with the obtained 13 full genomes. It showed pair-wise nucleotide similarities of 71.9% to 82.7% over the entire genome length. Notably, the nucleotide similarities among these four pairs of sequences were higher than the 75%-80% range that was previously defined to classify different HCV subtypes. To investigate further, four prototype isolates were also entirely sequenced. Consistently, analyses of the resulted full genome sequences gave a same range of nucleotide similarities, providing new information for HCV subtype classification. Phylogenetic analysis demonstrated that these 13 isolates were all classified into HCV genotype 6. Further analysis in conjunction with partial sequences from the Los Alamos HCV database led to the identification of many other closely related HCV isolates that were exclusively derived from south-eastern Asia or immigrants from that region. Common source of HCV infection was therefore strongly suggested. The second purpose of this study was to validate the hypothesis of interspousal HCV transmission. Three other HCV complete genomes from two infected Thai blood donors and their spouses were further entirely sequenced. The similarities firstly provided the complete genome information to support the interspousal HCV transmission. Two other HCV genotype 6 variants co-infection with HIV-1 from IDUs were also entirely sequenced. Analysis of the two sequences suggests that there is a trend for subtype 6e to change from a previous endemic pattern to a current epidemic pattern in southern China. This was likely promoted by network transmission of HCV among injection drug users. Conclusively, in this study a total of 21 new HCV variants were entirely sequenced, each from a 100 μl of serum sample remained. The strategy can be simply described as DNA walking over “bridges” and “islands” using conventional PCR with degenerate primers combined with strain specific primers. While this was efficient in HCV genomic study, the strategy would be also applicable to other studies of molecular epidemiology in which the viral sequence information is critical but the sample volume is small. Furthermore, These genomes represent a full panel of 17 subtypes within the oldest, the most diverse, the endemic, and possibly the zoonotic genotype 6. The utility of this panel of complete sequences for accurate detection and classification of infection, and for estimating the origin and evolution of this genotype of HCV, and for finding new variants, and for predicting the future epidemic trend and health burden, and for improving HCV diagnostic, treatment, and vaccination, is very important. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6116]  |
| 专题 | 昆明动物研究所_生殖与发育生物学 |
| 推荐引用方式 GB/T 7714 | 李春华. 丙型肝炎病毒(HCV)株的全基因组序列测定: 完成HCV 基因型6 全套17 个亚型基因组的测序[D]. 北京. 中国科学院研究生院. 2007. |
入库方式： OAI收割
来源：昆明动物研究所
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