【作者】 孟胜利；

【导师】 严家新；

【作者基本信息】 武汉生物制品研究所 ， 免疫学， 2010， 博士

【摘要】 本实验研究对分离于中国的6株狂犬病病毒街毒株毒株进行全基因组测序,并对基因组的5个结构基因(N、P、M、G和L)核苷酸和氨基酸序列以及及非编码区序列进行了全面的分析与比较,与来自GenBank的40株毒株从全基因组水平进行了分子进化研究。所测6株中国狂犬病病毒株全基因组核苷酸序列长度介于11907 nt(CQ92)和11924 nt(SH06和gg4)之间,结构相同,用全基因组和不同的结构基因构建的进化树拓扑结构相似,基因组3’和5’末端高度保守而且末端11个核苷酸互补配对,5个结构基因保守性依次是N>L>M>G>P,核苷酸同源性的最小值依次分别是81.9%、81.7%、80.7%、78.3%和76.7%。在全基因组分析的基础上,我们对从全国各地共分离到的60株街毒株和CTN疫苗株N基因进行序列测定,并与GenBank中得到毒株组成一个共计167株、分离时间介于1931-2009年,来源于全国20个省的毒株的N基因序列进行全面的系统进化分析,以探讨中国狂犬病病毒株的基因分型和分组情况、时间和空间的动态进化。我们发现目前分离于中国毒株都属于基因1型狂犬病病毒,可以分为2个进化分支共计8个组,分支Ⅰ包括1-4组,分支Ⅱ包括5-8组,组内核苷酸同源性≥93.2%,氨基酸同源性≥94.3%；组间核苷酸差异性≥8.0%,氨基酸差异性≥1.7%；选择压力分析表明中国狂犬病病毒处于较强的净化选择约束下,狂犬病病毒N蛋白中的核苷酸突变主要是同义突变；运用贝叶斯中的马尔科夫链的蒙特卡洛方法估计中国狂犬病病毒N基因核苷酸的平均碱基替代率为4×10-4取代／位点／年,共同祖先出现在250年前；迁移分析表明中国狂犬病病毒株存在跨地域传播；我国狂犬病高发区流行的毒株(分支Ⅰ)与泰国、越南、菲律宾、印度尼西亚、马来西亚等东南亚国家分离的狂犬病病毒株同属于一个进化分支；分支Ⅱ的毒株在全球分布。更多还原

【Abstract】 In order to study phylogeography, population dynamics and molecular evolution of rabies viruses (RABVs) isolates from China, especially spatio-temporal dynamics, the timescale of RABVs evolution and its pattern of migration. In total we have determined 6 new complete genome sequences of rabies field isolates from China and analysised the complete nucleotide and deduced amino acid sequence of the five structural genes (N, P, M, G and L) and non-structural genes, then we compared the complete genome and did evolutionary analysis with 40 available genome sequences from GenBank. All genomes of six omplete genome sequences of rabies field isolates from China have the same structural organization athough their lengths varied between 11907 nt (CQ92) and 11924 nt (SH06 and gg4). In phylogenetic analyses using the full genome dataset, N, P, M, G and L genes, similar topologies of the reconstructed trees were demonstrated. Our data also reveals a strict complementarity limited to the 11 terminal nucleotides of 3’and 5’UTR at both ends of the genome. The extent of genetic diversity, reflected in percentage identity, varies among proteins, in the order N>L>M>G>P (81.9%,81.7%,80.7%,78.3% and 76.7% nucleotide identity at a minimum value, respectively). At the same time, we determined the N gene sequence of CTN vacine strain and 60 street isolates of rabies viruses, and were collected from various areas in China. We performed an extensive comparative analysis of RABV N gene sequence data, representing 167 isolates sampled from 20 provinces in a 78-year period (from 1931 through 2009). The available Chinese isolates could be divided into two distinct clades:Phylogroup clades I comprises Chinese 1-4 group; Phylogroup cladesⅡcontains Chinese 5-8 group.Complete N gene sequences determined for these isolates indicated intra-group identities of nucleotide and amino acid sequences of≧ 93.2% and 94.3%, respectively; Intre-group divergence revealed up to 8.0% at the nucleotide level and 1.7% at the amino acid level. We found no evidence for positive selection (dN/dS>1) acting at any codon and found strong selective constraints for N gene. Bayesian Markov chain Monte Carlo (MCMC) analysis suggested that the Chinese rabies viruses arose 250 ago and the mean rates of nucleotide substitution for the N gene were approximatelately 4×10-4 substitutions per site year-1. The analyses of the spatial and spatio-temporal evolution indicated that RABV isolates from China migrated among different provinces. Phylogenetic analysis of the N gene sequence indicated that the isolates belonging to phylogroup cladesⅠwith isolates from Thailand, Vietnam, Indonesia, Philippines, Malaysia etal. were in the same branch in phylogenetic clades and the isolates belonging to phylogroup cladesⅡare found in many parts of the world.更多还原