摘要
[研究背景]
狂犬病是由狂犬病毒引起的人畜共患病,表现为急性、进行性、几乎不可逆转的脑脊髓炎。该病宿主动物广泛、呈世界性分布。中国是世界上仅次于印度的狂犬病第二高发国家。从1996年至今,中国的狂犬病疫情进入新的流行高峰,2007年中国狂犬病疫情达到顶峰,全国报告病例3302例。与此同时,狂犬病地理分布的范围和宿主动物的种类也逐步扩大和增加。全国31个省(直辖市)中除西藏、青海之外均有狂犬病疫情的报告,近几年野生动物伤人引发狂犬病的报告也逐步增多。尽管狂犬病在中国已成为严重的公共卫生问题,但与狂犬病流行密切相关的诸多理论和实际问题并没有得到很好的研究。近年来虽有不同的学者开展了狂犬病分子流行病学的研究,但多数局限于系统发生分析。因此,本研究在全国狂犬病主要流行区的七个省(直辖市)收集宿主动物的脑组织标本并进行狂犬病毒核蛋白基因测序,利用核蛋白核苷酸序列进行了包括系统发生分析、空间动力学分析和种群历史分析的综合的分子流行病学研究。本研究从分子水平上了解中国狂犬病毒的时空分布特征、分布分化机制,探讨其与病毒流行、扩散的内在联系,为有效地预防和控制狂犬病在我国的蔓延提供科学依据。
[研究目的]
1.了解中国狂犬病毒N基因遗传变异特点和进化特征,为研究其与病毒毒力的关系提供基础数据。
2.研究中国狂犬病毒间系统发生关系,描述中国狂犬病毒的时空分布特征。
3.研究中国狂犬病毒的分布分化机制,探讨其在中国的播散方式和路径。
4.重构中国狂犬病毒的进化历史和种群动态,探讨其与疫情流行和扩散的关系,为有效地预防和控制狂犬病在我国的蔓延提供科学依据。
[研究方法]
1.疫情资料的整理和分析
狂犬病疫情资料来自于中国疾病预防控制中心疾病监测信息系统。通过Excel对疫情资料进行整理、分析,了解1996-2008年中国狂犬病流行特征。
2.标本的采集、检测和测序
本研究在覆盖中国狂犬病主要流行区的7个省、直辖市收集动物(犬、猫)的脑组织标本。利用直接免疫荧光法(DFA)和RT-PCR法对标本进行抗原和核酸检测,获得阳性标本。利用PCR方法对阳性标本进行N基因编码区核苷酸序列的扩增和测序,获得完整的N基因核苷酸序列。
3.构建Lyssavirus基因序列信息数据库
下载Genbank上所有Lyssavirus序列信息,以PostgreSQL交互式数据库为平台,利用Perl编程语言编制脚本程序,构建包含序列信息、流行病学信息等的Lyssavirus基因序列信息数据库,并利用SQL语言实现对数据的输入、更新、查询、下载等应用。
4.序列的整理和分析
(1)序列的整理
测序结果用ATGC软件拼接后,ClustalX软件进行完全比对；BioEdit软件进行序列的分析和剪切。DNAStar(5.01)软件中MegAlign分别进行核苷酸和氨基酸序列间同源性比较；GeneDoc软件制成核苷酸及氨基酸差异显示图。
(2)系统发生分析
利用本次试验获得的34条序列和GenBank中的中国代表序列的N基因构造系统发生树。首先用Modeltest检验数据,获得最适模型(HKY)和各种参数值；然后利用PHYLIP软件包的最大似然法(ML)构建特征法系统发生树,bootstrap值设定为100。
(3)空间动力学分析
对选自中国狂犬病主要流行区的具有明确分离地点的221条N基因序列,利用PAUP的最大简约法(MP)构建系统发生树,结果保存为不带分支长度的无根树；同时用a-z单个字母来表示每条序列的分离地点,再利用MigraPhyla和PAUP的MP法计算迁移事件距离矩阵。
利用UniFrac,以带分支长度的有根树和表明分离地点的序列名字作为输入文件,进行主成分PCA (Principal component analysis)分析。根据分析结果判定不同环境(省份)之间的、在进化上的距离、异质性,同时绘制三维空间矩阵。
(4)种群动态与进化历史分析
基于蒙特卡洛马尔科夫链(MCMC)随机算法,利用BEAST软件对选择的有明确分离年代的我国211条N基因序列进行碱基替换速率、最近共祖先(Tmrca)、种群增长模型和skyline plot分析。根据Modeltest分析结果选择HKY模型,分别选择严格分子钟(strict clock)和松弛分子钟(relaxed clock)模型下的三种种群增长模型一常数模型(constant population size)、指数模型(exponential growth)、对数模型(logistic growth),并计算各参数值和likelihood值。利用likelihood值进行比较,确定种群的增长模型和各参数的值。
[结果]
1.本研究在广西、贵州、山东、安徽、浙江、江苏、上海收集动物脑组织标本987份,经DFA和RT-PCR法检测后共获得阳性标本34份。对34份标本进行N基因编码区核苷酸序列的扩增和测序,获得长度为1353bp的完整的N基因核苷酸序列。
2.基因序列同源性分析显示,34个病毒标本N基因的1353个核苷酸序列同源性为88.9%-100%,推导的氨基酸序列(N蛋白212-450位)同源性为97.6%-100%,说明N基因核苷酸序列的变异主要是同义突变。N蛋白四个抗原位点及Th细胞表位都高度保守。
3.ML系统发生树显示：中国狂犬病病毒形成四个独立的进化分支(Clade I-Clade V),每个分支又可细分为不同的进化群。其中CladeⅠ和CladeⅡ包含近年来分离的多数毒株,为优势分支。每个进化分支同时具有地域分布和不同地区毒株混合特征。
4.87%的配对地点间没有发生迁移事件。中国狂犬病毒存在分别以江苏、上海和湖南为中心的东部和西南部两个循环圈。白面鼬狂犬病毒存在从江西向浙江的传播。
5.基于马尔科夫链的贝叶斯分析显示,CladeⅠ和CladeⅡ分别起源于1992和1960前后,进化速率分别为：1.274×10-3(C195%：8.3705-4-1.2515E-3)和9.629×10-4(CI95%：3.519-4-1.628E-3)。CladeⅠ和CladeⅡ的最适种群模型均为分别为指数模型(exponential population growth)和常数模型(constant population size)。
6. CladeⅠ的基因多样性在从1994年开始到1996年上升迅速然后维持稳定期,随后在2001-2003年又形成一个快速上升期,并在2003年达到最高。2004-2005年经历了迅速下降并维持平缓下降的趋势。CladeⅡ基因多样性一直维持稳定的状态,2000年起到2003年经历快速上升,然后2004明显下将并在2005年前后下降至最低点后维持稳定状态。
[结论]
1.N基因具有高度保守的特性,可能与其结构和功能密切相关。
2.目前中国四个进化分支的狂犬病毒(CladeⅠ-CladeⅣ),已形成广泛的地理分布。其分布分化同时存在着地理分散和迁移事件(即基因漂移)两种机制,其中地理分散为中国狂犬病毒播散的主要方式。
3. CladeⅠ狂犬病毒起源于1992年,在1994-1996和2001-2003期间以湖南为中心发生广泛的地理分散,并于2004-2008年尤其是2004-2005年在华东地区以江苏为中心发生迁移事件。
4.CladeⅡ狂犬病毒起源于1960年,在2000-2003期间发生地理分散,在2004年有迁移事件的发生。
5.优势分支CladeⅠ和CladeⅡ在中国相应地区发生的广泛地理播散,对中国的狂犬病疫情发展发挥作用,造成狂犬病疫情从1996年进入新的流行高峰并在2001年和2003年经历了疫情的最快增长。
【BACKGROUND】
Rabies is a zoonotic disease caused by rabies virus and is manifested as acute, progressive and almost irreversible encephalomyelitis. It is also known as hydrophobia (Hydrophobia) for its clinical features of fear of water. Rabies was distributed worldwide with a wide range of host animals and China reported the seconded highest incidence only to India over the world. The rabies epidemic in China has been experiencing a new prevalence since 1996, especially in 2001 and 2003, with increase rate of 75% and 71%, respectively and reached peak with 3,302 reported cases nationwide in 2007. At the same time, the scope of geographic distribution and host animal species of rabies have gradually expanded and increased. Among all the 31 provinces (municipality), only Tibet and Qinghai did not report human rabies cases; the reported human rabies cases caused by injuries of wild animals have also increased gradually in recent years. Although rabies in China is a serious public health problem, many theoretical and practical issues that are closely related to rabies have not been well studied. Although different scholars in recent years have conducted molecular epidemiology of rabies in China, most are limited in phylogenetic analysis using sequences from local area. Therefore, a comprehensive analysis of molecular epidemiology including phylogenetic analysis, spatial analysis and population dynamics were conducted in this study using N gene sequences isolated from different host animals in rabies epidemic areas in China, in order to understand the characteristic of spatial and temporal distribution of rabies virus in China, the mechanism of its differentiation and its relationship with rabies epidemic and therefore provide scientific basis for the effective prevention and control of the spread of rabies in China.
【OBJECTIVES】
1. To learn the characteristics of genetic variation and evolution of rabies virus N gene in China and provide the basic data for the study of its relationships with virulence, pathogenicity;
2. To study phylogenetic relationships between rabies virus in China and describe characteristics of its spatial and temporal distribution;
3. To study the differentiation mechanism of rabies virus in China and explore its spread patterns in China;
4. Reconstructing the evolutionary history of rabies virus in China and population dynamics, to explore the prevalence and spread of rabies in China relationship, for the effective prevention and control of the spread of rabies in China provide the scientific basis
【METHODS】
1. Collection and analysis of epidemiological data
Epidemiological data were collected from Chinese center for Disease Control and Prevention. The data was arranged and analyzed by Excel to understand characteristics of rabies in China during 1996-2008.
2. Sample collection, detection and sequencing
Brain tissues of animals (dogs and cats) were collected in seven provinces or municipalities covering the major epidemic areas of rabies in China. The samples were detected by dFA and RT-PCR and positive samples were obtained. The complete nucleoprotein gene sequences of positive samples were amplified and sequenced by RT-PCR.
3. Construction of database of Lyssavirus genetic information
Lyssavirus sequence information was downloaded from GenBank, the interactive platform of PostgreSQL system and Perl programming language were used to construct database which contains sequence information, epidemiological information. SQL language was used to input, update, query and download information.
4. Sequence collation and analysis
(1) Sequence collation
The sequencing results were spliced by ATGC, aligned by ClustalX, analyzed and trimmed by BioEdit. The nucleotide and amino acid sequence homology were calculated by MegAlign in DNAStar software (5.01); GeneDoc software was used to make nucleotide and amino acid differences Figure.
(2) Phylogenetic analysis
Phylogenetic tree was constructed using N gene of 34 sequences obtained in this study and the representative ones from GenBank. First data sets were tested with Modeltest to obtain the best-fit model (HKY) and various parameter values; then PHYLIP package was used to construct phylogenetic tree by maximum likelihood (ML) method with bootstrap values set by 100.
(3) Analysis of spatial dynamics
Maximum parsimony (MP) phylogenetic tree was constructed by PAUP using 211 N gene sequence isolated from the major rabies epidemic areas in China, and was saved as non-root tree without branch length o; the isolated location of the sequences was indicated with a single a-z letter, and migration events were inferred with MP method using PAUP and MigraPhyla.
Using UniFrac, PCA (Principal component analysis) analysis was conducted won the input files of a rooted tree with branch length and sequence name with isolated location. The evolutionary distance, heterogeneity, and three-dimensional matrix were obtained based on the PCA result.
(4) Analysis of population dynamics and evolutionary history
The most recent common ancestors (Tmrca), substitution rate, population growth model and the skyline plot analysis were conducted by MCMC random algorithm in BEAST software based on 211 N gene sequences with specific isolated location and time. The HKY model was selected according to Modeltest results. The three models of population growth (constant population size, exponential growth and logistic growth) under strict clock and relaxed clock were chosen. The parameter values and likelihood values were calculated and compared to determine what the most fit model is.
【RESULTS】
1.987 samples were collected in Guangxi, Guizhou, Shandong, Anhui, Zhejiang, Jiangsu and Shanghai,of which,34 samples were positive detected by both DFA and RT-PCR. The 1353bp complete nucleotide N gene of the 34 positive samples were amplified and sequenced.
2. Sequences comparison showed that 34 samples share a high degree of homology in nucleotide acid(88.9%-100%) and deduced amino acid(97.6%-100%). This indicates that the variations in this region are synonymous mutations. Antigenic sites and Th cell epitopes on N gene were highly conserved.
3. ML tree showed that all of the 211 sequences from China the can be divided into four separate evolutionary branch (CladeⅠ-CladeⅤ), and each branch can be further divided into different evolutionary groups. CladeⅠand CladeⅡcontains the majority of strains isolated in recent years, indicating that they are the dominant ones. Each evolutionary branch is characterized by both geographical distribution and mixture of strains from different regions.
4.87% of the pairwise locations were not found with migration events. There are two spread circles in east and southwest of China, with Jiangsu/Shanghai and Hunan as centers, respectively. Migration events of ferret rabies virus occurred from Jiangxi to Zhejiang.
5. The BEAST analysis based on MCMC showed that CladeⅠand CladeⅡcan be dated back to 1992 and 1960, respectively. Evolution rates were:1.274×10-3 (CI95%:8.3705-4-1.2515 E-3) for CladeⅠ, and 9.629×10-4(CI95%:3.519-4-1.628 E-3) for CladeⅡ.The optimal population model for CladeⅠand CladeⅡare exponential population growth and constant population size, respectively.
6. Genetic diversity in CladeⅠrose rapidly from 1994 to 1996 and then maintain stable, followed by another rapid rise during 2001-2003, and reached peak in 2003. During 2004-2005 there was a rapid decline and then maintained smooth downward trend. Genetic diversity in CladeⅡhas been in long steady state until 2000. From 2000 to 2003, it rapidly increased. There was a significant decrease in 2004 and reached the lowest point at around 2005.
【Conclusion】
1. N gene is highly conserved, which may be closely related to its structure and function.
2. The four different Clades of the rabies virus in China have been forming a broad geographical distribution, with two differentiation mechanisms of geographic dispersal and gene flow, the former of which are the dominant one contributing to the spread of rabies virus.
3. Rabies virus in CladeⅠwas dated back to about 1992 and experienced extensive geographic spread during 1994-1996 and 2001-2003 with Hunan as the center, and migration events in this Clade occurred during 2004-2008, especially from 2004 to 2005 with the center of Jiangsu in eastern China.
4. Rabies virus in CladeⅡwas dated back to about 1960 and experienced geographical dispersion and migration events in 2000-2003 and 2004, respectively.
5. The dominant ones-CladeⅠand CladeⅡcontributed to spread of rabies virus of corresponding region and the fact that rabies epidemic entered a new peak from 1996 and experienced the fastest growth in 2001 and 2003.
引文
[1]唐青,杨为松.狂犬病毒分子生物学及遗传变异性研究进展[J].中国人兽共患杂志,1999；15(2)：77-81.
[2]Tordo N., Poch O., Ermine A., et al. Walking along the rabies genome:is the large G-L intergenic region a remnant gene? [J]. Proc Natl Acad Sci U S A.1986 June; 83(11):3914-3918.
[3]俞永新.狂犬病和狂犬病疫苗.[M]北京：中国医药科技出版社,2009.140-151.
[4]World Health Organization.2005.WHO Expert Committee on Rabies.2004.First Report; WHO technical report series 931. World Health Organization. Geneva, Switzland.
[5]Zhang Y.Z., Xiong C.L., Xiao D.L., et al. Human rabies in China [J]. Emerg Infect Dis,2005; 11:1983-1984.
[6]唐青,赵秀芹,陶晓霞．中国人间狂犬病流行近况分析[J].中华流行病学杂志,2001；22(1)：8-10.
[7]宋淼,唐青,徐真,等.中国2005年狂犬病流行相关因素分析[J]．中华流行病学杂志,2006；27(11)：956-959.
[8]Zhang SF, Tang Q, Wu XF, et al. Rabies in Ferret Badgers, Southeastern China [J]. Emerg Infect Dis,2009; 15(6):946-949.
[9]Bourhy, H., Reynes, J.M., Dunham, et al. The origin and phylogeography of dog rabies virus [J]. J Gen Virol,2008;89:2673-81.
[10]Bourhy, H., Kissi, B., Audry, L., et al. Ecology and evolution of rabies in Europe [J]. J Gen Virol,1999; 80 (10):2545-57.
[11]K.L. Mansfield, V. Racloz, L.M. McElhinney, et al. Molecular epidemiological study of Arctic rabies virus isolates from Greenland and comparison with isolates from throughout the Arctic and Baltic regions[J]. Virus Research,2006; 116:1-10.
[12]Talbi, C., Holmes, E. C., de Benedictis, et al. Evolutionary history and dynamics of dog rabies virus in western and central Africa[J]. J Gen Virol,2009; 90:783-91.
[13]N. Johnson, L.M. McElhinney, Y.H. Ali, et al. Molecular epidemiology of canid rabies in Sudan:evidence for a common origin of rabies with Ethiopia [J]. Virus Research,2004; 104:201-205.
[14]T. Nagarajan, B. Mohanasubramanian, E. V. Seshagiri, et al. Molecular Epidemiology of Rabies Virus Isolates in India[J]. J CLIN MICROBIOL,2006; 44: 3218-3224.
[15]李浩,陶晓燕,唐青,等.狂犬病高发地区犬只感染情况调查分析[J].中华实验和临床病毒学杂志,2008；22(3)：161-164
[16]Larkin M.A., Blackshields G., Brown N.P., et al. Clustal Wand X Version 2.0[J]. Bioinformatics,2007.23:2947-2948.
[17]Hall T.A. BioEdit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser.1999; 41:95-98.
[18]Kelly L. W., Steven B. B, Jay W, et al. Development and Characterization of a Mouse Model for Marburg Hemorrhagic Fever[J]. J Virol,2009; 83:6404-6415.
[19]Cecilia L, Sabino L, Flavio, et al. Update of AMmtDB:a database of multi-aligned Metazoa mitochondrial DNA sequences [J].J Nucleic Acids Res.2000; 28(1):153-154.
[20]Swofford, D. L.PAUP 4.0 User's Manual:Phylogenetic Analysis Using Parsimony. Sunderland, MA,2003:Sinauer Associates.
[21]FELSENSTEIN J. PHYLIP:Phylogenetic inference package (version 3.5c).1993; Department of Genetics, University of Washington, Seattle, Washington.
[22]Kumar S, Tamura K, Jakobsen IB.MEGA2, molecular evolutionary genetics analysis softwar [J]. Bioinformatics,2001; 17:1244-1245.
[23]D.Posada, k.A.Crandall. MODELTEST:testing the model of DNA substitution [J]. Bioinformatics Applications Note,1998; 14(9):817-818.
[24]Robert G. W, Walter M. F. Influenza A H5N1 Immigration Is Filtered Out at Some International Borders [J].PLoS ONE.2008; 3(2):e1697.
[25]Wallace RG, HoDac H, Lathrop RH, et al. A statistical phylogeography of influenza A H5N1 [J]. Proc Natl Acad Sci U S A,2007 104:4473-4478.
[26]HoDac H, Lathrop RH, and Wallace RG, et al. (2007) MigraPhyla:Statistical Analysis of Migration Events through a Phylogeny. Version 1.0b manual.Currently available at http://pd.bio.uci.edu/ee/WallaceR/MigraPhyla.html
[27]Lozupone C, Knight R.UniFrac:A new phylogenetic method for comparing microbial communities [J].Applied and Environmental Microbiology,2005; 71: 8228-8235.
[28]Lozupone C, Hamady M, Knight R. UniFrac-an online tool for comparing microbial community diversity in a phylogenetic context [J].BMC Bioinformatics, 2006;7:371.
[29]Drummond A. J., Rambaut A. BEAST:Bayesian evolutionary analysis by sampling trees [J]. BMC Evol Biol,2007; 7,214.
[30]Goto H, Minamoto N, Ito H, et al. Mapping of epitopes and structural analysis of antigentic sites in the nucleoprotein of rabies virus[J].J Gen Virol,2000;81:119-127.
[31]Lafon M, Wikot T J. Antigenic sites on the ERA rabies virus nucleoprotein and non-structural protein[J].J Gen Virol.1985;66 (Pt10):2125-2133.
[32]吴小红,刘景华,董关木.中国狂犬病疫苗CTN-1-V株糖蛋白基因的克隆及序列分析[J].中国生物制品学杂志,2003；16(3)：321-323
[33]Ertl, H.C.J.Induction of rabies virus-specific T-helper cells by synthetic peptides that carrydominant T-helper-cell epitopes of the viral nucleoprotein [J]. J Virol.,1989; 63:2885-2892.
[34]Yang J, Koprowski H, Dietzscholk B, et al. Phosphorylation of rabies virus nucleoprotein regulates viral RNA transcription and replication by modulating leader RNA encapsidation [J]. J Virol,1999; Feb:1661-1664.
[35]Warrilow D.Australian bat lyssavirus:a recently discovered new rhabdovirus [J]. Curr Top Microbiol Immunol.2005; 292:25-44.
[36]WHO. Export Committee on Rabies.8th Report[R], Geneva:WHO.1992.9,22.
[37]李浩,陶晓燕,唐青,等.狂犬病高发地区犬只感染情况调查分析.中华实验和临床病毒学杂志,2008,22(3)：161-164
[38]熊成龙,邵中军,姜庆五,等.不同疫区家犬携带狂犬病毒的比较研究.中国热带医学,2008,8(3)：14-15.
[39]刘浩,江禹,王莉莉,等.湖南湘西地区外观健康犬携带狂犬病病毒的调查研究.中国动物检疫,2007,24(9)：17-19.
[40]Biek, R., Drummond, A. J.& Poss, M. A virus reveals population structure and recent demographic history of its carnivore host [J]. Science,2006;311,538-541.
[41]Grenfell B. T., Pybus O. G., Gog J. R., et al. Unifying the epidemiological and evolutionary dynamics of pathogens [J]. Science,2004; 303:327-332.
[42]Holmes, E. C. The phylogeography of human viruses [J]. J Mol Ecol,2004; 13:745-756.
[43]Davis P. L., Holmes E.C., Larrous F., et al. Phylogeography, population dynamics, and molecular evolution of European bat lyssaviruses [J]. J Virol,2005; 79:10487-10497.
[44]Holmes E. C., Woelk C. H., Kassis R, et al. Genetic constraints and the adaptive evolution of rabies virus in nature[J]. Virology,2002; 292:247-257.
[45]Kissi, B., Tordo, N.& Bourhy, H. Genetic polymorphism in the rabies virus nucleoprotein gene [J]. Virology,1995; 209:526-537.
[46]Sacrame D, Badrane H, Bourhy H, et al. Molecular epidemiology of rabies virus in France:comparison with vaccine strain [J].J Gen Virol.1992; 73(Pt5):1149-58.
[47]von Teichman BF, Thomson GR, Meredith CD, et al. Molecular epidemiology of rabies virus in South Africa:evidence for two distinct virus group[J]. J Gen Virol.1995;76(Ptl):73-82.
[48]Criscione C. D.& Blouin, M. S. Effective sizes of macroparasite populations:a conceptual model [J]. Trends Parasitol,2005; 21:212-217.
[49]Tao XY, Tang Q, Li H, et al.Molecular epidemiology of rabies in Southern People's Republic of China[J]. Emerg Infect Dis.2009; 15(8):1192-8.