狂犬病病毒准种适应性进化研究

发布时间：2018-04-09 00:02

本文选题：狂犬病病毒　切入点：G基因　出处：《吉林农业大学》2017年硕士论文

【摘要】：狂犬病是一种严重影响世界公共卫生的人兽共患病。一旦被感染而不采取有效措施,出现症状后死亡率几乎100%。据世界卫生组织报道,全世界每年因狂犬病导致的死亡人数为5~6万人,而绝大多数疫情出现在发展中国家,其中主要在亚洲和非洲。我国在世界上属狂犬病高发地区,发病和死亡人数仅次于印度,居世界第二位。狂犬病病毒(rabies virus,RABV)几乎感染所有温血动物,是目前发现宿主感染谱最为广泛的一种RNA病毒。鉴于狂犬病的巨大危害,狂犬病的预防控制日益得到关注。目前,世界各地都采取了一系列的措施来控制狂犬病,但是距离彻底控制狂犬病还需一定的时间。要想成功控制狂犬病的蔓延态势,针对狂犬病的特点进行控制才能达到更好的效果。狂犬病病毒的宿主十分广泛,几乎能感染所有的温血动物,这是其它任何病毒都不具备的特点,但是RABV如此广宿主感染谱的原因目前尚不清楚。包括RABV在内的RNA病毒是由一系列基因序列相近而又不完全相同的突变体构成,这称为病毒准种现象。病毒在动物或细胞内连续传代后会产生适应性,而RABV的准种在病毒感染谱中及宿主适应的过程中是否发挥作用,尚没有详细的研究。为研究狂犬病病毒街毒株在新宿主中的传播及其在适应新宿主过程中准种的变化规律,本研究首先选取处于不同进化分支的5株街毒株(HuND03、SXLF03、FJ02、CQQJD09、927A)和固定毒株CVS-11,将采集的感染这6株病毒致死的动物脑组织悬液脑内接种乳鼠,此为F1代,在乳鼠水平连续传至15代。选取乳鼠水平传代获得的HuND03、SXLF03、FJ02、CQQJD09、927A的F2、F7、F10、F15代鼠脑和上述毒株的原始脑组织(F0代),分别进行各代次的狂犬病病毒G基因全长序列的扩增,将扩增后的样品使用Illumina Hiseq 2500测序方法进行测序,使用Vivan软件进行多序列比较分析。结果表明:毒株在适应性传代过程,准种变异体始终是动态变化的,6株毒株F0、F2、F7、F10、F15代G基因上共有有302个位点出现突变,突变的类型只发现了点突变,并没有发现缺失突变、插入突变、提前或是终止突变的发生。出现频率最高的是A→G,其次是T→C。总体来说,同义突变变异体的数量大于非同义突变。研究发现RABV准种受到了宿主阳性选择的作用,阳性选择位点主要分布在G蛋白膜外区,但是变异体的数量和遭受阳性选择的位点随着毒株在乳鼠脑内传代代次的改变而改变。准种变异体在新宿主体内适应性传代过程中表现出3种不同的机制:1.适应新宿主的变异体出现2.不适应的变异体被淘汰3.变异体始终处于竞争状态。研究发现RABV序列较为保守,在新宿主(乳鼠)体内适应性传代过程中只出现了少数变异体,但是可能这少数变异体就足以使RABV完成跨宿主传播过程。与其他RNA病毒不同,RABV具有广泛宿主谱的特性并非后天逐步进化而来,而是RABV在形成之初,就具备适应不同宿主的特性。RABV这种高度保守的特性,可能是其迅速完成适应性进化的基础。本研究通过研究RABV准种,阐明了其在宿主适应性过程所表现出的动力学特征,初步了解了狂犬病病毒具有广泛宿主感染谱的原因,研究结果对于最终阐明RABV在宿主跨种传播分子遗传机制提供了新的视角,为狂犬病科学防控奠定了理论基础。
[Abstract]:Rabies is a serious impact on world public health Amphixenosis. Once infected and do not take effective measures, symptoms of mortality after almost 100%. WHO reported around the world each year the number of deaths caused by rabies caused by 5~6 million people, while the vast majority of the epidemic situation in developing countries, mainly in Asia and Africa. China is a high incidence of rabies regions in the world, the incidence and deaths after India, ranked second in the world. The rabies virus (rabies virus, RABV) infect almost all warm blooded animal, is found in the host spectrum of infection as a RNA virus. In view of the great harm of rabies, rabies prevention and control have been paid more and more attention at present, around the world have taken a series of measures to control rabies, rabies control completely but the distance needed for some time. In order to successfully control The trend of the spread of rabies, according to the characteristics of rabies control in order to achieve better results. The rabies virus host is very wide, almost can infect all warm blooded animal, which is characteristic of any other virus does not have, but RABV is so broad host spectrum of infection reason is unclear. Including RABV, RNA virus by a series of gene sequences are similar but not identical mutants, this is called a virus quasispecies. The virus was subcultured in animal cells or will produce adaptability, while RABV quasispecies infection does play a role in adaptation and host spectrum in the process of the virus, there is no detailed research for communication research. Rabies street virus strains in the host and in the adaptation process quasi variation of new host species, this paper selects 5 strains of street strains in different evolutionary branches (HuND03 SXLF03, FJ02, CQQJD09927A), and a fixed strain CVS-11, the collected brain tissue infected animal of the 6 strains of the virus lethal suspension intracerebral inoculation of suckling mice, the F1 generation in neonatal rat level 15 passages. Select the rat level obtained HuND03 passage SXLF03, FJ02, CQQJD09927A, F2. F7, F10, the original brain tissue F15 generation rat brain and the strain (F0), were amplified the full-length sequence of rabies virus G gene in each generation, the amplified samples using Illumina Hiseq 2500 sequencing methods were sequenced using Vivan software for multi sequence analysis. The results showed that the strains in the adaptability passage, quasi variants is always dynamic, 6 strains F0, F2, F7, F10, F15 G gene has 302 loci mutation, mutation types found only point mutations and found no deletion mutation, insertion mutation, mutation is terminated in advance or . the highest frequency is A to G, followed by T, C. overall, the number of synonymous mutation variants than non synonymous mutations. The study found that the RABV quasispecies is host of positive selection, positive selection sites mainly distributed in the outer membrane protein G, but the number of variants and subjected to positive selection sites with the passage of time changes in the generation of rat brain strain change. Quasispecies variants in the new host adaptive passage process showed 3 different mechanisms: 1. to adapt to the new host to the 2. variants appear variants were eliminated 3. variation of body is always in a competitive state. Research found that RABV sequence is more conservative, in a new host (rat) only a few variants appear in the process of adaptive passage, but perhaps this is enough to make a few variants of RABV to complete the process of inter host transmission. Different from other RNA virus, RABV Is not a characteristic of a broad host spectrum acquired gradually evolved, but RABV at the beginning of the formation, is to adapt to the characteristics of different host characteristics of.RABV this highly conserved, likely is the basis of the rapid completion of adaptive evolution. Through the study on the quasispecies of RABV, illustrates the show in the host adaptive process dynamics characteristics., a preliminary understanding of the rabies virus with a broad host spectrum of infection, the results of the study for clarifying the cross species transmission of RABV in host molecular genetic mechanism provides a new perspective, lays a theoretical foundation for the prevention and control of rabies.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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