盖塔病毒分子特征及其感染的相关研究
发布时间：2018-07-21 12:45
【摘要】：目的:测定新分离病毒株(YN12031,YN12042)的全基因组序列,验证其是否为盖塔病毒(Getah virus,GETV),研究新分离病毒株(YN12031)的生物学表型和分子遗传学特征;构建盖塔病毒基因序列分析数据集,对盖塔病毒的种群特征和时空动力学进行分析;最后分析盖塔病毒在中国大陆的感染情况。方法:第一部分将新分离的病毒株(YN12031)接种在BHK-21细胞上观察细胞病变效应(CPE),并在透射电子显微镜下鉴定该病毒的形态,用噬斑测定法检测病毒增殖的动态变化。设计用于扩增和测定全基因组序列的引物,使用PCR方法进行基因序列扩增。使用DNASTAR软件包中的Seqman软件对测序结果进行拼接和质量分析,使用Clustal X 1.8软件进行序列比对,使用MEGA 6.0进行系统发育和分子进化分析。第二部分在基因库中下载盖塔病毒序列,结合新分离的盖塔病毒(YN12031,YN12042病毒株)基因序列,以及15株本实验室此前在中国分离和鉴定、但未在Gen Bank登录的盖塔病毒毒株基因序列信息,用生物信息学方法构建GETV序列分析数据集。采用蒙特卡洛马尔科夫链(MCMC)统计算法分析盖塔病毒数据集。利用BEAET软件包进行碱基替换速率、最近共同祖先(Time of most recent Ancestor,t MRCA)以及种群动态分析(Beast skyride plot,BSP);采用BEAST v 1.8.1软件包里的Bayesian Stochastic Search Variable Selection(BSSVS)程序进行盖塔病毒的空间动力学研究;使用VMD软件和YASARA软件分析GETV E2蛋白的结构。第三部分利用空斑减少中和实验的方法检测人和多种动物(鸡、鸭、牛、猪、马)血清标本盖塔病毒中和抗体水平。结果:第一部分YN12031病毒株感染BHK-21细胞32小时出现明显的细胞病变。病毒粒子呈圆形,直径70nm左右,有包膜,表面有纤突。分子遗传分析显示,YN12031病毒与甲病毒属病毒如基孔肯雅病毒,辛德毕斯病毒密切相关,并与GETV原型株MM2021处于同一进化分枝中,盖塔病毒E2基因和Capsid基因序列的系统进化分析进一步显示,YN12031病毒与俄罗斯分离盖塔病毒处于同一进化分枝,此外盖塔病毒编码区基因及E2基因同源性分析显示,YN12031病毒与俄罗斯分离盖塔病毒同源性关系最近。第二部分盖塔病毒E2基因的氨基酸差异分析显示,与1955年马来西亚原始株(MM2021)相比,其他盖塔病毒分离株均存在7个共同的氨基酸差异位点,这7个共同位点的突变,导致GETV流行株与原始毒株在结构和电荷分布有差异。盖塔病毒最早出现在距今约145年前,随后逐渐演化出四个明显的进化种群,Group III进化种群是目前国际上在多种蚊虫和宿主动物中流行的,并能引起动物疾病的优势病毒进化种群。盖塔病毒种群进化动态分析显示,盖塔病毒种群动态经过平缓—增长—下降—平缓的种群变化过程。盖塔病毒的播散存在频繁的迁徙事件,马来西亚、日本和云南是盖塔病毒的重要迁徙源泉地区,迁徙事件也是盖塔病毒重要的分布分化机制。第三部分共检测了中国大陆895份人、畜动物血清标本,其中发热患者血清中GETV中和抗体阳性率为8.4%(37/443);禽类动物GETV中和抗体阳性率明显低于猪、马;同样为牛(cattle),奶牛和菜牛对GETV中和抗体阳性率存在巨大差异。发热患者血清标本GETV中和抗体水平从1:10至1:320不等;鸡、鸭和奶牛血清标本中仅可以检测到低滴度(1:10至1:20)盖塔病毒中和抗体;而绝大多数猪、马、菜牛血清标本可以检测到1:640至大于1:2560滴度的盖塔病毒中和抗体。结论:1测定了新分离病毒株YN12031,YN12042的全基因组序列,并证实了病毒株为GETV;生物学表型显示:GETV(YN12031分离株)可引起明显的细胞病变,病毒粒子呈圆形,直径70nm左右,有包膜,表面有纤突;分子遗传分析显示:YN12031病毒与俄罗斯分离盖塔病毒同源性关系最近。2成功构建了GETV序列分析数据集,生物信息学分析显示:GETV流行株和原始株之间存在结构和电荷的变化;GETV最早出现在距今约145年前,随后逐渐演化出四个明显的进化种群,Group III进化种群是主要的优势病毒进化种群;GETV种群动态经过平缓—增长—下降—平缓的种群变化过程;马来西亚、日本和云南是盖塔病毒的重要迁徙源泉地区。3中国大陆人和多种动物血清标本中存在盖塔病毒中和抗体,并且马、猪和菜牛盖塔病毒中和抗体水平显著高于人和其它动物,提示中国大陆人、畜动物存在盖塔病毒的感染,猪、马和菜牛很可能是GETV的扩增宿主。
[Abstract]:Objective: to determine the whole genome sequence of YN12031 (YN12042), to verify whether it is Getah virus (GETV), to study the biological and molecular genetic characteristics of the newly isolated virus strain (YN12031), and to construct the sequence analysis data set of the Getter virus gene, and to divide the population characteristics and spatiotemporal dynamics of the Getter virus. Analysis and final analysis of the infection of Getter virus in the mainland of China. Method: in the first part, the newly isolated virus strain (YN12031) was inoculated on the BHK-21 cell to observe the cytopathic effect (CPE), and the virus morphology was identified under the transmission electron microscope, and the dynamic changes of the virus proliferation were detected by plaque assay. The design was designed for amplification and determination. The primers of the whole genome sequence were amplified by the PCR method. The sequencing results were spliced and quality analyzed using the Seqman software in the DNASTAR software package. The sequence alignment was compared with the Clustal X 1.8 software, and the phylogenetic and molecular evolution analysis was carried out by MEGA 6. The second part downloaded the Getter virus sequence in the gene pool. According to the gene sequence of the newly separated Getter virus (YN12031, YN12042 virus strain), and the sequence information of the Getter virus strain which was isolated and identified in China before the laboratory, but not on the Gen Bank, the GETV sequence analysis data set was constructed by bioinformatics method. The Montecarlo Markoff chain (MCMC) statistical algorithm was used. Analysis of the Getter virus data set. Using the BEAET software package for the base substitution rate, the recent common ancestor (Time of most recent Ancestor, t MRCA) and the population dynamic analysis (Beast Skyride plot, BSP). Mechanical research; using VMD software and YASARA software to analyze the structure of GETV E2 protein. The third part uses the method of plaque reduction neutralization test to detect the level of Getter virus neutralization antibody in human and a variety of animals (chickens, ducks, cattle, pigs, horses). Results: the first part of the YN12031 virus strain infected with BHK-21 cells showed obvious cytopathic lesions for 32 hours. The virus particles are round and around 70nm, with a membrane and a fibrinolytic surface. Molecular genetic analysis shows that the YN12031 virus is closely related to the methoinvirus, such as kjkja virus, cindenbius virus, and is in the same evolutionary branch with the GETV prototype strain MM2021, and the phylogenetic analysis of the Getter's E2 gene and the Capsid gene sequence is analyzed. Step shows that YN12031 virus and Russian isolated Getter virus are in the same evolutionary branch. In addition, the homology analysis of the Getter virus coding region gene and the E2 gene shows that the YN12031 virus has the closest homology with the Russian separation of Getter virus. The analysis of the amino acid difference of the second part of the Getter virus E2 gene shows that the 1955 Malaysia original Compared to MM2021, there were 7 common amino acid difference sites in the other Getter virus isolates, and the mutation of the 7 common loci resulted in the difference in the structure and charge distribution between the GETV epidemic and the original strain. The first appearance of the Getter virus was about 145 years ago, and then gradually evolved four distinct evolutionary populations, and the evolution of Group III The population is an evolutionary population of dominant viruses in many mosquitoes and host animals, which can cause animal diseases. The dynamic analysis of Getter virus population evolution shows that the dynamics of the population of Getter virus has gone through the slow growth decline - the slow population change process. There are frequent migratory events in the dissemination of Getter virus. Western Asia, Japan and Yunnan are the important source of migration of Getter virus, and migration events are also the important distribution and differentiation mechanism of Getter virus. The third part examined 895 human and animal serum specimens in Chinese mainland, of which the positive rate of GETV neutralization antibody in serum of fever patients was 8.4% (37/443), and the positive rate of GETV neutralization antibody in poultry animals. There was a significant difference in the positive rates of neutralizing antibodies against GETV in cattle and cattle (cattle). The levels of neutralization antibodies in serum samples from fever patients ranged from 1:10 to 1:320, and in chickens, ducks and cows, only the low titer (1:10 to) could be detected in the serum of chickens, ducks and cows; the vast majority of pigs, horses, and beef cattle blood were detected in the serum samples of chickens, ducks and cows. The clear specimen can detect the Getter virus neutralization antibody of 1:640 to more than 1:2560 titer. Conclusion: 1 the whole genome sequence of the newly isolated virus strain YN12031, YN12042 is determined and the virus strain is GETV. The biological phenotype shows that the GETV (YN12031 isolate) can cause obvious cytopathic lesions, the virus particles are round, the diameter 70nm is around and there are bags. Membrane, surface with fibrous process; molecular genetic analysis showed that YN12031 virus and Russian separation of Getter virus homology recently.2 successfully constructed a GETV sequence analysis data set. Bioinformatics analysis showed that there was a change in structure and charge between the GETV epidemic and the original plant; GETV first appeared about 145 years ago and then gradually evolved. Four distinct evolutionary populations, the Group III evolution population is the dominant dominant virus evolution population; the GETV population dynamic through the slow growth - decline - the slow population change process; Malaysia, Japan and Yunnan are the important source of migration of Getter virus in.3 Chinese large land and a variety of animal serum specimens of the existence of Getter's disease Neutralizing antibodies, and the levels of neutralizing antibodies in horses, pigs and beef cattle Getter virus are significantly higher than those in humans and other animals, suggesting that the Chinese mainland and animal animals are infected with Getter virus, and pigs, horses and beef cattle are likely to be GETV's amplification hosts.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373;S852.65
[Abstract]:Objective: to determine the whole genome sequence of YN12031 (YN12042), to verify whether it is Getah virus (GETV), to study the biological and molecular genetic characteristics of the newly isolated virus strain (YN12031), and to construct the sequence analysis data set of the Getter virus gene, and to divide the population characteristics and spatiotemporal dynamics of the Getter virus. Analysis and final analysis of the infection of Getter virus in the mainland of China. Method: in the first part, the newly isolated virus strain (YN12031) was inoculated on the BHK-21 cell to observe the cytopathic effect (CPE), and the virus morphology was identified under the transmission electron microscope, and the dynamic changes of the virus proliferation were detected by plaque assay. The design was designed for amplification and determination. The primers of the whole genome sequence were amplified by the PCR method. The sequencing results were spliced and quality analyzed using the Seqman software in the DNASTAR software package. The sequence alignment was compared with the Clustal X 1.8 software, and the phylogenetic and molecular evolution analysis was carried out by MEGA 6. The second part downloaded the Getter virus sequence in the gene pool. According to the gene sequence of the newly separated Getter virus (YN12031, YN12042 virus strain), and the sequence information of the Getter virus strain which was isolated and identified in China before the laboratory, but not on the Gen Bank, the GETV sequence analysis data set was constructed by bioinformatics method. The Montecarlo Markoff chain (MCMC) statistical algorithm was used. Analysis of the Getter virus data set. Using the BEAET software package for the base substitution rate, the recent common ancestor (Time of most recent Ancestor, t MRCA) and the population dynamic analysis (Beast Skyride plot, BSP). Mechanical research; using VMD software and YASARA software to analyze the structure of GETV E2 protein. The third part uses the method of plaque reduction neutralization test to detect the level of Getter virus neutralization antibody in human and a variety of animals (chickens, ducks, cattle, pigs, horses). Results: the first part of the YN12031 virus strain infected with BHK-21 cells showed obvious cytopathic lesions for 32 hours. The virus particles are round and around 70nm, with a membrane and a fibrinolytic surface. Molecular genetic analysis shows that the YN12031 virus is closely related to the methoinvirus, such as kjkja virus, cindenbius virus, and is in the same evolutionary branch with the GETV prototype strain MM2021, and the phylogenetic analysis of the Getter's E2 gene and the Capsid gene sequence is analyzed. Step shows that YN12031 virus and Russian isolated Getter virus are in the same evolutionary branch. In addition, the homology analysis of the Getter virus coding region gene and the E2 gene shows that the YN12031 virus has the closest homology with the Russian separation of Getter virus. The analysis of the amino acid difference of the second part of the Getter virus E2 gene shows that the 1955 Malaysia original Compared to MM2021, there were 7 common amino acid difference sites in the other Getter virus isolates, and the mutation of the 7 common loci resulted in the difference in the structure and charge distribution between the GETV epidemic and the original strain. The first appearance of the Getter virus was about 145 years ago, and then gradually evolved four distinct evolutionary populations, and the evolution of Group III The population is an evolutionary population of dominant viruses in many mosquitoes and host animals, which can cause animal diseases. The dynamic analysis of Getter virus population evolution shows that the dynamics of the population of Getter virus has gone through the slow growth decline - the slow population change process. There are frequent migratory events in the dissemination of Getter virus. Western Asia, Japan and Yunnan are the important source of migration of Getter virus, and migration events are also the important distribution and differentiation mechanism of Getter virus. The third part examined 895 human and animal serum specimens in Chinese mainland, of which the positive rate of GETV neutralization antibody in serum of fever patients was 8.4% (37/443), and the positive rate of GETV neutralization antibody in poultry animals. There was a significant difference in the positive rates of neutralizing antibodies against GETV in cattle and cattle (cattle). The levels of neutralization antibodies in serum samples from fever patients ranged from 1:10 to 1:320, and in chickens, ducks and cows, only the low titer (1:10 to) could be detected in the serum of chickens, ducks and cows; the vast majority of pigs, horses, and beef cattle blood were detected in the serum samples of chickens, ducks and cows. The clear specimen can detect the Getter virus neutralization antibody of 1:640 to more than 1:2560 titer. Conclusion: 1 the whole genome sequence of the newly isolated virus strain YN12031, YN12042 is determined and the virus strain is GETV. The biological phenotype shows that the GETV (YN12031 isolate) can cause obvious cytopathic lesions, the virus particles are round, the diameter 70nm is around and there are bags. Membrane, surface with fibrous process; molecular genetic analysis showed that YN12031 virus and Russian separation of Getter virus homology recently.2 successfully constructed a GETV sequence analysis data set. Bioinformatics analysis showed that there was a change in structure and charge between the GETV epidemic and the original plant; GETV first appeared about 145 years ago and then gradually evolved. Four distinct evolutionary populations, the Group III evolution population is the dominant dominant virus evolution population; the GETV population dynamic through the slow growth - decline - the slow population change process; Malaysia, Japan and Yunnan are the important source of migration of Getter virus in.3 Chinese large land and a variety of animal serum specimens of the existence of Getter's disease Neutralizing antibodies, and the levels of neutralizing antibodies in horses, pigs and beef cattle Getter virus are significantly higher than those in humans and other animals, suggesting that the Chinese mainland and animal animals are infected with Getter virus, and pigs, horses and beef cattle are likely to be GETV's amplification hosts.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373;S852.65
【参考文献】
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1 郑雅匀;曹玉玺;付士红;程t熛，
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