蓋塔病毒分子特征及其感染的相關(guān)研究
發(fā)布時(shí)間：2018-07-21 12:45
【摘要】：目的:測(cè)定新分離病毒株(YN12031,YN12042)的全基因組序列,驗(yàn)證其是否為蓋塔病毒(Getah virus,GETV),研究新分離病毒株(YN12031)的生物學(xué)表型和分子遺傳學(xué)特征;構(gòu)建蓋塔病毒基因序列分析數(shù)據(jù)集,對(duì)蓋塔病毒的種群特征和時(shí)空動(dòng)力學(xué)進(jìn)行分析;最后分析蓋塔病毒在中國(guó)大陸的感染情況。方法:第一部分將新分離的病毒株(YN12031)接種在BHK-21細(xì)胞上觀察細(xì)胞病變效應(yīng)(CPE),并在透射電子顯微鏡下鑒定該病毒的形態(tài),用噬斑測(cè)定法檢測(cè)病毒增殖的動(dòng)態(tài)變化。設(shè)計(jì)用于擴(kuò)增和測(cè)定全基因組序列的引物,使用PCR方法進(jìn)行基因序列擴(kuò)增。使用DNASTAR軟件包中的Seqman軟件對(duì)測(cè)序結(jié)果進(jìn)行拼接和質(zhì)量分析,使用Clustal X 1.8軟件進(jìn)行序列比對(duì),使用MEGA 6.0進(jìn)行系統(tǒng)發(fā)育和分子進(jìn)化分析。第二部分在基因庫中下載蓋塔病毒序列,結(jié)合新分離的蓋塔病毒(YN12031,YN12042病毒株)基因序列,以及15株本實(shí)驗(yàn)室此前在中國(guó)分離和鑒定、但未在Gen Bank登錄的蓋塔病毒毒株基因序列信息,用生物信息學(xué)方法構(gòu)建GETV序列分析數(shù)據(jù)集。采用蒙特卡洛馬爾科夫鏈(MCMC)統(tǒng)計(jì)算法分析蓋塔病毒數(shù)據(jù)集。利用BEAET軟件包進(jìn)行堿基替換速率、最近共同祖先(Time of most recent Ancestor,t MRCA)以及種群動(dòng)態(tài)分析(Beast skyride plot,BSP);采用BEAST v 1.8.1軟件包里的Bayesian Stochastic Search Variable Selection(BSSVS)程序進(jìn)行蓋塔病毒的空間動(dòng)力學(xué)研究;使用VMD軟件和YASARA軟件分析GETV E2蛋白的結(jié)構(gòu)。第三部分利用空斑減少中和實(shí)驗(yàn)的方法檢測(cè)人和多種動(dòng)物(雞、鴨、牛、豬、馬)血清標(biāo)本蓋塔病毒中和抗體水平。結(jié)果:第一部分YN12031病毒株感染BHK-21細(xì)胞32小時(shí)出現(xiàn)明顯的細(xì)胞病變。病毒粒子呈圓形,直徑70nm左右,有包膜,表面有纖突。分子遺傳分析顯示,YN12031病毒與甲病毒屬病毒如基孔肯雅病毒,辛德畢斯病毒密切相關(guān),并與GETV原型株MM2021處于同一進(jìn)化分枝中,蓋塔病毒E2基因和Capsid基因序列的系統(tǒng)進(jìn)化分析進(jìn)一步顯示,YN12031病毒與俄羅斯分離蓋塔病毒處于同一進(jìn)化分枝,此外蓋塔病毒編碼區(qū)基因及E2基因同源性分析顯示,YN12031病毒與俄羅斯分離蓋塔病毒同源性關(guān)系最近。第二部分蓋塔病毒E2基因的氨基酸差異分析顯示,與1955年馬來西亞原始株(MM2021)相比,其他蓋塔病毒分離株均存在7個(gè)共同的氨基酸差異位點(diǎn),這7個(gè)共同位點(diǎn)的突變,導(dǎo)致GETV流行株與原始毒株在結(jié)構(gòu)和電荷分布有差異。蓋塔病毒最早出現(xiàn)在距今約145年前,隨后逐漸演化出四個(gè)明顯的進(jìn)化種群,Group III進(jìn)化種群是目前國(guó)際上在多種蚊蟲和宿主動(dòng)物中流行的,并能引起動(dòng)物疾病的優(yōu)勢(shì)病毒進(jìn)化種群。蓋塔病毒種群進(jìn)化動(dòng)態(tài)分析顯示,蓋塔病毒種群動(dòng)態(tài)經(jīng)過平緩—增長(zhǎng)—下降—平緩的種群變化過程。蓋塔病毒的播散存在頻繁的遷徙事件,馬來西亞、日本和云南是蓋塔病毒的重要遷徙源泉地區(qū),遷徙事件也是蓋塔病毒重要的分布分化機(jī)制。第三部分共檢測(cè)了中國(guó)大陸895份人、畜動(dòng)物血清標(biāo)本,其中發(fā)熱患者血清中GETV中和抗體陽性率為8.4%(37/443);禽類動(dòng)物GETV中和抗體陽性率明顯低于豬、馬;同樣為牛(cattle),奶牛和菜牛對(duì)GETV中和抗體陽性率存在巨大差異。發(fā)熱患者血清標(biāo)本GETV中和抗體水平從1:10至1:320不等;雞、鴨和奶牛血清標(biāo)本中僅可以檢測(cè)到低滴度(1:10至1:20)蓋塔病毒中和抗體;而絕大多數(shù)豬、馬、菜牛血清標(biāo)本可以檢測(cè)到1:640至大于1:2560滴度的蓋塔病毒中和抗體。結(jié)論:1測(cè)定了新分離病毒株YN12031,YN12042的全基因組序列,并證實(shí)了病毒株為GETV;生物學(xué)表型顯示:GETV(YN12031分離株)可引起明顯的細(xì)胞病變,病毒粒子呈圓形,直徑70nm左右,有包膜,表面有纖突;分子遺傳分析顯示:YN12031病毒與俄羅斯分離蓋塔病毒同源性關(guān)系最近。2成功構(gòu)建了GETV序列分析數(shù)據(jù)集,生物信息學(xué)分析顯示:GETV流行株和原始株之間存在結(jié)構(gòu)和電荷的變化;GETV最早出現(xiàn)在距今約145年前,隨后逐漸演化出四個(gè)明顯的進(jìn)化種群,Group III進(jìn)化種群是主要的優(yōu)勢(shì)病毒進(jìn)化種群;GETV種群動(dòng)態(tài)經(jīng)過平緩—增長(zhǎng)—下降—平緩的種群變化過程;馬來西亞、日本和云南是蓋塔病毒的重要遷徙源泉地區(qū)。3中國(guó)大陸人和多種動(dòng)物血清標(biāo)本中存在蓋塔病毒中和抗體,并且馬、豬和菜牛蓋塔病毒中和抗體水平顯著高于人和其它動(dòng)物,提示中國(guó)大陸人、畜動(dòng)物存在蓋塔病毒的感染,豬、馬和菜牛很可能是GETV的擴(kuò)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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R373;S852.65
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 鄭雅勻;曹玉璽;付士紅;程t熛，
本文編號(hào)：2135572
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2135572.html
最近更新
教材專著
