甲型肝炎病毒在人源二倍体细胞的分子进化研究

发布时间：2018-05-19 22:12

本文选题：分子进化 + 病毒进化　；参考：《浙江大学》2007年博士论文

【摘要】： 分子进化研究可以帮助病毒学家了解病毒在已知限定条件下进化时所发生的分子事件。我们计划以甲肝病毒为模型，研究病毒在人源二倍体细胞长期连续传代过程中的进化热点、进化时序、进化方向等。本文中，我们将甲肝病毒野毒H_2W在人胚肺二倍体细胞KMB_(17)连续传代至30代，在传代的中问代次应用冷适应法变异病毒，然后挑选六个子代病毒进行基因序列分析。对H_2W及六个子代病毒的基因组RNA进行RT-PCR扩增，并通过PCR产物直接测序法测定其全长基因组序列。通过对所获序列进行同源性分析，总结甲肝病毒野毒在人源二倍体细胞连续传代过程中的分子进化信息。同时我们将所获序列与HM175、LA等标准病毒株，以及L-A-1、MBB、HAV7等细胞适应病毒株进行进化树分析，以研究甲肝病毒在人源二倍体细胞中的进化方向等。实验结果显示第五代之前绝大多数变异已完成，第5代相比于原代野毒株，共有648个核苷酸发生变异，其中40个导致了氨基酸改变。从第5代到第10代仅发生一个核苷酸变异，位于5’NCR区；第10代到第14代，仅发生一个核苷酸变异，即4222位U／C突变导致Phe／Ser的氨基酸变异；第14代到第15代仅发生一个变异，位于5’NCR区；从第15代到第30代，再未观察到任何变异发生。从变异的热点看，许多与病毒适应细胞相关的热点变异均发生于第5代附近，如128至136位的连续9bp缺失、176至177位的11bp插入序列，特别是3889位的C／U变异是病毒适应细胞的标志性变异。与毒力相关的热点变异则发生第10代到第14代之间。从病毒基因变异的时序来看，大多数变异完成于第5代附近，其中包括与细胞适应相关的关键变异；第5代到第15代之间仅发生极少量的变异，其中包括位于4222点的U／C变异，这一变异与毒力高度相关，其发生时序为第10到第14代之间；第15代之后的连续传代中病毒基因组表现为高度稳定。本文所获序列与文献序列进行进化树分析表明，H_2W在进化树上趋近于GBM系列，而他的六个子代病毒在进化树上距离其亲本较远，更加接近于L-A-1、MBB等细胞适应株。本文所获得的分子进化的时序、热点、基因稳定性等资料有助于解决活疫苗病毒株育种中相关技术问题，同时对于监测活病毒疫苗株的遗传稳定性等方面的研究均可提供理论支持和技术储备。第一部分甲型肝炎病毒野毒在人源二倍体细胞连续传代中的分子进化研究目的以甲肝病毒为模型，研究RNA病毒在人源二倍体细胞连续适应传代培养中的分子进化，包括进化热点、时序、方向等。方法将甲肝病毒野毒H_2W在人源二倍体细胞KMg_(17)中连续传代培养至30代。每隔5代选取其细胞适应子代病毒共六个，用PCR产物直接测序法测定H_2W及选定的六个子代病毒的全基因序列。然后用序列分析软件对所获序列进行分析，总结甲肝病毒野毒在长期的细胞连续适应培养中的进化热点、进化时序。通过对所获基因序列与其他已知甲肝病毒株特别是细胞适应株的进化树分析，研究甲肝病毒在人源二倍体细胞中的进化方向。结果 1．获得了H_2W及其六个子代病毒的接近全长的基因序列，并存入GenBank，录入号分别为：H_2W：EF406357；H_2K_5：EF406358；H_2K_(10)：EF406359；H_2K_(15)：EF406360；H_2K_(20)：EF406361；H_2K_(25)：EF406362；H_2K_(30)：EF406363 2．基于VP1／2A区168bp核苷酸片段的差异性比较，结果表明甲肝病毒野毒在适应KMB_(17)细胞的过程中，其基因亚型由IA转变为IB。 3．在H_2W适应细胞的前5代，一共有648个核苷酸发生变异，其中有40个导致氨基酸改变。这些变异中包含以下热点变异，如第128至136位的9bp缺失、第176至177位的11bp插入、第5018到5023位的6bp缺失(导致二个亲水氨基酸Asn、Asp缺失)、第3889位的C／U突变(导致Ala／Val的氨基酸变异)等是甲肝病毒野毒在适应细胞过程中普遍发生的热点变异。在第10代到第15代之间，，发生一个毒力相关的热点变异，即4222位发生U／C点突变，该变异导致Phe／Ser的氨基酸突变。 4．从原代到第5代，共发生648个核苷酸变异。从第5代到第10代，仅一个核苷酸发生变异，203位缺失1bp。从第10代到第15代，仅二个核苷酸发生变异，其一位于4222位的U／C点突变，导致Phe／Ser的氨基酸突变。另一个则位于5’NCR区。第15代到第30代，无任何突变发生。绝大多数的变异完成于第5代附近，其中包括与细胞适应相关的热点变异，其后长达25代的连续传代中仅累积发生三个变异，其中包含一个与毒力相关的变异，发生于第10代至第15代之间。 5．进化树分析结果显示，H_2W在进化树上比较趋向于GBM系列，而他的六个子代病毒则更加趋向于L-A-1、MBB等细胞适应株。他们在进化树上的距离较远。结论 1．首次观察到病毒在细胞中的完整进化时序：获得了甲肝病毒野毒在人二倍体细胞的进化时序，细胞适应相关基因的变异大约完成于第5代，毒力相关基因的变异介于第10代至15代之间。 2．甲肝病毒野毒适应人二倍体细胞连续传代的特点表现为：快速变异后的稳定遗传。甲肝病毒基因组在人二倍体细胞连续传代中的稳定性极高。进化方向上趋近于其他细胞适应株如MBB、L-A-1。 3．分子进化可用于甲肝病毒的毒力基因定位研究、毒力基因变异条件的研究以及甲肝病毒基因组遗传稳定性研究。 4．甲肝病毒是一个研究病毒分子进化的极有效的模型：可在多种细胞系培养(猴肾、肝瘤细胞、非肝二倍体细胞等等)；基因组较小，可方便地进行全基因组序列测定。第二部分甲型肝炎减毒活疫苗病毒株H_2K的培育研究目的将甲肝病毒野毒直接适应于人源二倍体细胞以选育一株毒力弱化且免疫原性良好的甲肝减毒活疫苗病毒株。方法将甲肝病毒野毒株H_2W适应到人源二倍细胞KMB_(17)，并连续传代培养至30代，在传代培养的中间代次(第15至20代)使用低温适应法变异毒力基因。以分子进化的相关研究方法研究毒力基因的定位、毒力基因的变异条件、毒力基因的变异时序以及基因组的稳定性特别是变异后的毒力基因的稳定性。根据毒力基因的变异时序选择合适代次的细胞适应株进行猴体动物实验，评价减毒株的毒力及免疫原性。结果 1．H_2W在细胞传代过程的第5代开始，其繁殖滴度稳步增长至较高水平，其后不再增长。 2．通过序列比较研究，发现4222位点是甲肝病毒的主要毒力决定位点，该位点的变异发生于第10至第14代，其后在长达15代(第16至30代)的连续传代中该变异一直稳定遗传。 3．低温适应不是甲肝病毒毒力基因变异的主要原因，这一点与流感病毒、呼吸道合胞病毒等不同。在特定细胞进行长达10代以上的适应传代可能是甲肝病毒减毒的主要原因，其中细胞系的来源尤为重要。 4．甲肝病毒野毒在适应细胞生长后，其后的连续25代(第5代到第30代)培养中仅积累三个变异，基因组表现出极高的稳定性。 5．猴体毒力试验结果表明实验组均抗体阳转且维持较高抗体水平，ALT转氨酶水平无异常，肝组织未见充血、水肿、变性和坏死等病理改变。H_2K_(25)是一株较理想的活疫苗毒种候选株。结论 1．HAV野毒在KMB17细胞连续传代后可获减毒，动物试验证实免疫原性佳、毒力弱化。 2．经KMB17细胞适应后的基因组高度稳定，毒力基因变异后稳定遗传。 3．成功培育了一株减毒活疫苗毒种候选株，其具备以下特征：繁殖滴度高、毒力弱化、免疫原性佳、基因组高度稳定。
[Abstract]:The molecular evolution of hepatitis A virus in human diploid cells was studied by RT - PCR .
In the 10th to 14th passages , only one nucleotide mutation occurred , that is , 4222 U / C mutation resulted in the amino acid variation of Phe / Ser ;
Only one variation occurred in the 14th to 15th generations ,

Located in the 5 ' NCR area ;
From the 15th to 30th generations , no variation has been observed . From the point of view of variation , many of the hotspot variations associated with the virus - adapted cells occur in the vicinity of the 5th generation , such as the deletion of 9 bp in the 128 - 136 position , the deletion of the 11bp insertion sequence in the 176 - 177 position , especially the 3889 - bit C / U mutation , which occurs between the 10th and 14th generations . Most of the variation is near the 5th generation , including the key variation related to cell adaptation .
Only a small amount of variation occurs between the 5th generation and the 15th generation , including U / C variation at 4222 points , which is highly correlated with the virulence height , with a timing of between 10th and 14th generations ;
The results show that H _ 2W approaches the series in the evolution tree , and the six progeny viruses are distant from their parents in the evolution tree , which is closer to the cells of L - A - 1 and MBB . The timing , hotspot and gene stability of the molecular evolution obtained in this paper can help to solve the related technical problems in the breeding of live vaccine virus strains , and provide theoretical support and technical reserve for the research of monitoring the genetic stability of live virus vaccine strains .

the first portion

Molecular evolution of hepatitis A virus wild virus in the continuous generation of human diploid cells

Using hepatitis A virus as a model , the molecular evolution of RNA viruses in human diploid cells was studied , including evolutionary hot spots , timing , direction and so on .

method

Human diploid cells KMg _ ( 17 ) were passaged continuously for 30 generations . The sequences of all genes were determined by direct sequencing of PCR products . The evolution of hepatitis A virus in human diploid cells was analyzed by means of sequencing analysis .

Results

1 . The gene sequences near full length of H _ 2W and its six progeny viruses were obtained and stored in GenBank . The entry numbers are : H _ 2W : EF406357 ;
H _ 2K _ 5 : EF406358
H _ 2K _ ( 10 ) : EF406359 ;
H _ 2K _ ( 15 ) : EF406360 ;
H _ 2K _ ( 20 ) : EF406361 ;
H _ 2K _ ( 25 ) : EF406362 ;
H _ 2K _ ( 30 ) : EF406363

2 . Based on the difference of the nucleotide fragments in VP1 / 2A region , the results showed that the gene subtype of hepatitis A virus was changed from IA to IB during the adaptation of KMB _ ( 17 ) cells .

3 . There were 648 nucleotides in the first 5 generations of H _ 2W - adapted cells . Among them , there were 40 mutations in amino acids , such as the deletion of 9 bp in the 128 - 136 position , the deletion of 6bp at the 176 - 177 position , the C / U mutation at position 5018 to 5023 ( resulting in the amino acid variation of Ala / Val ) , etc . , which resulted in a virus - related hotspot variation between the 10th and 15th generations , that is , 4222 - bit U / C - point mutation , which resulted in the amino acid mutation of Phe / Ser .

4 . A total of 648 nucleotide variations occurred from the first to the 5th generation . From the 5th to the 10th generation , only one nucleotide had been mutated , and the 203 - bit deletion was 1bp . The mutation occurred in only two nucleotides from the 10th to 15th generations . One of the mutations was located in the vicinity of the 5th generation , including the hotspot variation associated with the cell adaptation .

5 . The results of evolutionary tree analysis showed that H _ 2W tended to be more popular in evolutionary trees , while his six progeny were more likely to adapt to cells such as L - A - 1 , MBB , etc . They were far away from the evolutionary tree .

Conclusion

1 . The complete evolution timing of the virus in cells was observed for the first time : the evolution timing of the hepatitis A virus wild virus in human diploid cells was obtained , and the variation of the cell adaptation related gene was approximately completed in the 5th generation , and the variation of the virulence - related genes was between the 10th and 15th generations .

2 . The characteristics of the continuous passaging of the human diploid cell in the hepatitis A virus are as follows : stable inheritance after rapid variation . The stability of the hepatitis A virus genome in the continuous passage of human diploid cells is extremely high . The evolution direction approaches other cell - adapted strains such as MBB , L - A - 1 .

3 . Molecular evolution can be used for the study of virulence gene localization of hepatitis A virus , the research on mutation conditions of virulence genes and the study on genetic stability of hepatitis A virus genome .

4 . Hepatitis A virus is a very effective model to study the evolution of virus molecules : it can be cultured in a variety of cell lines ( monkey kidney , liver tumor cell , non - hepatic diploid cell , etc . ) ;
The genome is small , and the whole genome sequence determination can be carried out conveniently .

the second part

Cultivation of attenuated hepatitis A virus strain H _ 2K

Purpose of study

the hepatitis A virus wild virus is directly applicable to human diploid cells to breed a hepatitis A virus attenuated live vaccine virus strain which is attenuated and has good immunogenicity .

method

The strain H _ 2W of hepatitis A virus strain was adapted to human diploid cell KMB _ ( 17 ) and cultured continuously for 30 generations . The virulence gene was studied by means of molecular evolution .

Results

1.H _ 2W increased steadily to a high level in the 5th passage of the cell passage process , and then no longer increased .

2 . Through a sequence comparison study , it was found that 4222 sites were the main virulence determinants of hepatitis A virus , the variation of which occurred in the 10th to 14th passages , followed by stable inheritance in successive passages up to 15 passages ( 16th to 30th generation ) .

3 . Low temperature adaptation is not the main cause of hepatitis A virus virulence gene mutation , which is different from influenza virus , respiratory cell virus and so on . It may be the main reason for virus shedding of hepatitis A virus in the specific cell for more than 10 generations , in which the source of cell line is very important .

4 . After adaptation to cell growth , hepatitis A virus wild virus accumulates only three variants in the subsequent 25 - generation ( 5th generation to 30th generation ) culture , and the genome exhibits extremely high stability .

5 . The results of toxicity test of monkey body showed that in the experimental group , the antibody was positive and maintained higher antibody level , ALT transaminase level was not abnormal , the liver tissue did not see congestion , edema , degeneration and necrosis . H _ 2K _ ( 25 ) was the ideal candidate for live vaccine virus .

Conclusion

1 . After continuous passage of KMB17 cells , HAV wild virus can be attenuated , and animal experiments prove that the immunogenicity is good and the virulence is weakened .

2 . After KMB17 cell adaptation , the genome was highly stable and the virulence gene was stable after mutation .

3 . A candidate strain of attenuated live vaccine is successfully bred , which has the following characteristics : high reproductive titer , weakened virulence , good immunogenicity and stable genome height .
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R373

【参考文献】