柯萨奇病毒B组3型在ECV304中持续感染模型的建立及其持续感染机制的初步研究
发布时间：2018-07-26 10:04
【摘要】： 尽管CVB3是常见的病毒性心肌炎等急性疾病的病原体，然而也有许多证据表明CVB3也参与了许多严重的慢性疾病的病理过程。有研究表明CVB3与扩张型心肌病关系密切，说明病毒可能在心血管系统中持续存在，本文旨在通过建立CVB3(Nancy)在ECV304细胞中持续感染的模型，初步探索CVB3持续感染的机制。 以10~(-1) TCID50，10~(-2) TCID50，10~(-3) TCID50 CVB3感染ECV304，建立持续感染细胞模型，稳定传代120天以上，上清液及细胞裂解液接种敏感细胞Vero没有CPE，核酸检测结果阳性，从细胞裂解液中提取病毒RNA，进行RT反应，反转录出基因组cDNA，将整个基因组划分为5段进行PCR扩增，连接目的片段至PGM-T载体，进行测序，分析可能存在的变异是否与持续感染状态的形成有关。 结果发现持续感染细胞中CVB3与建模前的CVB3存在16处核苷酸变异，1处位于5’NTR：16R-G，其余15处位于病毒的ORF，其中有7个核苷酸变异造成了氨基酸的改变，分别是：P1-A区16P-G、18K-N，P3-AB区1486H-Y，P3-C区1696E-K，P3-D区1745R-K、1849A-T、2098P-L。经过对文献中病毒基因的分析P3-AB区、P3-D区的改变最可能影响病毒的毒力，致病毒表现出一种低复制的表型，这一结论尚需要反向遗传学验证。
[Abstract]:Although CVB3 is a common pathogen of acute diseases such as viral myocarditis, there is a lot of evidence that CVB3 is also involved in the pathological process of many serious chronic diseases. Some studies have shown that CVB3 is closely related to dilated cardiomyopathy, indicating that the virus may persist in the cardiovascular system. This paper aims to explore the mechanism of CVB3 persistent infection by establishing a model of CVB3 (Nancy) persistent infection in ECV304 cells. ECV304 was infected with 10 ~ (-1) TCID _ (50) ~ (-2) TCID _ (50) ~ (-3) TCID50 CVB3. The cell model of persistent infection was established. The supernatant and cell lysate were inoculated with sensitive cell Vero for more than 120 days. The results of nucleic acid detection were positive, virus RNAs were extracted from the cell lysis fluid and RT reaction was performed. The whole genome was divided into 5 segments for PCR amplification. The target fragment was ligated into PGM-T vector and sequenced to analyze whether the possible mutation was related to the formation of persistent infection. The results showed that there were 16 nucleotide mutations in CVB3 and CVB3 before modeling, one was located at 5 NTR: 16R-Gand the other 15 were located at ORF of virus, among which 7 nucleotide mutations caused amino acid changes. They are respectively: 1: P1-A region 16P-Gnn 18K-NCU P3-AB region 1486H-YP3-C region 1696E-KN P3-C region 1745R-Knn 1849A-Tnn 2098P-L. By analyzing the viral genes in the literature, the change of the P3-D region in the P3-AB region is most likely to affect the virulence of the virus, and the virus exhibits a low replication phenotype. This conclusion needs to be verified by reverse genetics.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373
本文编号：2145645
[Abstract]:Although CVB3 is a common pathogen of acute diseases such as viral myocarditis, there is a lot of evidence that CVB3 is also involved in the pathological process of many serious chronic diseases. Some studies have shown that CVB3 is closely related to dilated cardiomyopathy, indicating that the virus may persist in the cardiovascular system. This paper aims to explore the mechanism of CVB3 persistent infection by establishing a model of CVB3 (Nancy) persistent infection in ECV304 cells. ECV304 was infected with 10 ~ (-1) TCID _ (50) ~ (-2) TCID _ (50) ~ (-3) TCID50 CVB3. The cell model of persistent infection was established. The supernatant and cell lysate were inoculated with sensitive cell Vero for more than 120 days. The results of nucleic acid detection were positive, virus RNAs were extracted from the cell lysis fluid and RT reaction was performed. The whole genome was divided into 5 segments for PCR amplification. The target fragment was ligated into PGM-T vector and sequenced to analyze whether the possible mutation was related to the formation of persistent infection. The results showed that there were 16 nucleotide mutations in CVB3 and CVB3 before modeling, one was located at 5 NTR: 16R-Gand the other 15 were located at ORF of virus, among which 7 nucleotide mutations caused amino acid changes. They are respectively: 1: P1-A region 16P-Gnn 18K-NCU P3-AB region 1486H-YP3-C region 1696E-KN P3-C region 1745R-Knn 1849A-Tnn 2098P-L. By analyzing the viral genes in the literature, the change of the P3-D region in the P3-AB region is most likely to affect the virulence of the virus, and the virus exhibits a low replication phenotype. This conclusion needs to be verified by reverse genetics.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373
【参考文献】
相关期刊论文 前1条
1 苏琦华,訾自强,张霞,李晓眠,徐燕;柯萨奇B组3型、5型病毒在人脐静脉血管内皮细胞中持续感染模型的建立[J];中华微生物学和免疫学杂志;2004年10期
，本文编号：2145645
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