肠道病毒71型上海流行株基因组序列分析及感染性克隆构建

发布时间：2018-05-04 18:10

本文选题：EV71 + 上海流行株　；参考：《复旦大学》2012年硕士论文

【摘要】：导致婴幼儿手足口病(hand, foot and mouth disease, HFMD)的病原体主要是肠道病毒71型(Enterovirus type71, EV71)和柯萨奇病毒A组16(Coxsackievirus A16, Cox A16)。EV71引起的手足口病,部分患者出现无菌性脑膜炎(asepicmeningitis)、脑炎(encephalitis)和类脊髓灰质炎的麻痹性疾病(poliomyelitis-like paralysis)等多种与神经系统相关的疾病,甚至死亡 EV71属小RNA病毒科肠道病毒属成员之一,其基因组为约7.4kb的单股正链RNA,两端分别为5’非编码区(untranslated region, UTR)和3’UTR,仅含一个开放阅读框(open reading frame, ORF),编码约2194个氨基酸的多聚蛋白(polyprotein),可进一步被水解成P1、P2、P3三种前体蛋白。P1进一步裂解成4种病毒外壳蛋白VP1-VP4；P2和P3分别裂解成7种非结构蛋白(2A、2B、2C和3A、3B、3C、3D)。利用上海手足口病患儿粪便标本,本研究分离出7株EV71病毒,其中4株来源于重症患者,3株来源于非重症患者。RT-PCR和3'-RACE将此7株EV71的基因组分为相互重叠的6个特异性片段进行扩增和测序,获得了全基因组序列(GenBank登陆号码为HQ891923、HQ891924、HQ891925、HQ891926、HQ891927、HQ891928、HQ891929)。同源性比较及遗传进化分析发现,上海流行株与中国大陆其他地方大部分流行株相似,均属于C4亚型。VP1进化树分析发现EV71上海流行株与北京、阜阳、广东分离株亲缘关系密切。全基因组进化树分析发现上海流行性株与阜阳、北京和浙江分离株亲缘关系密切,与以VP1所建进化树有细微差异。将分离的EV71上海流行株与其他基因亚型及Cox A16基因组用Simplot基因相似度和重组分析发现,EV71C4亚型从5’UTR到2A区域(约3600bp位置)可能源于EV71C2亚型；3C区域(从5800bp开始)到3’UTR可能源于Cox A16,表明EV71C4亚型可能是EV71C2亚型及Cox A16重组形成。将7株EV71上海流行株感染人横纹肌肉瘤细胞(human rhabdomyosarcomacells,RD),通过检测病毒的TCIDs0及病毒基因组拷贝数,发现临床上属于重症病例的HQ891923(27号)、HQ891924(28号)、HQ891925(36号)、HQ891928(117号),只有HQ891923病毒无明显复制及CPE出现。而在临床上属于非重症病例的HQ891926(51号)、HQ891927(64号)、HQ891929(118号)中,HQ891926和HQ891927病毒在RD细胞上复制效率很高,CPE明显且出现较早。因此,上海流行株感染细胞后表现出来的复制及其诱导细胞CPE的能力,与临床症状没有直接相关性。由于所分离的上海流行株间的基因组序列差异不明显,但其复制能力和致细胞病变能力差异较大,为进一步研究影响病毒基因组复制控制元件和主要毒力因子,选择高复制能力的HQ891927分离株进行感染性克隆的构建,将全基因组分3段克隆到载体中,并于3'UTR后加上多聚A尾。重组载体经过线性化后,利用噬菌体T7启动子聚合酶系统起始合成RNA转录本并转染RD细胞。转染后细胞出现病变反应,通过RT-PCR和间接免疫荧光反应检测到拯救病毒的基因和蛋白质表达。HQ891927感染性克隆的构建成功,为进一步研究EV71基因组结构和功能研究奠定了基础。
[Abstract]:The pathogens that cause hand (foot and mouth disease, HFMD) are mainly the enterovirus 71 (Enterovirus type71, EV71) and the coxsackievirus A group 16 (Coxsackievirus A16), which are caused by hand foot and mouth disease. A variety of nervous system related diseases, such as poliomyelitis-like paralysis, and even death.
EV71 is one of the members of the enteroviruses of the small RNA family, whose genome is about 7.4kb of single strand RNA. The two ends are 5 'non coding region (untranslated region, UTR) and 3' UTR, containing only one open reading frame (open reading frame, ORF), and about 2194 amino acids. The three precursor protein.P1 was further cleavaged into 4 viral coat proteins VP1-VP4, P2 and P3 respectively cleaved into 7 non structural proteins (2A, 2B, 2C and 3A, 3B, 3C, 3D).
7 EV71 viruses were isolated from the stool specimens of children with hand foot and mouth disease in Shanghai. 4 of them were from severe patients. 3 of them were derived from non severe patients.RT-PCR and 3'-RACE. The genome of 7 EV71 strains were divided into 6 specific fragments of each other to be amplified and sequenced, and the whole genome sequence (GenBank landing number was HQ891923). HQ891924, HQ891925, HQ891926, HQ891927, HQ891928, HQ891929). Homology comparison and genetic evolution analysis found that Shanghai epidemic strains are similar to most popular strains in other parts of mainland China, all belong to the C4 subtype.VP1 evolution tree analysis found that EV71 Shanghai epidemic strains are closely related to Beijing, Fuyang and Guangdong isolates. Whole genome evolution tree. The analysis found that Shanghai epidemic strains were closely related to the isolates of Fuyang, Beijing and Zhejiang, and had slight differences with the evolutionary trees built by VP1. The similarity and recombination analysis of the isolated EV71 Shanghai strains and other gene subtypes and the Cox A16 genome by Simplot gene found that the EV71C4 subtype from 5 'UTR to 2A region (about 3600bp position) may be possible. From the EV71C2 subtype, the 3C region (from 5800bp) to 3 'UTR can be derived from Cox A16, suggesting that EV71C4 subtypes may be EV71C2 subtypes and Cox A16 recombination.
7 strains of EV71 Shanghai strain infected human rhabdomyosarcoma cells (human rhabdomyosarcomacells, RD). By detecting the virus TCIDs0 and the copy number of the virus genome, it was found that HQ891923 (No. 27), HQ891924 (28), HQ891925 (36), HQ891928 (117), and only HQ891923 virus did not have obvious replication and CPE appeared. In HQ891926 (No. 51), HQ891927 (No. 64) and HQ891929 (118), the replication efficiency of HQ891926 and HQ891927 virus is very high in RD cells, and the CPE is obvious and appears earlier. Therefore, the replication of the infected cells in Shanghai and the ability to induce cell CPE are not directly related to the clinical symptoms.
The diversity of the genome sequences of the isolated Shanghai epidemic strains is not obvious, but their replication ability and cytopathic ability differ greatly. In order to further study the control elements and major virulence factors affecting the replication of the virus genome, the HQ891927 isolates with high replication ability are selected to construct the infectious clones, and the whole genome is divided into 3 segments. It was cloned into the carrier and added to the A tail after 3'UTR. The recombinant vector was linearized and the phage T7 promoter polymerase system was used to begin to synthesize the RNA transcript and transfect the RD cells. The transfected cells showed the lesion reaction. The genes and proteins that saved the virus were detected by RT-PCR and indirect immunofluorescence. The.HQ891927 sense of the gene and protein was detected. The successful construction of the dye clone laid the foundation for further research on the structure and function of EV71 genome.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373;R3416

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