猪瘟病毒2.1基因亚型野毒株的体外细胞传代适应与全基因组序列分析

发布时间：2018-09-07 11:08

【摘要】：猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)引起的一种严重危害全球养猪业的烈性传染病。CSF在全球范围内流行,给全世界各地的养猪业造成了巨大的经济损失。CSFV只有一种血清型,根据病毒基因组5’NTR、E2和NS5B等基因区段的核苷酸序列的差异可将CSFV分为3个基因型,其可进一步分为11个基因亚型(1.1~1.4;2.1~2.3;3.1~3.4)。涂长春等于上世纪九十年代对全国30个省市CSFV临床样品进行病原生态学和分子流行病学调查,研究发现在我国流行的CSFV主要是2.1、2.2、2.3和1.1亚型,而2.1亚型和2.2亚型作为优势毒株在我国的流行范围更广,呈全国性分布。由于2.1基因亚型存在较大的遗传多样性,进而又将其分为2.1a、2.1b、2.1c和2.1d基因亚亚型。陈宁等人对2004至2007年采集自我国华南地区的35份CSFV阳性样品调查时发现,其中34份样品为2.1b亚亚型,只有1份采集于2004年的阳性样品为2.2亚型。这说明2.1亚型是华南地区的主要流行毒株。2009年在我国陕西省首次分离获得了一株2.1a亚亚型毒株SXCDK,蒋大良等报道在湖南和广东等地区流行CSFV 2.1c亚亚型的毒株。目前,由于CSF流行在有些国家和地区得到有效控制,有些国家已宣布彻底消灭了CSF,这也使得一些当年的流行毒株成为历史,比如曾在欧洲流行的基因1.1亚型和曾在亚洲流行的3.3和3.4亚型,近年来已鲜有报道。然而,基因2型悄然成为在世界各地广泛流行的优势毒株。20世纪90年代欧洲许多国家曾暴发2.1亚型毒株引起的CSF疫情,而近年来的报道则以2.2亚型和2.3亚型居多。在我国,2.1型毒株也是主要流行毒株。本实验利用RT-PCR从三个广东某猪场的疑似CSF病例中检测出CSFV,并对扩增的E2全长基因进行了序列测定和系统进化分析。结果表明,引发这3起猪瘟疫情的毒株分别属于CSFV 2.1b基因亚亚型,命名为GD176;CSFV 2.1c基因亚亚型,命名为GD53;CSFV 2.1d基因亚亚型,命名为GD19。为了进一步了解该些毒株的复制特点,实验利用PK-15细胞对三株流行毒株进行了体外细胞适应性传代,并对获得的细胞适应毒F46进行了全基因组测序。结果表明三株流行毒株通过在PK-15上不断传代,最终获得了高滴度的适应毒株,GD176的滴度从第6代的102.6TCID50/mL提高至第46代的108.17TCID50/mL,GD53的滴度从第6代的103.39TCID50/mL提高至第46代的108.53TCID50/mL,GD19的滴度从第6代的102.54TCID50/mL提高至第46代的107.88TCID50/mL。病毒生长动力学结果显示三株流行毒株在感染后72h病毒滴度达到最高值。为了进一步分析三株流行毒株在细胞传代适应过程中的稳定性,实验对不同代次细胞毒的E2全长基因进行了扩增和序列测定,结果发现三株流行毒株的F0与F6、F10、F15、F20、F25、F30、F35、F40和F46的E2基因序列完全一致,这表明囊膜蛋白E2在病毒适应PK-15细胞过程中非常稳定。通过比对三株流行毒株和其他2.1亚型毒株各蛋白的氨基酸序列发现,不同毒株之间同源性最小的病毒蛋白是NS5A,低于病毒囊膜糖蛋白E2。本实验获得了高度适应PK-15细胞的CSFV 2.1亚型毒株细胞毒,为以后进行病毒毒力评价、复制和致病特性研究奠定了重要基础。
[Abstract]:Classical swine fever (CSF) is a severe infectious disease caused by Classical swine fever virus (CSFV) that seriously endangers the global pig industry. The prevalence of CSF worldwide has caused huge economic losses to pig industry worldwide. CSFV has only one serotype, according to the virus genome 5'NTR, E2 and NS5B. The difference of nucleotide sequence in isogenic regions can be divided into three genotypes, which can be further divided into 11 genotypes (1.1-1.4; 2.1-2.3; 3.1-3.4). Tu Changchun was the same as in 1990s. Pathogenic ecology and molecular epidemiology of CSFV in 30 provinces and cities in China were investigated. 2.1, 2.2, 2.3 and 1.1 subtypes, while 2.1 and 2.2 subtypes, as dominant strains, are more prevalent in China and are distributed nationwide. The results showed that 34 of the positive samples were 2.1b subtype and only one positive sample was 2.2 subtype collected in 2004. This indicated that 2.1 subtype was the main epidemic strain in South China. In 2009, a 2.1a subtype SXCDK strain was isolated from Shaanxi Province for the first time, and Jiang Daliang reported that it was prevalent in Hunan and Guangdong provinces. At present, due to the effective control of CSF epidemic in some countries and regions, some countries have declared the total elimination of CSF. This also makes some of the epidemic strains become history, such as the 1.1 subtype prevalent in Europe and the 3.3 and 3.4 subtype prevalent in Asia, which have been rarely reported in recent years. Genotype 2 has quietly become the dominant strain in the world. In the 1990s, many European countries had an outbreak of CSF caused by 2.1 subtype strain. In recent years, reports were mostly about 2.2 subtype and 2.3 subtype. CSFV was detected in SF cases, and the full-length E2 gene was amplified and sequenced and phylogenetic analysis was carried out. The results showed that the * 3 strains of swine plague belong to the CSFV 2.1b gene sub type, named GD176, CSFV 2.1c gene sub type, named GD53, CSFV 2.1d gene sub type, named 2.1c, in order to further understand this. According to the replication characteristics of some strains, three strains of epidemic virus were subcultured by PK-15 cells in vitro and the genome of F46 was sequenced. TCID50/mL increased to 108.17TCID50/mL in the 46th generation, the titer of GD53 increased from 103.39TCID50/mL in the 6th generation to 108.53TCID50/mL in the 46th generation, and the titer of GD19 increased from 102.54TCID50/mL in the 6th generation to 107.88TCID50/mL in the 46th generation. Further analysis of the stability of the three epidemic strains in the process of cell passage adaptation showed that the sequence of E2 gene of the three epidemic strains was identical with that of F6, F10, F15, F20, F25, F30, F35, F40 and F46, suggesting that the envelope protein E2 was adapted to PK-6. By comparing the amino acid sequences of the proteins of the three epidemic strains and the other 2.1 subtypes, it was found that the homology between the three strains was the smallest, which was NS5A, lower than the envelope glycoprotein E2. In this experiment, the CSFV 2.1 subtype virus strain which was highly adapted to the PK-15 cells was obtained, and it was used as the virus for further study. Virulence evaluation, replication and pathogenicity studies laid an important foundation.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

【共引文献】