【作者】 王承宝；

【导师】 薛飞；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2010， 博士

【摘要】 猪传染性胃肠炎(Porcine transmissible gastroenteritis, TGE)是由猪传染性胃肠炎病毒(Porcine transmissible gastroenteritis virus, TGEV)引起的一种高度接触传染性肠道疾病,以引起一周龄以下仔猪呕吐、水样腹泻和高死亡率(通常为100 %)为主要特征。TGEV主要感染仔猪的小肠上皮细胞,从而最终导致发生致死性的腹泻。近几十年来,各种不同毒力的TGEV已经被各国的研究人员成功分离,同时对其特性进行了详细的研究。一些现地分离的强毒株在长期的体外培养过程中其毒力逐渐降低,并且被培育成优良的弱毒疫苗株。我国马思奇等人成功培育了我国TGE华毒弱毒株。该弱毒株是由TGE华毒强毒株经过5次蚀斑克隆、经PK-15细胞系体外连续培养165代培育而成。目前已广泛应用于我国TGE的防控中。至今为止,TGE华毒弱毒株作为我国具有独立自主知识产权的优秀疫苗株其完整的全基因组背景目前仍不清楚,其分子致弱机制更不清楚,因此,亟待完成TGE华毒强、弱毒株的全基因组的序列测定工作,其全基因组序列的完成将为我国TGE的防控提供分子基础。本研究首先通过常规的RT-PCR方法分别克隆完成了我国TGE华毒强、弱毒株的全基因组序列(登录号分别为:FJ755618和EU074218)。TGE华毒强、弱毒株的全基因组序列全长均为28, 569 bp(包含30 bp的poly(A)尾巴)。序列分析发现:在长期的体外培养传代过程中,TGE华毒弱毒株在序列组成上没有插入、缺失等现象发生,只存在27个核苷酸的突变,最终导致16个氨基酸发生突变。因此,我们得出结论:氨基酸的突变是导致TGE华毒强毒株致弱的原因。迄今为止,GenBank数据库中只有3对TGEV强弱毒株完整的全基因组序列存在,研究发现,国外的Miller M6强毒株在长期的体外传代致弱过程中,其基因3基因片段存在缺失;而我国TGE华毒强毒株和国外Purdue强毒株在长期的体外传代过程中,在该部位未发现存在缺失现象。同时,不同毒力的TGEV毒株在S基因和(或)基因3区域往往存在大片段的缺失或插入现象。然而至今没有关于我国TGEV毒株基因3基因片段的相关研究报道。本研究通过建立的RT-PCR方法对我国部分现地TGEV毒株的基因3的基因片段进行检测,结果显示:我国现地TGEV毒株基因3存在较大差异,其中,CH/SDQ/08毒株的3a基因其ATG上游的31个碱基以及包含ATG在内的50个碱基发生缺失;CH/JLY1/08的3a基因起始密码子ATG突变为ACG,导致CH/JLY1/08毒株不能形成完整的ORF 3a。CH/JLY1/08的3b基因的终止密码子TAG突变为TTT,导致其3b基因出现了移码突变,与其它毒株相比多了7个氨基酸。同时,同一猪群中存在多种不同基因型TGEV毒株的感染。通过对TGE华毒强、弱毒株的全基因组序列及GenBank数据库中登录的所有相关TGEV毒株的基因序列进行分析比较发现:TGE华毒弱毒株在全基因组序列上存在6个独有的核苷酸突变(G6014TORF1a、T12388CORF1b、T21937CS、T21969AS、A26025CE,和C27507TN),可以作为区分TGE疫苗株与现地野毒感染的分子标签。本研究针对TGEV N基因核苷酸突变(C27507TN)所产生的Acl I限制性内切酶位点分子标签建立了区分TGEV疫苗免疫和野毒感染的RT-PCR-RFLP鉴别诊断方法。利用建立的RT-PCR-RFLP鉴别诊断方法对我国部分现地TGEV毒株的N基因片段进行检测,结果显示:我国现地TGEV毒株处于不断的遗传演化中,现地TGEV毒株分为3个群:不同群中的TGEV毒株N基因差异较大,与以往报道的TGEV毒株相比,9株我国现地TGEV毒株形成一个独立的新群。本研究所中所采用的TGE华毒弱毒株是由本实验室成功培育的弱毒株,具有安全、稳定、免疫原性好和遗传背景清楚等特点,为了阐述我国TGE华毒强毒株致弱的分子机制,本研究拟希望采用西班牙马德里大学的Luis Enjuanes教授馈赠的pBAC-5`- 3`载体为骨架构建我国TGE华毒弱毒株的反向遗传系统。目前,我们已经在对TGE华毒弱毒株全基因组序列的第3,012位和26,925位的Pml I位点、第24,699位的ApaLI位点进行了无义突变的基础上,构建完成了涵盖TGE华毒弱毒株全基因组的5个中间质粒(A、B、C、D和E中间质粒);同时已经将获得准确的序列E、A和B质粒(大约14 Kb)依次亚克隆到pBAC载体中,下游的工作还在继续进行当中。更多还原

【Abstract】 Porcine transmissible gastroenteritis virus (TGEV) was initially identified as the etiological agent of transmissible gastroenteritis (TGE) in swine in 1946 in the United States. In neonates, TGEV infects the epithelial cells of the small intestines, leading to potentially fatal gastroenteritis. The virus can also lead to infection in the upper respiratory tract and less often, in the lungs. In adults, TGEV causes mild disease. In swine, it is the major cause of viral enteritis and fetal diarrhea in neonates, resulting in significant economic losses. TGEV was reported in many swine-producing countries between the late 1980s and the 1990s. TGEV strains of varying virulence have been isolated and characterized worldwide. Some strains have been used to develop modified live vaccines with limited success. In China, a TGE outbreak was first reported in the 1970s. Since then it has been prevalent in many provinces and has become one of the most important viral diarrhea diseases in China. The Chinese TGEV vaccine strain H165 was derived from a virulent field strain H16 by 165 passages in PK15 cells. Vaccines based on the H165 strain are currently commercially available to prevent and control TGEV infections in China. H165 virus was proven to be safe in piglets and pregnant sows and efficacious against TGEV infection. Whole genome sequences of strains H165 and H16 will help us to understand the genetic basis of TGEV attenuation and enhance the geographic differentiation information among TGEV strains.Up to now, there were only seven TGEV strains and one PRCV strain PRCV-ISU-1 had been fully sequenced, though partial sequences of TGEV strains were available in the GenBank. Moreover, only two virulent and attenuated TGEV pairs were reported with difference in the gene 3 region. Previously studies have shown that there is a genetic diversity in the genomes of TGEV, especially in the gene 3. However, this report is a first one dealing with the genetic diversity in the gene 3 of Chinese field TGEV strains and reference TGEV strains. Our findings showed that 8 Chinese TGEV strains were genetically diverse in the gene 3 among themselves as well as in comparison with the reference strains. The nucleotide sequence data revealed genetic diversity in the gene 3 region of the Chinese field TGEV strains, even though the viruses were isolated from the same place. These results also indicated that different strains were present in the same farm.There were a total of 27 nt mutations identified in strain H165, resulting in a total of 16 aa mutations mainly located within proteins 1a, 1ab, S, 3a, 3b, and E. Furthermore, six nt mutations (G6014TORF1a, T12388CORF1b, T21937CS, T21969AS, A26025CE, and C27507TN) could be the makers used to differentiate the Chinese vaccine strain from other strains of TGEV in the GenBank. Vaccines based on passages 155 to 165 in cell cultures are available commercially as vaccines for the prevention and control of infections with TGEV in China. Nucleoprotein (N) sequences of the virus at passages 155 and 165 were aligned and compared using a computer software program. The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiation of the vaccine strain from the other TGEVs was investigated. The RFLP analysis identified a change in the cleavage sites of Acl I at passages 155 and 165. This RFLP pattern of the N gene differentiated the Chinese vaccine strain from its parental strain, the TGEVs studied and the other reported TGEVs in the GenBank. Phylogenetic and sequence analysis results showed that the Chinese TGEVs were divided into three groups with several specific nucleotides and amino acids among them. These findings suggest that Chinese strains of TGEV are evolving continuously.In order to determine the genetic basis of TGEV attenuation for virulent H, we would like to construction the reverse genetics system based on the pBAC-5`- 3` vector, which was kindly provided by the Prof. Luis Enjuanes in Campus Universidad Autonoma. Five Intermediate vector plasmid with the corrected sequences were prepared to insert into the pBAC-5`- 3` vector. Up to now, three fragments had been inserted into the vector, In order to construct the reverse genetics system of attenuated H, it is necessary to insert the left two fragments into the vector in the following study.更多还原