猪瘟病毒分子流行病学研究及流行毒株全基因组序列分析
发布时间：2018-09-12 19:41
【摘要】：猪瘟(Classical swine fever, CSF)是由属于黄病毒科(Flaviviridae)瘟病毒属(Pestivirus)的猪瘟病毒(Classical swine fever virus, CSFV)引发的猪的一种急性、发烧和接触性传染传染病。每年对我国养猪业造成重大经济损失,被国际兽疫防治局(OIE)列为重大动物疫病。近些年来,全国范围内猪瘟兔化弱毒疫苗的使用有效地控制了猪瘟的大规模流行,但猪瘟在我国仍有散布流行,尚不能完全被消灭。一些学者认为,现在流行的毒株偏离弱毒疫苗株,是造成猪瘟疫苗对我国猪瘟病毒免疫保护作用有限的主要原因,也是疫苗免疫失败的一个重要原因。为了解我国猪瘟病毒流行及遗传变异情况,为有效地防治猪瘟提供理论依据,本论文进行了如下内容的研究： (1) CSFVE2基因遗传变异研究,采用RT-nestPCR方法,对2012-2014年来自我国12个省份的234份猪瘟阳性病料中的E2基因高变区的190片段进行序列克隆和测序,并对部分代表毒株的E2全基因进行克隆和测序,通过MEGA和DNAstar软件利用E2基因高变区的190片段序列构建了234株流行毒株的系统进化树,对部分具有代表性的毒株的E2全基因与参考毒株核苷酸及氨基酸的同源性进行比较,分析CSFV流行毒株E2蛋白的遗传变异情况。 (2)分离株HuN23/2013和GXF29/2013全基因组的研究：参考Genbank上已发表CSFV全基因序列,设计引物,通过RT-PCR成功的分4段扩增了CSFV分离株HuN23/2013和GXF29/2013全基因组,并对两个分离株的同源性及细胞表位变异进行了分析。 结果表明：我国流行的CSFV仍然以2.1亚群中的2.1b类占绝对优势,但也出现了一些新的变化,一是以石门株为代表的1.1亚群强毒株在我国可能已经消失,出现了新的2.1c类病毒,该类病毒于2011年后在我国流行的区域和流行强度有上升的趋势。2.1b类优势流行毒株和新出现的2.1c类毒株在某些重要的抗原表位位点和糖基化位点与临床上使用的猪瘟兔化疫苗存在差异,可能影响疫苗的免疫效果,也可能是目前非典型CSF长期存在的原因。对2013年两株流行毒株HuN23/2013和GXF29/2013的全基因序列分析表明,测定HuN23/2013和GXF29/2013全长分别为12295bp和12296bp,HuN23/2013是我国主要流行的2.1b类毒株,GXF29/2013是我国新近分化出来的2.1c类毒株,为建立CSFV反向遗传操作平台打下基础。
[Abstract]:Classical swine fever (Classical swine fever, CSF) is an acute, feverish and contagious disease in pigs caused by (Classical swine fever virus, CSFV) of (Flaviviridae) cholera virus belonging to (Pestivirus). It causes great economic loss to the pig industry of our country every year and is listed as a major animal disease by the (OIE) of the International Bureau of Animal Disease Prevention and Cure. In recent years, the use of hog fever rabbit attenuated vaccine in China has effectively controlled the large-scale epidemic of swine fever, but the spread of classical swine fever in China has not been completely eliminated. Some scholars believe that the main reason for the limited protection of swine fever vaccine against swine fever virus is that the prevalent strain deviates from the attenuated vaccine strain, and it is also an important reason for the failure of vaccine immunization. In order to understand the prevalence and genetic variation of CSFV in China and to provide theoretical basis for the effective control of CSFV, the following contents were studied in this paper: (1) genetic variation of CSFVE2 gene was studied by RT-nestPCR method. The 190 fragments of E2 gene in 234 samples of CSFV from 12 provinces of China from 2012 to 2014 were cloned and sequenced. Some of the E2 genes representing the virulent strains were cloned and sequenced. The phylogenetic tree of 234 epidemic strains was constructed by using the 190fragment sequence of E2 gene hypervariable region by MEGA and DNAstar software. The homology of nucleotide and amino acid of E2 gene of some representative strains was compared with that of reference strain. The genetic variation of E2 protein of CSFV epidemic strain was analyzed. (2) study on the whole genome of HuN23/2013 and GXF29/2013: referring to the whole CSFV gene sequence published on Genbank, the primers were designed. The whole genome of CSFV isolate HuN23/2013 and GXF29/2013 were amplified successfully by RT-PCR, and the homology and cell epitope variation of the two isolates were analyzed. The results show that the prevalence of CSFV in China is still dominated by 2.1 b subgroup, but there are some new changes. One is that the strong subgroup 1.1 strain, represented by Shimen strain, may have disappeared in China, and a new type 2.1c virus has appeared. After 2011, the epidemic area and the epidemic intensity of this kind of virus have an increasing trend. The dominant epidemic strain of class 2.1b and the new strain of class 2.1c are used in some important epitopes, glycosylation sites and clinical use in pigs. There are differences in rabbit-specific vaccines, It may affect the immune effect of the vaccine and may be the reason for the long-term existence of atypical CSF. The whole gene sequence analysis of HuN23/2013 and GXF29/2013 showed that the total length of HuN23/2013 and GXF29/2013 were 12295bp and 12296bpHN23 / 2013, respectively, and GXF29 / 2013 was the newly differentiated type 2.1c strain in China. For the establishment of CSFV reverse genetic operation platform lay the foundation.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.651
本文编号：2240035
[Abstract]:Classical swine fever (Classical swine fever, CSF) is an acute, feverish and contagious disease in pigs caused by (Classical swine fever virus, CSFV) of (Flaviviridae) cholera virus belonging to (Pestivirus). It causes great economic loss to the pig industry of our country every year and is listed as a major animal disease by the (OIE) of the International Bureau of Animal Disease Prevention and Cure. In recent years, the use of hog fever rabbit attenuated vaccine in China has effectively controlled the large-scale epidemic of swine fever, but the spread of classical swine fever in China has not been completely eliminated. Some scholars believe that the main reason for the limited protection of swine fever vaccine against swine fever virus is that the prevalent strain deviates from the attenuated vaccine strain, and it is also an important reason for the failure of vaccine immunization. In order to understand the prevalence and genetic variation of CSFV in China and to provide theoretical basis for the effective control of CSFV, the following contents were studied in this paper: (1) genetic variation of CSFVE2 gene was studied by RT-nestPCR method. The 190 fragments of E2 gene in 234 samples of CSFV from 12 provinces of China from 2012 to 2014 were cloned and sequenced. Some of the E2 genes representing the virulent strains were cloned and sequenced. The phylogenetic tree of 234 epidemic strains was constructed by using the 190fragment sequence of E2 gene hypervariable region by MEGA and DNAstar software. The homology of nucleotide and amino acid of E2 gene of some representative strains was compared with that of reference strain. The genetic variation of E2 protein of CSFV epidemic strain was analyzed. (2) study on the whole genome of HuN23/2013 and GXF29/2013: referring to the whole CSFV gene sequence published on Genbank, the primers were designed. The whole genome of CSFV isolate HuN23/2013 and GXF29/2013 were amplified successfully by RT-PCR, and the homology and cell epitope variation of the two isolates were analyzed. The results show that the prevalence of CSFV in China is still dominated by 2.1 b subgroup, but there are some new changes. One is that the strong subgroup 1.1 strain, represented by Shimen strain, may have disappeared in China, and a new type 2.1c virus has appeared. After 2011, the epidemic area and the epidemic intensity of this kind of virus have an increasing trend. The dominant epidemic strain of class 2.1b and the new strain of class 2.1c are used in some important epitopes, glycosylation sites and clinical use in pigs. There are differences in rabbit-specific vaccines, It may affect the immune effect of the vaccine and may be the reason for the long-term existence of atypical CSF. The whole gene sequence analysis of HuN23/2013 and GXF29/2013 showed that the total length of HuN23/2013 and GXF29/2013 were 12295bp and 12296bpHN23 / 2013, respectively, and GXF29 / 2013 was the newly differentiated type 2.1c strain in China. For the establishment of CSFV reverse genetic operation platform lay the foundation.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.651
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