豬瘟病毒分子流行病學(xué)研究及流行毒株全基因組序列分析
發(fā)布時間：2018-09-12 19:41
【摘要】：豬瘟(Classical swine fever, CSF)是由屬于黃病毒科(Flaviviridae)瘟病毒屬(Pestivirus)的豬瘟病毒(Classical swine fever virus, CSFV)引發(fā)的豬的一種急性、發(fā)燒和接觸性傳染傳染病。每年對我國養(yǎng)豬業(yè)造成重大經(jīng)濟損失,被國際獸疫防治局(OIE)列為重大動物疫病。近些年來,全國范圍內(nèi)豬瘟兔化弱毒疫苗的使用有效地控制了豬瘟的大規(guī)模流行,但豬瘟在我國仍有散布流行,尚不能完全被消滅。一些學(xué)者認為,現(xiàn)在流行的毒株偏離弱毒疫苗株,是造成豬瘟疫苗對我國豬瘟病毒免疫保護作用有限的主要原因,也是疫苗免疫失敗的一個重要原因。為了解我國豬瘟病毒流行及遺傳變異情況,為有效地防治豬瘟提供理論依據(jù),本論文進行了如下內(nèi)容的研究： (1) CSFVE2基因遺傳變異研究,采用RT-nestPCR方法,對2012-2014年來自我國12個省份的234份豬瘟陽性病料中的E2基因高變區(qū)的190片段進行序列克隆和測序,并對部分代表毒株的E2全基因進行克隆和測序,通過MEGA和DNAstar軟件利用E2基因高變區(qū)的190片段序列構(gòu)建了234株流行毒株的系統(tǒng)進化樹,對部分具有代表性的毒株的E2全基因與參考毒株核苷酸及氨基酸的同源性進行比較,分析CSFV流行毒株E2蛋白的遺傳變異情況。 (2)分離株HuN23/2013和GXF29/2013全基因組的研究：參考Genbank上已發(fā)表CSFV全基因序列,設(shè)計引物,通過RT-PCR成功的分4段擴增了CSFV分離株HuN23/2013和GXF29/2013全基因組,并對兩個分離株的同源性及細胞表位變異進行了分析。 結(jié)果表明：我國流行的CSFV仍然以2.1亞群中的2.1b類占絕對優(yōu)勢,但也出現(xiàn)了一些新的變化,一是以石門株為代表的1.1亞群強毒株在我國可能已經(jīng)消失,出現(xiàn)了新的2.1c類病毒,該類病毒于2011年后在我國流行的區(qū)域和流行強度有上升的趨勢。2.1b類優(yōu)勢流行毒株和新出現(xiàn)的2.1c類毒株在某些重要的抗原表位位點和糖基化位點與臨床上使用的豬瘟兔化疫苗存在差異,可能影響疫苗的免疫效果,也可能是目前非典型CSF長期存在的原因。對2013年兩株流行毒株HuN23/2013和GXF29/2013的全基因序列分析表明,測定HuN23/2013和GXF29/2013全長分別為12295bp和12296bp,HuN23/2013是我國主要流行的2.1b類毒株,GXF29/2013是我國新近分化出來的2.1c類毒株,為建立CSFV反向遺傳操作平臺打下基礎(chǔ)。
[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.
【學(xué)位授予單位】：海南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.651
本文編號：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.
【學(xué)位授予單位】：海南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.651
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