發(fā)布時間：2018-03-03 03:20

  本文選題：E種腸道病毒　切入點：全長基因組序列　出處：《吉林大學》2017年博士論文　論文類型：學位論文

【摘要】：牛腸道病毒(Bovine enteroviruses)屬于小RNA病毒科腸道病毒屬的成員,該病毒主要引起動物的呼吸道和消化道疾病,給養(yǎng)殖業(yè)帶來極大的危害。2012年,本實驗室從吉林省暴發(fā)的一種臨床上以消化道和呼吸道為主要特征、發(fā)病率及死亡率高達50%以上的病牛群分離到國內首株E種腸道病毒HY12毒株。由于牛腸道病毒感染在國內為新發(fā)疫病,有關該病毒的生物學特性、致病機理等方面缺乏深入研究。本研究應用RT-PCR方法對HY12毒株的全長基因組序列進行擴增,獲得的HY12毒株全長基因組序列為7 469 bp,編碼一個2 176個氨基酸的前體多聚蛋白。序列分析顯示,HY12毒株的全基因組序列與SL305毒株的親緣關系最近,為E種腸道病毒。HY12結構蛋白基因(VP1、VP2、VP3和VP4)的核苷酸序列與14/3/96毒株的親緣關系最近;而5′UTR及3D基因與SL305毒株的親緣關系最近,表明HY12毒株可能為重組病毒,屬于E種腸道病毒的E3基因亞型。與其它牛腸道病毒比較,HY12毒株的VP1和VP4蛋白存在多個特有的氨基酸位點。對同一代次的HY12毒株的序列測定發(fā)現(xiàn),HY12毒株基因組的多位點在同一核苷酸位置出現(xiàn)不同的堿基序列,表明HY12毒株以病毒的準種形式存在。對HY12準種的進化與變異的研究發(fā)現(xiàn),不同代次的HY12傳代病毒其VP1和VP2基因序列與HY12病毒的主序列不完全相同,為不同的克隆類型,確定HY12準種存在明顯的多樣性和復雜性。同時發(fā)現(xiàn),HY12病毒準種在傳代過程中其突變率、復雜性隨著傳代次數(shù)的增加逐漸增大,主序列的比例及序列間的同源性隨著傳代次數(shù)的增加逐漸降低的進化規(guī)律。生物學特性的比對結果顯示,不同代次的HY12毒株的生物學特性存在明顯差異。與第2代的HY12病毒比較,第40代次HY12病毒的TCID50增高,蝕斑大小明顯增大。對不同代次的HY12主序列分析發(fā)現(xiàn),第40代次VP1基因272位核苷酸的點突變(272 GA),導致其91位氨基酸由精氨酸突變?yōu)榻M氨酸(R91H),該氨基酸的突變使其抗原性及三級結構發(fā)生改變。應用分子生物學、反向遺傳學、病毒學等技術手段成功構建出E種腸道病毒HY12毒株的全長感染性克隆Dp OK12-HY12,并成功拯救出HY12病毒(r HY12)和建立HY12病毒的反向遺傳學平臺,并利用該拯救平臺,拯救出VP1突變株病毒r HY12(H),證實了VP1蛋白(R91H)的突變導致HY12病毒生物學特性顯著改變和病毒復制的增強。與親本毒HY12和拯救毒r HY12相比,r HY12(H)的病毒滴度增高,蝕斑大小增大,且r HY12(H)病毒的基因組拷貝數(shù)和蛋白表達水平顯著升高,對3日齡的ICR乳鼠的致病性較強,表明VP1蛋白91位氨基酸由精氨酸突變?yōu)榻M氨酸(R91H)顯著改變HY12毒株生物學特性,影響病毒復制及其致病性。該結果為進一步深入研究病毒與宿主之間的相互作用、病毒的復制機理、致病機理及病毒準種的進化機制打下基礎。
[Abstract]:Bovine enterovirusesis a member of the family RNA viridae, which causes respiratory and digestive tract diseases in animals and causes great harm to the breeding industry. A clinical outbreak in Jilin Province was characterized by digestive tract and respiratory tract. The first HY12 strain of enterovirus E was isolated from cattle with morbidity and mortality of more than 50%. Because bovine enterovirus infection is a new epidemic disease in China, the biological characteristics of this virus are concerned. In this study, RT-PCR method was used to amplify the full-length genomic sequence of HY12 strain. The full-length genomic sequence of HY12 strain was 7 469 BP, encoding a 2,176 amino acid precursor protein. Sequence analysis showed that the whole genome sequence of HY12 strain was most closely related to SL305 strain. The nucleotide sequences of VP1VP2VP3 and VP4) were most closely related to 14 / 3 / 96 strains of enterovirus, while 5- and 3D genes were most closely related to SL305 strains, suggesting that HY12 strains might be recombinant viruses. Compared with other bovine enterovirus strains, VP1 and VP4 proteins of HY12 have many unique amino acid loci. Sequence analysis of the same generation of HY12 strains revealed that the genome of HY12 strain. Different nucleotide sequences appeared at the same nucleotide site at multiple loci. The results showed that the HY12 strains existed in the form of quasispecies of the virus. The study on the evolution and variation of HY12 quasispecies showed that the sequences of VP1 and VP2 genes in different generations of HY12 subculture viruses were not identical to the main sequences of HY12 viruses, and they were different clone types. At the same time, it was found that the mutation rate and complexity of quasispecies of HY12 increased with the increase of passage times. The evolutionary law that the proportion of the main sequence and the homology among the sequences gradually decrease with the increase of the number of generations. Compared with the second generation of HY12 virus, the TCID50 of the 40th generation of HY12 virus increased and the size of the plaque increased obviously. The main sequence analysis of HY12 of different generations showed that there was a significant difference in the biological characteristics of different generations of HY12 virus strains, and compared with the second generation of HY12 virus, the TCID50 of the 40th generation of HY12 virus increased and the size of plaque increased obviously. The point mutation of 272-nucleotide at the 40th generation of VP1 gene led to the amino acid mutation of 91 amino acids from arginine to histidine R91H. The mutation of this amino acid changed its antigenicity and tertiary structure. The full-length infectious clone DpOK12-HY12 of E species enterovirus HY12 strain was successfully constructed by virology, and the reverse genetic platform of HY12 virus was successfully saved by using DpOK12-HY12) and the reverse genetic platform of HY12 virus was established. It was proved that the mutation of VP1 protein R91H) resulted in a significant change in the biological characteristics of HY12 virus and the enhancement of virus replication. The titer of rHY12H1H) was higher and the size of plaque was larger than that of parent virus HY12 and rescue virus r HY12. Moreover, the genomic copy number and protein expression of r HY12 / H) virus were significantly increased, and the pathogenicity of rHY12 / HV was stronger in ICR suckling mice at 3 days of age, indicating that the amino acid mutation of VP1 protein 91 from arginine to histidine R91H significantly changed the biological characteristics of HY12 strain. The results laid a foundation for further study on the interaction between virus and host, the mechanism of virus replication, the mechanism of pathogenicity and the evolution mechanism of virus quasispecies.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S852.65

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