【作者】 谢金鑫；

【导师】 侯喜林；

【作者基本信息】 黑龙江八一农垦大学 ， 预防兽医学， 2010， 硕士

【摘要】 A群轮状病毒(Rotavirus, RV)是新生牛病毒性腹泻的主要病原之一。牛轮状病毒(Bovine Rotavirus, BRV)属于呼肠孤病毒科(Reoviridae),轮状病毒属(Rotavirus)。牛轮状病毒性腹泻多发生在1月龄以内的犊牛,腹泻犊牛严重脱水,甚至有些犊牛因电解质紊乱引起死亡。为了解A群轮状病毒在大庆牧场中的流行情况及优势流行株血清型,本研究自2008年12月至2009年6月在大庆某牧场共收集117份新生牛腹泻标本,其中10份标本(8.5%)经A群轮状病毒抗原检测试剂盒确认为阳性。应用RT-PCR方法对阳性标本进行分型鉴定,得知10份标本均为G10P[11]型轮状病毒。应用MA104细胞成功的从编号为75的阳性标本中分离出一株轮状病毒,命名为DQ-75。分析分离株DQ-75的全序列:根据以轮状病毒11节段基因编码区核苷酸同源性为基础的分类原则,我们发现G10P[11]型牛轮状病毒基因节段VP7、VP4、VP1、VP2、VP3、VP6、NSP1、NSP2、NSP3、NSP4和NSP5的基因型分别为G10、P[11]、R2、C2、M2、I2、A3、N2、T6、E2和H3。分离株DQ-75 11段基因编码区氨基酸的进化树分析发现:分离株DQ-75为重配株,其中基因节段NSP2来源于G6P[5]型牛轮状病毒株,基因节段NSP1和NSP5来源于G6P[1]型牛轮状病毒,基因节段VP1、VP2、VP3和VP6分别来源于G8P[14]型羊轮状病毒株OVR762,基因节段NSP3来源于G10P[14]型人轮状病毒株A64,基因节段VP7、VP4和NSP4在分离株DQ-75中稳定进化。分离株DQ-75与同为G10P[11]型07年大庆分离株DQ-07,08年安达分离株AD-08以及我国分离株HM26的VP7核苷酸和氨基酸同源性分别为96.5%、96.5%、96.8%和97.5%、97.5%、98.5%。分析4株轮状病毒的5个主要抗原表位,我们推测近期G10型牛轮状病毒通过抗原表位氨基酸的突变来逃避宿主的免疫压力。本研究在大庆发现了G10P[11]型牛轮状病毒,应用MA104细胞进行成功分离。首次对G10P[11]型牛轮状病毒进行了全序列分析。为大庆地区牛轮状病毒的研究奠定理论基础。更多还原

【Abstract】 Group A rotavirus are one of the most frequently detected viral agents associated with the acute diarrhea in neonatal calves. Bovine rotavirus belongs to a genus rotavirus in the Reoviridea family. A complete viral particle contains a segments of 11 double-stranded RNA (dsRNA) that encode 6 structural (VP1-4, 6 and 7) and 6 nonstructural (NSP1-6) protein. Neonatal calves are susceptible to rotavirus, rotavirus cause disease including asymptomatic, mild , severe or fatal infection with excessive fluid loss and severe electrolyte imbalance. In order to investigate the situation of rotavirus strains circulating and popular strain in diary farm, Daqing region.A total of 117 fecal specimens were collected from diarrhea calves under 4 weeks-age on diary farm in Daqing region in China from 2008 to 2009. Ten specimens were detected to be positive by a Rotavirus Group A Diagnose Kit, which confirm that the rotavirus was important viral agent associated with diarrhea in this farm. According to RT-PCR, only G10P[11] rotavirus were found in positive samples. A G10P[11] rotavirus, designed as the DQ-75 strain, was isolated successfully from a positive specimen numbered 75. Full genomic analysis of DQ-75 strain based on nucleotide percentage identity cut-off values defining genotypes for 11 rotavirus gene segments was proposed, we found that genotypes of gene segments of VP7、VP4、VP1、VP2、VP3、VP6、NSP1、NSP2、NSP3、NSP4 and NSP5 of DQ-75 strain were G10、P[11]、R2、C2、M2、I2、A3、N2、T6、E2 and H3, respectively. Phylogenetic analyzing found that DQ-75 strain was a reassortment strain including intraspecies and interspecies, gene segments of NSP1 and NSP5 could originate from genotype G6P[1] bovine rotavirus; gene segment of NSP2 could originate from genotype G6P[5] bovine rotavirus; gene segment of NSP3 could originate from genotype G10P[14] bovine rotavirus A64 strain; gene segments of VP1、VP2、VP3 and VP6 could originate from lamb, gene segments of VP7、VP4 and NSP4 are evoluting among genotype G10 bovine rotavirus.Homology of the nucleotide and deduced amino acid sequences of the genome segments encoding VP7 of DQ-75 strain, DQ-07 strain isolated in Daqing 2007, AD-08 strain isolated in Anda 2008 and HM26 strain were 96.5%、96.5%、96.8% and 97.5%、97.5%、98.5%, respectively. Analysis of antigen sites of four G10P[11] bovine rotavirus above, we proposed that G10 bovine rotavirus strain protected themselves by amino mutation of 5 main antigenic regions.In this study we detected G10P[11] bovine rotavirus in a diary farm, Daqing region and isolated a strain DQ-75. Analysis of the first full genomic sequence of G10P[11] bovine rotavirus. and understood molecular characteristic of G10P[11] bovine rotavirus. Giving some theoretical background about reseaching G10P[11] bovine rotavirus.更多还原