肉食动物细小病毒流行株分子特征与致病性分析

发布时间：2018-01-06 18:41

本文关键词：肉食动物细小病毒流行株分子特征与致病性分析　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

【摘要】：肉食动物细小病毒属于细小病毒科、原细小病毒属成员,包括犬细小病毒(Canine parvovirus,CPV),猫细小病毒(Feline parvovirus,FPV)和水貂肠炎病毒(Mink enteritis virus,MEV)等。该病毒能感染猫科、犬科、鼬科、浣熊科及灵猫科等家养及野生多种动物发病,肉食动物细小病毒引起的疾病存在于世界各地,是危害上述肉食目动物的主要传染病之一,每年都造成巨大的经济损失。CPV、FPV和MEV等肉食动物细小病毒关系密切,本文从分子诊断学、流行病学、免疫原性及遗传进化、转录组学等方面对我国肉食动物细小病毒进行了研究和探讨。首先建立了肉食动物细小病毒通用的纳米PCR检测方法,为我国肉食动物细小病毒流行病学调查提供了有效的手段。该方法敏感性可以检测到8.75×101个拷贝的重组质粒,是传统PCR的100倍;特异性试验表明本方法适合于CPV、FPV和MEV等肉食动物细小病毒的检测,而与其他相关病毒均无交叉反应性;临床样本检测表明本方法适用性良好,对于肉食动物细小病毒感染动物检出率较高。采用分子生物学方法对我国北方主要省市的CPV分子流行病学进行调查,对收集于2013~2016年间的221份CPV病料进行纳米PCR初筛,克隆测序病毒的VP2基因和NS1基因,分析氨基酸位点突变情况,进行同源性比对及系统发育树构建。结果表明我国CPV-2型、2a型、2b型和2c型共同流行,主要流行CPV-2a型,2014年以后北京和河北等地区开始出现CPV-2c型;首次发现VP2上Ala5Gly新的突变,NS1上Val160Gly,Ala586Thr,Ala587Thr和Leu600Arg4个位点发生新的突变;完成了114株病毒VP2基因和49株病毒NS1基因序列测定。进一步对我国流行的不同亚型的CPV进行分离鉴定,对CPV-2c型代表毒株进行免疫原性研究。结果表明,分别分离得到CPV-2型、2a型、2b型及2c型病毒2株、4株、5株和9株,鉴定表明分离毒株具有CPV典型特征,动物回归试验表明试验组比格犬呈现出细小病毒感染的典型症状,免疫原性试验表明分离得到的CPV-2c型毒株能够诱导比格犬产生中和抗体。序列分析表明分离的CPV-2a和CPV-2b型毒株都是Ser297Ala突变的新2a/2b型毒株,CPV-2c具有Ala5Gly突变的变异毒株,国内首次分离鉴定2c型CPV。为研制不同亚型源CPV疫苗提供了基础。为了更好的了解我国MEV的流行情况,采用F81细胞从疑似患有肠炎的水貂粪便样品中分离出2株病毒,经形态学、血清学、动物回归试验和分子生物学鉴定,分离的病毒为MEV,分别命名为MEV/LN-10和SD12/01。对分离毒株VP2基因及其全基因组进行遗传进化和基因重组分析表明,MEV/LN-10株病毒发生了自然重组,其主要与次要亲本病毒分别是MEV-SDNH株和cpv/nj01/06株,首次发现CPV和MEV的自然重组现象,并且分离到重组毒株。选取遗传背景明确的MEVB株做为肉食动物细小病毒代表,利用RNA-Seq技术分析F81细胞感染MEV前后宿主基因转录组的变化,共设感染0 h、3 h、6 h、12 h和24 h五组。结果表明在感染前后共筛选出614个差异基因,其中上调基因123个,下调基因491个;选取了14个差异基因进行了qRT-PCR验证,结果表明测序差异显著的组别与qRT-PCR验证结果一致。功能分析表明这些差异基因与细胞增值和细胞周期、免疫反应、肿瘤发生或抑制、细胞凋亡、信号通路、受体活性、分子传感器活性等相关。本研究构建了F81细胞感染MEV前后的mRNA数据库。
[Abstract]:Small carnivorous animal virus belongs to the Parvoviridae, the original members of the genus parvovirus, including canine parvovirus (Canine parvovirus, CPV), feline parvovirus (Feline parvovirus, FPV) and mink enteritis virus (Mink enteritis, virus, MEV). The virus can infect feline, canine, Mustelidae, the incidence of the raccoon family and civet a variety of domestic and wild animal, carnivorous animal caused by parvovirus disease in the world, is one of the major infectious diseases of the carnivorous animal,.CPV has caused huge economic losses each year, FPV and MEV, a carnivorous animal parvovirus is closely related to the molecular diagnostics, epidemiology, immunogenicity and genetic evolution, transcriptomics and other aspects of the study and discussion of carnivorous parvovirus animal in China. Firstly, nano PCR inspection carnivorous animal parvovirus general measuring method for China's fine carnivorous animal The investigation of virus epidemiology provides an effective method. This method can detect the sensitivity of 8.75 * 101 recombinant plasmid copy, is 100 times the traditional PCR; the specificity test showed that this method is suitable for the detection of CPV, FPV and MEV and other carnivorous animal parvovirus, and other related viruses had no cross reactivity the detection of clinical samples showed that; the method is good for carnivorous animal parvovirus infection. The higher detection rate of animal molecular biology methods of molecular epidemiology of CPV in northern China's major cities were investigated on 221 samples of 2013~ collected in CPV disease 2016 years material nano PCR screening, VP2 gene and NS1 gene cloning and sequencing virus, mutation analysis, homology comparison and phylogenetic tree construction. The results show that China's CPV-2 type, 2 type, 2b type and 2C type common popular, mainly for CPV-2 a, 2014 Years after the Beijing and Hebei areas began to appear CPV-2c type; first discovered the new mutation Ala5Gly VP2, NS1 Val160Gly, Ala586Thr, Ala587Thr and Leu600Arg4 loci with new mutations; the determination of gene 114 strains and 49 strains of VP2 virus NS1 gene sequence. Further to different subtypes of CPV epidemic in China identification of immunogenicity of CPV-2c strains. The results showed that the isolated type CPV-2, 2 type, 2b type and 2C type 2 virus strains, 4 strains, 5 strains and 9 strains of CPV, identified with typical characteristics, animal regression test showed that the experimental group showed in beagle dogs the typical symptoms of parvovirus infection, immunity test showed that CPV-2c strain isolated could induce canine neutralizing antibody. Sequence analysis showed that CPV-2a and CPV-2b strains isolated are new type 2a/2b virus Ser297Ala mutation CPV-2c is a variant strain strain, Ala5Gly mutation, isolation and identification of 2C CPV. for the first time in China to provide a basis for the development of different types of source CPV vaccine. In order to better understand the prevalence of MEV, the F81 cells isolated from 2 strains of virus, suspected of mink enteritis from fecal samples by morphology, serology. Animal regression test and molecular identification, the isolated virus was MEV, which were named MEV/LN-10 and SD12/01. genetic evolution and gene recombination of genes of isolates VP2 and genomic analysis showed that the natural recombinant MEV/LN-10 virus, its primary and secondary parent virus were MEV-SDNH and cpv/nj01/06 strains, first discovered natural recombination CPV and MEV, and isolated recombinant strains. Select clear genetic background of MEVB strain for carnivorous animal parvovirus, analysis of F81 cells using RNA-Seq Technology The changes of MEV before and after infection of the host gene transcription group, a total of 0 infected h, 3 h, 6 h, 12 h and 24 h. The results show that in the five groups before and after infection were screened out 614 genes, including 123 up-regulated genes and 491 down regulated genes; selected 14 differentially expressed genes were verified by qRT-PCR the results show that, significant differences between the groups and qRT-PCR sequencing results. Functional analysis showed that the value of these differences of gene and cell and cell cycle, immune response, tumor or inhibition of apoptosis, signal transduction, receptor activity, molecular sensor activity. This study constructed F81 cells infected with MEV and mRNA database.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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