本文關(guān)鍵詞：肉食動(dòng)物細(xì)小病毒流行株分子特征與致病性分析　出處：《中國農(nóng)業(yè)科學(xué)院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肉食動(dòng)物細(xì)小病毒 流行株 分子特征 致病性

【摘要】：肉食動(dòng)物細(xì)小病毒屬于細(xì)小病毒科、原細(xì)小病毒屬成員,包括犬細(xì)小病毒(Canine parvovirus,CPV),貓細(xì)小病毒(Feline parvovirus,FPV)和水貂腸炎病毒(Mink enteritis virus,MEV)等。該病毒能感染貓科、犬科、鼬科、浣熊科及靈貓科等家養(yǎng)及野生多種動(dòng)物發(fā)病,肉食動(dòng)物細(xì)小病毒引起的疾病存在于世界各地,是危害上述肉食目動(dòng)物的主要傳染病之一,每年都造成巨大的經(jīng)濟(jì)損失。CPV、FPV和MEV等肉食動(dòng)物細(xì)小病毒關(guān)系密切,本文從分子診斷學(xué)、流行病學(xué)、免疫原性及遺傳進(jìn)化、轉(zhuǎn)錄組學(xué)等方面對(duì)我國肉食動(dòng)物細(xì)小病毒進(jìn)行了研究和探討。首先建立了肉食動(dòng)物細(xì)小病毒通用的納米PCR檢測(cè)方法,為我國肉食動(dòng)物細(xì)小病毒流行病學(xué)調(diào)查提供了有效的手段。該方法敏感性可以檢測(cè)到8.75×101個(gè)拷貝的重組質(zhì)粒,是傳統(tǒng)PCR的100倍;特異性試驗(yàn)表明本方法適合于CPV、FPV和MEV等肉食動(dòng)物細(xì)小病毒的檢測(cè),而與其他相關(guān)病毒均無交叉反應(yīng)性;臨床樣本檢測(cè)表明本方法適用性良好,對(duì)于肉食動(dòng)物細(xì)小病毒感染動(dòng)物檢出率較高。采用分子生物學(xué)方法對(duì)我國北方主要省市的CPV分子流行病學(xué)進(jìn)行調(diào)查,對(duì)收集于2013~2016年間的221份CPV病料進(jìn)行納米PCR初篩,克隆測(cè)序病毒的VP2基因和NS1基因,分析氨基酸位點(diǎn)突變情況,進(jìn)行同源性比對(duì)及系統(tǒng)發(fā)育樹構(gòu)建。結(jié)果表明我國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區(qū)開始出現(xiàn)CPV-2c型;首次發(fā)現(xiàn)VP2上Ala5Gly新的突變,NS1上Val160Gly,Ala586Thr,Ala587Thr和Leu600Arg4個(gè)位點(diǎn)發(fā)生新的突變;完成了114株病毒VP2基因和49株病毒NS1基因序列測(cè)定。進(jìn)一步對(duì)我國流行的不同亞型的CPV進(jìn)行分離鑒定,對(duì)CPV-2c型代表毒株進(jìn)行免疫原性研究。結(jié)果表明,分別分離得到CPV-2型、2a型、2b型及2c型病毒2株、4株、5株和9株,鑒定表明分離毒株具有CPV典型特征,動(dòng)物回歸試驗(yàn)表明試驗(yàn)組比格犬呈現(xiàn)出細(xì)小病毒感染的典型癥狀,免疫原性試驗(yàn)表明分離得到的CPV-2c型毒株能夠誘導(dǎo)比格犬產(chǎn)生中和抗體。序列分析表明分離的CPV-2a和CPV-2b型毒株都是Ser297Ala突變的新2a/2b型毒株,CPV-2c具有Ala5Gly突變的變異毒株,國內(nèi)首次分離鑒定2c型CPV。為研制不同亞型源CPV疫苗提供了基礎(chǔ)。為了更好的了解我國MEV的流行情況,采用F81細(xì)胞從疑似患有腸炎的水貂糞便樣品中分離出2株病毒,經(jīng)形態(tài)學(xué)、血清學(xué)、動(dòng)物回歸試驗(yàn)和分子生物學(xué)鑒定,分離的病毒為MEV,分別命名為MEV/LN-10和SD12/01。對(duì)分離毒株VP2基因及其全基因組進(jìn)行遺傳進(jìn)化和基因重組分析表明,MEV/LN-10株病毒發(fā)生了自然重組,其主要與次要親本病毒分別是MEV-SDNH株和cpv/nj01/06株,首次發(fā)現(xiàn)CPV和MEV的自然重組現(xiàn)象,并且分離到重組毒株。選取遺傳背景明確的MEVB株做為肉食動(dòng)物細(xì)小病毒代表,利用RNA-Seq技術(shù)分析F81細(xì)胞感染MEV前后宿主基因轉(zhuǎn)錄組的變化,共設(shè)感染0 h、3 h、6 h、12 h和24 h五組。結(jié)果表明在感染前后共篩選出614個(gè)差異基因,其中上調(diào)基因123個(gè),下調(diào)基因491個(gè);選取了14個(gè)差異基因進(jìn)行了qRT-PCR驗(yàn)證,結(jié)果表明測(cè)序差異顯著的組別與qRT-PCR驗(yàn)證結(jié)果一致。功能分析表明這些差異基因與細(xì)胞增值和細(xì)胞周期、免疫反應(yīng)、腫瘤發(fā)生或抑制、細(xì)胞凋亡、信號(hào)通路、受體活性、分子傳感器活性等相關(guān)。本研究構(gòu)建了F81細(xì)胞感染MEV前后的mRNA數(shù)據(jù)庫。
[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.

【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65

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