禽肿瘤病混感病例中REV基因序列分析及LTR重组MDV的鉴定

发布时间：2018-03-18 23:15

本文选题：鸡马立克氏病病毒　切入点：禽网状内皮组织增生病病毒　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：鸡马立克氏病(MD)是由鸡马立克氏病病毒(MDV)引起的一种鸡的传染性、肿瘤性疾病,可导致其主要宿主鸡的免疫抑制和以淋巴细胞增生为特征的肿瘤发生,直至死亡。禽网状内皮组织增生病(RE)是另一种致瘤性的疾病,其病原为禽网状内皮组织增生症病毒(REV),能引起鸡、火鸡、鸭和其他禽类的慢性肿瘤、矮小综合征及免疫器官萎缩等临床症状。近些年来,我国MDV和REV混合感染的病例逐渐增加,二者混合感染导致宿主更早的发病,临床症状更加严重。此外,污染REV的MD疫苗也是RE流行的一个重要来源。这些问题增加了MD和RE的防控难度。疱疹病毒MDV与逆转录病毒REV在体内和体外混合感染,会发生REV基因组,尤其是它的LTR序列片段在MDV基因组的整合。LTR具有启动子和增强子的活性,可调控MDV某些的基因表达,导致MDV的致病性和水平传播能力等生物学特性的改变,对MDV的演化具有重要意义。本研究从山东省和辽宁省2个发生肿瘤病的养鸡场采集病料,经琼脂扩散试验和PCR检测证实2个鸡群发生的均为MD。进一步的检测显示,2个鸡群存在REV的混合感染。为了进一步研究混感病料中REV的分子特征,我们在DF-1细胞上进行了REV的病毒分离,获得了2株REV病毒株。获得的毒株利用PCR、间接免疫荧光(IFA)和电镜观察等方法进行了鉴定。分离到的2株REV分别命名为CY1111株和SY1209株。在此基础上,对CY1111株和SY1209株进行了前病毒基因组测序,并与其他已发表的REV分离株进行序列比对和遗传进化分析。根据基因组不同区域的序列构建了LTR、gag基因、pol基因、env基因和全基因的遗传进化树。遗传进化树分析显示:我们获得的2株REV分离株和其他中国分离株,除GD1210株外,均位于同一个进化分枝,表明我国REV分离株之间具有较高的遗传进化相关性。值得注意的是,我们分离的REV CY1111株和SY1209株,与来源于MD商品疫苗中的MD-2毒株之间序列相似性达到99.8%,遗传进化分析也显示它们位于同一个分枝,推测两个鸡场中的REV可能来源于疫苗污染,这提示我们应加强对MD疫苗的质量控制。此外,进化分析的结果现实REV分离株并没有表现出宿主或地域的特异性,在自然条件下,REV可能能够自由地在不同宿主以及不同地域之间传播。为了研究REV在MDV基因组中整合的发生,我们采用一种检测REV LTR在MDV基因组中整合的HS-cPCR(Hot Spot Combined PCR)方法,用于检测REV LTR在MDV中重组的热点位置。应用该方法对REV和MDV在体内和体外混合感染过程中的病毒之间的插入重组进行了检测。MDV LCY-BAC株为本实验室在混感REV的MDV野毒株分离过程中获得的毒株,该毒株经细菌人工染色体技术(BAC)进行了病毒纯化。对其检测发现,MDV LCY-BAC株基因组中存在两个REV的完整LTR序列插入,插入位置分别在基因组的短独特区(Us)两侧与内部短重复区(IRs)和末端短重复区(TRs)交接处,而且这两个LTR的插入方向相同,均为反向插入到MDV基因组。为研究这种重组病毒的遗传稳定性,将LCY-BAC株体外传至15代,对第2、5、8、12和15代分别进行了检测,结果发现LTR插入的位置、方向和拷贝数均没有发生变化,证明具有这种REV LTR插入的重组MDV毒株能够在体外稳定遗传,对研究MDV和REV之间的重组具有重要意义。通过HS-cPCR检测来自8个不同鸡群的确诊为MDV和REV混合感染的病鸡样品,并没有发现有LTR重组的现象,说明自然发生LTR重组MDV的几率可能较低。本研究对混合感染病料中的REV进行了病毒分离、鉴定和遗传进化分析,发现我国REV毒株遗传进化关系接近,并且推测疫苗污染是REV的一种可能的来源。REV重组MDV的分析研究发现了REV LTR一种新的重组插入方式和插入位置,并且这种新的重组病毒在体外具有良好的遗传稳定性;对一定数量的自然混感病例的检测未发现自然重组的野毒株。本研究为两种主要禽肿瘤病原的重组、演化以及它们的防控提供了一些重要的研究数据和理论依据。
[Abstract]:Marek's disease (MD) is made of chicken Marek's disease virus (MDV) chicken caused by infectious and neoplastic diseases, can cause immune suppression and its main host chicken with lymphocyte proliferation of tumor, until death. Avian Reticuloendotheliosis (RE) is a kind of tumor of the disease, the pathogen of Avian Reticuloendotheliosis Virus (REV), can cause chickens, turkeys, ducks and other birds of short stature syndrome and chronic cancer, immune organ atrophy and other clinical symptoms. In recent years, our country gradually increased MDV and REV cases of mixed infection, cause host earlier mixed infection two, the clinical symptoms are more serious. In addition, an important source of pollution in the REV MD RE vaccine is also popular. These problems increased MD and RE prevention difficult. Herpes simplex virus MDV and retroviral REV in vivo and in vitro mixed infection occurs The genome of REV, especially LTR sequence with its promoter and enhancer activity in the integration of.LTR MDV genome, can regulate the expression of MDV gene leads to some biological characteristics of MDV, pathogenicity and transmissibility of the change, has important significance for the evolution of MDV. This study collected from 2 place tumor disease of chicken in Shandong province and Liaoning Province, the agar diffusion test and PCR test confirmed that the groups had 2 chickens were showed further detection of MD. mixed infection 2 chickens of REV. In order to further study the characteristics of mixed into susceptible material in REV, we performed a virus isolation REV in DF-1 cells, obtained 2 strains of REV virus strains. The strains using PCR, indirect immunofluorescence (IFA) and electron microscopy and other methods were identified. The isolated 2 strains of REV were named CY1111 and SY1209 strains. On the basis of CY111 1 and SY1209 strains of proviral genome sequencing, and sequence alignment and phylogenetic analysis with other published REV strains. According to the different regions of the genome sequence of LTR was constructed, gag gene, pol gene, phylogenetic tree of env gene and gene. Phylogenetic analysis showed that strain and other we have Chinese isolates from 2 strains of REV, in addition to GD1210 strain, were located in the same clade, showed that genetic evolution has high correlation between REV isolates in China. It is worth noting that we isolated REV CY1111 strain and SY1209 strain, and MD from the commodity in the vaccine MD-2 strain the sequence similarity is 99.8%, phylogenetic analysis also show that they are located in the same branch, that two farms in REV may be derived from the vaccine contamination, suggesting that we should strengthen the quality control of MD vaccine. In addition, phylogenetic analysis The results showed REV isolates did not show specific host or region, under natural conditions, REV may be able to spread freely between different hosts and different regions. In order to study the occurrence of REV in MDV genome integration, we adopt a REV LTR detection in MDV genome integrated HS-cPCR (Hot Spot Combined PCR) method for detection of REV LTR hot spots in MDV recombination. This method was used to detect the.MDV LCY-BAC strain of MDV field strains in the laboratory are mixed REV obtained in the separation process of recombinant strains inserted between REV and MDV in vivo and in vitro mixed infection process of the virus, the virus the bacterial artificial chromosome Technology (BAC) were found. The purification of virus detection, complete LTR sequence of two REV genomic MDV LCY-BAC strain insert, insert position respectively in the genome of the short unique Area (Us) on both sides and internal short repeats (IRs) and the end of the short repeat region (TRs) junction, and the two LTR into the same direction, are inserted into the MDV genome. Reverse genetic stability of the recombinant virus, LCY-BAC strain in vitro to the 15 generation of the 2,5,8,12, and the 15 generation were detected, the results showed that LTR inserted into the position, direction and copy number are not changed, this has proved REV LTR insertion of recombinant MDV strains in vitro genetic stability, is of great significance to the study of MDV and REV between the recombinant. Detected by HS-cPCR from 8 different chickens were diagnosed as MDV the mixture of REV and infected chicken samples, and found no LTR recombination phenomenon, that the probability of a naturally occurring LTR recombinant MDV may be lower. This study was carried out for virus isolation mixed virus infection in REV, identification and analysis of genetic evolution, I found Close to the REV strain genetic evolutionary relationship, and that the vaccine pollution is discussed a possible source of.REV recombinant MDV found REV REV LTR a new recombinant insert mode and insert position, and the new recombinant virus has good genetic stability in vitro; detection of a certain number of natural mixed case not found wild strains of natural recombinant. This study consists of two kinds of main pathogenic avian tumor recombinant, provides some important research data and theoretical basis for the evolution and their prevention and control.

【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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