本文選題：雞馬立克氏病病毒　切入點：禽網(wǎng)狀內(nèi)皮組織增生病病毒　出處：《中國農(nóng)業(yè)科學院》2015年碩士論文　論文類型：學位論文

【摘要】：雞馬立克氏病(MD)是由雞馬立克氏病病毒(MDV)引起的一種雞的傳染性、腫瘤性疾病,可導致其主要宿主雞的免疫抑制和以淋巴細胞增生為特征的腫瘤發(fā)生,直至死亡。禽網(wǎng)狀內(nèi)皮組織增生病(RE)是另一種致瘤性的疾病,其病原為禽網(wǎng)狀內(nèi)皮組織增生癥病毒(REV),能引起雞、火雞、鴨和其他禽類的慢性腫瘤、矮小綜合征及免疫器官萎縮等臨床癥狀。近些年來,我國MDV和REV混合感染的病例逐漸增加,二者混合感染導致宿主更早的發(fā)病,臨床癥狀更加嚴重。此外,污染REV的MD疫苗也是RE流行的一個重要來源。這些問題增加了MD和RE的防控難度。皰疹病毒MDV與逆轉(zhuǎn)錄病毒REV在體內(nèi)和體外混合感染,會發(fā)生REV基因組,尤其是它的LTR序列片段在MDV基因組的整合。LTR具有啟動子和增強子的活性,可調(diào)控MDV某些的基因表達,導致MDV的致病性和水平傳播能力等生物學特性的改變,對MDV的演化具有重要意義。本研究從山東省和遼寧省2個發(fā)生腫瘤病的養(yǎng)雞場采集病料,經(jīng)瓊脂擴散試驗和PCR檢測證實2個雞群發(fā)生的均為MD。進一步的檢測顯示,2個雞群存在REV的混合感染。為了進一步研究混感病料中REV的分子特征,我們在DF-1細胞上進行了REV的病毒分離,獲得了2株REV病毒株。獲得的毒株利用PCR、間接免疫熒光(IFA)和電鏡觀察等方法進行了鑒定。分離到的2株REV分別命名為CY1111株和SY1209株。在此基礎上,對CY1111株和SY1209株進行了前病毒基因組測序,并與其他已發(fā)表的REV分離株進行序列比對和遺傳進化分析。根據(jù)基因組不同區(qū)域的序列構(gòu)建了LTR、gag基因、pol基因、env基因和全基因的遺傳進化樹。遺傳進化樹分析顯示:我們獲得的2株REV分離株和其他中國分離株,除GD1210株外,均位于同一個進化分枝,表明我國REV分離株之間具有較高的遺傳進化相關性。值得注意的是,我們分離的REV CY1111株和SY1209株,與來源于MD商品疫苗中的MD-2毒株之間序列相似性達到99.8%,遺傳進化分析也顯示它們位于同一個分枝,推測兩個雞場中的REV可能來源于疫苗污染,這提示我們應加強對MD疫苗的質(zhì)量控制。此外,進化分析的結(jié)果現(xiàn)實REV分離株并沒有表現(xiàn)出宿主或地域的特異性,在自然條件下,REV可能能夠自由地在不同宿主以及不同地域之間傳播。為了研究REV在MDV基因組中整合的發(fā)生,我們采用一種檢測REV LTR在MDV基因組中整合的HS-cPCR(Hot Spot Combined PCR)方法,用于檢測REV LTR在MDV中重組的熱點位置。應用該方法對REV和MDV在體內(nèi)和體外混合感染過程中的病毒之間的插入重組進行了檢測。MDV LCY-BAC株為本實驗室在混感REV的MDV野毒株分離過程中獲得的毒株,該毒株經(jīng)細菌人工染色體技術(BAC)進行了病毒純化。對其檢測發(fā)現(xiàn),MDV LCY-BAC株基因組中存在兩個REV的完整LTR序列插入,插入位置分別在基因組的短獨特區(qū)(Us)兩側(cè)與內(nèi)部短重復區(qū)(IRs)和末端短重復區(qū)(TRs)交接處,而且這兩個LTR的插入方向相同,均為反向插入到MDV基因組。為研究這種重組病毒的遺傳穩(wěn)定性,將LCY-BAC株體外傳至15代,對第2、5、8、12和15代分別進行了檢測,結(jié)果發(fā)現(xiàn)LTR插入的位置、方向和拷貝數(shù)均沒有發(fā)生變化,證明具有這種REV LTR插入的重組MDV毒株能夠在體外穩(wěn)定遺傳,對研究MDV和REV之間的重組具有重要意義。通過HS-cPCR檢測來自8個不同雞群的確診為MDV和REV混合感染的病雞樣品,并沒有發(fā)現(xiàn)有LTR重組的現(xiàn)象,說明自然發(fā)生LTR重組MDV的幾率可能較低。本研究對混合感染病料中的REV進行了病毒分離、鑒定和遺傳進化分析,發(fā)現(xiàn)我國REV毒株遺傳進化關系接近,并且推測疫苗污染是REV的一種可能的來源。REV重組MDV的分析研究發(fā)現(xiàn)了REV LTR一種新的重組插入方式和插入位置,并且這種新的重組病毒在體外具有良好的遺傳穩(wěn)定性;對一定數(shù)量的自然混感病例的檢測未發(fā)現(xiàn)自然重組的野毒株。本研究為兩種主要禽腫瘤病原的重組、演化以及它們的防控提供了一些重要的研究數(shù)據(jù)和理論依據(jù)。
[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.

【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.31

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