禽痘病毒疫苗及野毒株重組禽網(wǎng)狀內(nèi)皮組織增生病病毒的研究
發(fā)布時(shí)間：2018-08-28 14:23
【摘要】：禽痘(Avian pox,AP)是由痘病毒科、脊椎動(dòng)物痘病毒亞科、禽痘病毒屬中的禽痘病毒引起的家禽的一種急性、接觸性傳染病。禽網(wǎng)狀內(nèi)皮組織增殖病(reticuloendotheliosis,RE)是由反轉(zhuǎn)錄病毒科的禽網(wǎng)內(nèi)皮組織增殖病病毒(reticuloendotheliosis virus,REV)引起的幾種禽類的一群病理綜合征,REV基因片段整合進(jìn)FPV疫苗、基因片段甚至完整的基因組整合進(jìn)FPV野毒株已成為普遍現(xiàn)象。研究表明,隨著FPV和REV整合的不斷發(fā)展,其對(duì)于痘病毒的增殖和感染都有促進(jìn)作用。而且這種整合對(duì)FPV的生物特性產(chǎn)生很大影響,其中最明顯的表現(xiàn)為FPV疫苗保護(hù)力的下降;野毒致病力的增強(qiáng)等。本研究通過(guò)對(duì)2012-2014年國(guó)內(nèi)外不同地區(qū)、不同批次的12個(gè)FPV疫苗株以及來(lái)自泰安某地區(qū)的3個(gè)火雞痘及1個(gè)雞痘野毒株中整合REV進(jìn)行分離鑒定及其序列比較,以評(píng)估國(guó)內(nèi)外當(dāng)前使用FPV疫苗的缺陷、潛在風(fēng)險(xiǎn)以及FPV中REV的污染狀況。12個(gè)禽痘疫苗株接種CEF傳代至出現(xiàn)明顯的細(xì)胞病變,提取細(xì)胞DNA,根據(jù)已發(fā)表的整合序列,PCR擴(kuò)增FPV-REV 5,LTR整合區(qū),同時(shí)分段擴(kuò)增REV的LTR、Env、Gag、Pol序列,結(jié)果表明,12個(gè)禽痘疫苗株均擴(kuò)增出含REV 5,LTR和FPV片段在內(nèi)的產(chǎn)物,但未擴(kuò)增到REV的Env、Gag、Pol序列。對(duì)FPV-REV 5,LTR整合區(qū)序列進(jìn)行測(cè)定,經(jīng)NCBI、DNA-star比對(duì)分析,其中2個(gè)國(guó)外疫苗株整合了446bp的REV-LTR序列,與中國(guó)近年分離到的REV野毒株HA9901的同源性為98%,與美國(guó)標(biāo)準(zhǔn)株SNV的同源性為94.3%;另外10個(gè)國(guó)內(nèi)疫苗株整合的REV LTR片段相對(duì)較小,僅為194bp,與中國(guó)近年分離到的REV野毒株HA1101、HA9901的同源性分別為98.4%和96.4%,與美國(guó)標(biāo)準(zhǔn)株SNV的同源性為94.3%。10個(gè)國(guó)內(nèi)疫苗株整合區(qū)與國(guó)外已發(fā)表的禽痘毒株AY255632、HP-438的同源性分別為98.6%和100%,2個(gè)國(guó)外疫苗株整合區(qū)與AY255632的同源性達(dá)到100%。結(jié)果表明所檢測(cè)的12個(gè)禽痘疫苗株只整合部分REV LTR序列,并且與REV的分離株同源性不高,但與國(guó)外禽痘分離株整合區(qū)的同源性較高。分離到的3個(gè)火雞痘與1個(gè)雞痘野毒株經(jīng)雞胚連續(xù)傳代至出現(xiàn)痘斑,研磨后接種CEF傳代至出現(xiàn)CPE,提取細(xì)胞DNA,結(jié)果除擴(kuò)增到485bp的FPV-REV 5,LTR整合區(qū)以外,同時(shí)擴(kuò)增到REV的Env基因(1700bp)、Gag基因(2050bp)及Pol基因(3450bp)。序列分析表明,4個(gè)野毒株的FPV-REV 5,LTR整合區(qū)同源性達(dá)99.8%-100%,且與國(guó)內(nèi)疫苗株的整合區(qū)高度同源。同時(shí)選取T1毒株人工感染20只SPF雞,使用ELISA方法檢測(cè)血清中REV抗體,攻毒前REV抗體陽(yáng)性率為0%(0/20),攻毒兩周后陽(yáng)性率為100%(20/20)。剖檢發(fā)現(xiàn)出現(xiàn)免疫器官及其他器官的病變,包法氏囊萎縮出血,胸腺萎縮出血,氣管出血并伴有粘液,腎臟腫大等癥狀,可能與FPV野毒中的REV有關(guān),但還需進(jìn)一步研究。選取3株不同廠家的整合REV-LTR的FPV疫苗(2個(gè)中國(guó)疫苗株,1個(gè)國(guó)外疫苗株)對(duì)SPF雞進(jìn)行免疫保護(hù),人工感染整合REV全基因組FPV野毒株,結(jié)果表明,三株疫苗的免疫保護(hù)性均達(dá)到100%(保護(hù)率分別為20/20、26/26、15/15),對(duì)照組發(fā)病率為85%(18/21)。
[Abstract]:Fowlpox (Avian pox,AP) is an acute, contact infectious disease in poultry caused by poxvirus, vertebrate poxvirus subfamily, and fowlpox virus. Avian reticuloendotheliosis (reticuloendotheliosis,RE) is a group of pathological syndrome-associated virus (reticuloendotheliosis virus,REV) gene fragments integrated into the FPV vaccine, which is caused by the avian reticuloendotheliosis virus (reticuloendotheliosis virus,REV) of the family Retroviridae. Integration of gene fragments and even complete genomes into FPV wild strains has become a common phenomenon. As the integration of FPV and REV develops, it can promote the proliferation and infection of poxvirus. Moreover, this integration has a great impact on the biological characteristics of FPV, the most obvious of which is the decrease of FPV vaccine protection and the enhancement of wild virulence, etc. In this study, REV was isolated and identified from 12 FPV vaccine strains from different regions and different batches, and from three turkey pox and one fowlpox wild strain from a certain area in Tai'an from 2012 to 2014, and their sequences were compared. In order to evaluate the defects, potential risks and REV contamination in FPV, 12 avian pox vaccine strains were inoculated with CEF to induce obvious cytopathia. According to the published integration sequence, DNA, was amplified from the FPV-REV 5T region, and the LTR,Env,Gag,Pol sequence of REV was amplified by segments. The results showed that all the 12 avian pox vaccine strains were amplified with REV 5G LTR and FPV fragments, but no REV Env,Gag,Pol sequence was amplified. The sequence of FPV-REV 5 FPV-REV integration region was determined. By NCBI,DNA-star alignment analysis, two foreign vaccine strains integrated the REV-LTR sequence of 446bp. The homology was 98 with HA9901 isolated from China in recent years and 94.33 with the standard SNV of the United States. The other 10 domestic vaccine strains had relatively small integrated REV LTR fragments. The homology was 98.4% and 96.4% with HA1101,HA9901 isolated from China in recent years. The homology with SNV was 94.33.10% for domestic vaccine strains and 98.6% for AY255632,HP-438, respectively. The homology between two foreign vaccine strains and AY255632 was 100%. The results showed that the 12 fowlpox vaccine strains only integrated part of REV LTR sequence, and did not share high homology with REV isolates, but had high homology with foreign fowlpox isolates. Three turkey pox strains and one chicken pox wild virus strain were continuously passed through chicken embryo to appear poxella spots. After grinding, they were inoculated with CEF to produce DNA, of CPE, extraction cells. The results showed that the DNA, was amplified to the FPV-REV 5T region of 485bp. At the same time, the Env gene (1700bp) and Pol gene (3450bp) of REV were amplified. The sequence analysis showed that the homology of the FPV-REV 5 FPV-REV integration region of the four wild strains was 99.8- 100, and it was highly homologous with the domestic vaccine strains. At the same time, 20 SPF chickens were artificially infected with T1 strain. The positive rate of REV antibody was 0% (0 / 20) before attack and 100% (20 / 20) after two weeks of attack with ELISA method. Pathological changes of immune organs and other organs, atrophy of bursa of Fabricius, atrophy of thymus, trachea hemorrhage with mucus and renal enlargement may be related to REV in FPV field, but need further study. Three FPV vaccines (2 Chinese vaccine strains and 1 foreign vaccine strain) integrated with REV-LTR from different manufacturers were selected to protect SPF chickens. The SPF chickens were artificially infected with the whole REV genome FPV field strain. The three vaccines were 100% immunized (20 / 20 / 26 / 2615 / 15, respectively) and the control group was 85% (18 / 21).
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3
本文編號(hào)：2209648
[Abstract]:Fowlpox (Avian pox,AP) is an acute, contact infectious disease in poultry caused by poxvirus, vertebrate poxvirus subfamily, and fowlpox virus. Avian reticuloendotheliosis (reticuloendotheliosis,RE) is a group of pathological syndrome-associated virus (reticuloendotheliosis virus,REV) gene fragments integrated into the FPV vaccine, which is caused by the avian reticuloendotheliosis virus (reticuloendotheliosis virus,REV) of the family Retroviridae. Integration of gene fragments and even complete genomes into FPV wild strains has become a common phenomenon. As the integration of FPV and REV develops, it can promote the proliferation and infection of poxvirus. Moreover, this integration has a great impact on the biological characteristics of FPV, the most obvious of which is the decrease of FPV vaccine protection and the enhancement of wild virulence, etc. In this study, REV was isolated and identified from 12 FPV vaccine strains from different regions and different batches, and from three turkey pox and one fowlpox wild strain from a certain area in Tai'an from 2012 to 2014, and their sequences were compared. In order to evaluate the defects, potential risks and REV contamination in FPV, 12 avian pox vaccine strains were inoculated with CEF to induce obvious cytopathia. According to the published integration sequence, DNA, was amplified from the FPV-REV 5T region, and the LTR,Env,Gag,Pol sequence of REV was amplified by segments. The results showed that all the 12 avian pox vaccine strains were amplified with REV 5G LTR and FPV fragments, but no REV Env,Gag,Pol sequence was amplified. The sequence of FPV-REV 5 FPV-REV integration region was determined. By NCBI,DNA-star alignment analysis, two foreign vaccine strains integrated the REV-LTR sequence of 446bp. The homology was 98 with HA9901 isolated from China in recent years and 94.33 with the standard SNV of the United States. The other 10 domestic vaccine strains had relatively small integrated REV LTR fragments. The homology was 98.4% and 96.4% with HA1101,HA9901 isolated from China in recent years. The homology with SNV was 94.33.10% for domestic vaccine strains and 98.6% for AY255632,HP-438, respectively. The homology between two foreign vaccine strains and AY255632 was 100%. The results showed that the 12 fowlpox vaccine strains only integrated part of REV LTR sequence, and did not share high homology with REV isolates, but had high homology with foreign fowlpox isolates. Three turkey pox strains and one chicken pox wild virus strain were continuously passed through chicken embryo to appear poxella spots. After grinding, they were inoculated with CEF to produce DNA, of CPE, extraction cells. The results showed that the DNA, was amplified to the FPV-REV 5T region of 485bp. At the same time, the Env gene (1700bp) and Pol gene (3450bp) of REV were amplified. The sequence analysis showed that the homology of the FPV-REV 5 FPV-REV integration region of the four wild strains was 99.8- 100, and it was highly homologous with the domestic vaccine strains. At the same time, 20 SPF chickens were artificially infected with T1 strain. The positive rate of REV antibody was 0% (0 / 20) before attack and 100% (20 / 20) after two weeks of attack with ELISA method. Pathological changes of immune organs and other organs, atrophy of bursa of Fabricius, atrophy of thymus, trachea hemorrhage with mucus and renal enlargement may be related to REV in FPV field, but need further study. Three FPV vaccines (2 Chinese vaccine strains and 1 foreign vaccine strain) integrated with REV-LTR from different manufacturers were selected to protect SPF chickens. The SPF chickens were artificially infected with the whole REV genome FPV field strain. The three vaccines were 100% immunized (20 / 20 / 26 / 2615 / 15, respectively) and the control group was 85% (18 / 21).
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 崔治中;;禽反轉(zhuǎn)錄病毒與DNA病毒間的基因重組及其流行病學(xué)意義[J];病毒學(xué)報(bào);2006年02期
2 孫淑紅;崔治中;邱玉玉;;網(wǎng)狀內(nèi)皮增生病病毒感染SPF雞對(duì)疫苗免疫反應(yīng)的抑制作用[J];Virologica Sinica;2006年01期
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