禽痘病毒疫苗及野毒株重组禽网状内皮组织增生病病毒的研究
发布时间：2018-08-28 14:23
【摘要】：禽痘(Avian pox,AP)是由痘病毒科、脊椎动物痘病毒亚科、禽痘病毒属中的禽痘病毒引起的家禽的一种急性、接触性传染病。禽网状内皮组织增殖病(reticuloendotheliosis,RE)是由反转录病毒科的禽网内皮组织增殖病病毒(reticuloendotheliosis virus,REV)引起的几种禽类的一群病理综合征,REV基因片段整合进FPV疫苗、基因片段甚至完整的基因组整合进FPV野毒株已成为普遍现象。研究表明,随着FPV和REV整合的不断发展,其对于痘病毒的增殖和感染都有促进作用。而且这种整合对FPV的生物特性产生很大影响,其中最明显的表现为FPV疫苗保护力的下降;野毒致病力的增强等。本研究通过对2012-2014年国内外不同地区、不同批次的12个FPV疫苗株以及来自泰安某地区的3个火鸡痘及1个鸡痘野毒株中整合REV进行分离鉴定及其序列比较,以评估国内外当前使用FPV疫苗的缺陷、潜在风险以及FPV中REV的污染状况。12个禽痘疫苗株接种CEF传代至出现明显的细胞病变,提取细胞DNA,根据已发表的整合序列,PCR扩增FPV-REV 5,LTR整合区,同时分段扩增REV的LTR、Env、Gag、Pol序列,结果表明,12个禽痘疫苗株均扩增出含REV 5,LTR和FPV片段在内的产物,但未扩增到REV的Env、Gag、Pol序列。对FPV-REV 5,LTR整合区序列进行测定,经NCBI、DNA-star比对分析,其中2个国外疫苗株整合了446bp的REV-LTR序列,与中国近年分离到的REV野毒株HA9901的同源性为98%,与美国标准株SNV的同源性为94.3%;另外10个国内疫苗株整合的REV LTR片段相对较小,仅为194bp,与中国近年分离到的REV野毒株HA1101、HA9901的同源性分别为98.4%和96.4%,与美国标准株SNV的同源性为94.3%。10个国内疫苗株整合区与国外已发表的禽痘毒株AY255632、HP-438的同源性分别为98.6%和100%,2个国外疫苗株整合区与AY255632的同源性达到100%。结果表明所检测的12个禽痘疫苗株只整合部分REV LTR序列,并且与REV的分离株同源性不高,但与国外禽痘分离株整合区的同源性较高。分离到的3个火鸡痘与1个鸡痘野毒株经鸡胚连续传代至出现痘斑,研磨后接种CEF传代至出现CPE,提取细胞DNA,结果除扩增到485bp的FPV-REV 5,LTR整合区以外,同时扩增到REV的Env基因(1700bp)、Gag基因(2050bp)及Pol基因(3450bp)。序列分析表明,4个野毒株的FPV-REV 5,LTR整合区同源性达99.8%-100%,且与国内疫苗株的整合区高度同源。同时选取T1毒株人工感染20只SPF鸡,使用ELISA方法检测血清中REV抗体,攻毒前REV抗体阳性率为0%(0/20),攻毒两周后阳性率为100%(20/20)。剖检发现出现免疫器官及其他器官的病变,包法氏囊萎缩出血,胸腺萎缩出血,气管出血并伴有粘液,肾脏肿大等症状,可能与FPV野毒中的REV有关,但还需进一步研究。选取3株不同厂家的整合REV-LTR的FPV疫苗(2个中国疫苗株,1个国外疫苗株)对SPF鸡进行免疫保护,人工感染整合REV全基因组FPV野毒株,结果表明,三株疫苗的免疫保护性均达到100%(保护率分别为20/20、26/26、15/15),对照组发病率为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).
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
本文编号：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).
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
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相关期刊论文 前2条
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