【作者】 王波；

【导师】 孙淑红；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2014， 硕士

【摘要】 马立克氏病(Marek’s Disease,MD)、禽白血病（Avian Leukosis,AL）和网状内皮组织增生症(Reticuloendotheliosis,RE)是家禽常见的病毒性肿瘤病。它们不仅能够引发肿瘤和死亡，而且还能引起感染鸡群的生产性能下降、免疫应答水平降低、对其它疾病更易感等，给家禽养殖业带来巨大经济损失。本研究通过对疑似肿瘤病感染的泰山柴鸡进行病毒的分离鉴定，从同一只鸡体内分离到了马立克氏病病毒(Marek’s Disease Virus,MDV)、禽白血病病毒（Avian LeukosisVirus, ALV）及网状内皮组织增生症病毒(Reticuloendotheliosis Virus,REV)；通过ELISA方法检测ALV p27抗原确定鸡群的ALV感染情况；采用PCR方法扩增MDV的致瘤相关基因及ALV、REV的囊膜蛋白（env）基因，通过分析序列确定三种病毒的相关基因的特性及病鸡存在不同亚型的ALV的共感染；最后，为验证分离到的病毒是否具有生物学活性，本研究进行了动物回归实验，并从攻毒组的鸡血液中成功分离鉴定出了这三种肿瘤病病毒。具体研究结果如下：1三种肿瘤病病毒的分离鉴定本研究根据病鸡的临床及剖检症状，初步判定为肿瘤病感染，并进行了病毒的分离鉴定。将病鸡的白细胞接种鸡胚成纤维细胞（CEF）分离病毒，采用间接免疫荧光试验（IFA）鉴定出了MDV、ALV及REV。其中，用MDV单克隆抗体BA4、H19均得到了阳性的结果，其中H19单抗的IFA结果阳性证明分离到的为强毒株MDV；利用J亚群ALV（ALV-J）的单克隆抗体JE9确定分离到了ALV-J，同时，用ELISA方法检测采集的泰山柴鸡鸡群棉拭子及卵白样品的ALV p27抗原，阳性率为65.5%（553/844）,结果表明泰山柴鸡中存在较严重的ALV感染；用针对REV的单克隆抗体11B118检测确定有REV感染。通过病毒的分离及IFA鉴定发现同一只泰山柴鸡体内存在MDV、ALV及REV的共感染，命名为SD110503。同时扩增了三种肿瘤病病毒的相关基因，测序结果验证了上述IFA结论，并且证实了存在不同亚型ALV的共感染。2三种肿瘤病病毒相关基因序列分析MDV meq、pp24及pp38基因序列分析结果表明，MDV meq、pp24及pp38基因与国内外参考毒株序列的同源性分别为98.3%-99.0%、96.6%及98.1%-99.8%。分析碱基序列发现meq基因第575-577位的碱基为CAC，为强毒株的特征；pp24基因序列第81位碱基为C，证明此株源于国外，最可能来自CVI988；pp38基因第320位碱基为G，对应第107位氨基酸为精氨酸（R），第326位碱基为G，对应第109位氨基酸为甘氨酸（G），为疫苗株。综合上述碱基分析结果可判定此病毒为基因突变或者重组的强毒株。ALV env基因序列分析显示扩增出的ALV-J（SD110503J）env基因与已发表的国内外参考株env基因之间的同源性为84.1%-94.4%；进化树的结果显示SD110503J株与SD07LK01株的进化关系最近；扩增到的ALV-A （SD110503A、SD110503A1）env基因与已发表的ALV(A、B、C、D、E亚型)序列同源性为70.6-88.4%，基因进化树结果表明这扩增出的两个毒株与RSA-A的亲缘关系最近，均为ALV-A；扩增出来的ALV-C（SD110503C）毒株env基因与已发表的序列同源为82.5-95.2%，进化树结果显示此毒株TW-3593株同源性最近，本序列为ALV-C的gp85和ALV-E的gp37重组形成。上述结果表明，SD110503中存在不同亚型的ALV共感染，并且存在不同亚型之间基因重组现象。REV env基因序列分析表明扩增出来的SD110503REV env基因序列与已发表的REV序列同源性分别为94.4-99.3%，基因进化树结果表明此毒株与HLJR0901的亲缘关系最近。3动物回归实验SD110503混合毒攻毒试验结果可知，从攻毒组SPF鸡血液中均能分离到MDV、ALV及REV，经IFA试验可确定检测的样品为阳性，这表明SD110503可以感染SPF鸡，对下一步的研究奠定基础。更多还原

【Abstract】 Marek’s Disease Virus (MDV), Avian Leukosis Virus(ALV) and ReticuloendotheliosisVirus(REV) are widely considered to cause Avian Sarcoma. They cann not only causedifferent syndromes and deaths, but also tead to the decline of production performance,thereduction of immune response level and the susceptibility to other diseases in infectedchickens. They causes tremendous losses in the poultry industry.In this study, Marek’s Disease Virus, Avian Leukemia Virus and ReticuloendotheliosisVirus were isolated from the same chicken, by isolating and identifying of Taishan localchickens, which are are infected with suspected tumor disease. Determined the ALV infectionsituation of the chickens, by detecting ALV p27antigen through the method of ELISA.Amplify the tumor related genes of MDV and the envelope protein (env) gene of ALV, REVby PCR. By analyzing the sequences of genes, determined the characteristics of the threekinds of virus genes and found the presence of different subtypes of ALV infections in thesick chickens. Finally, to verify whether there are biological activities in the isolated virus,regression experiments on animals were carried out in the study. And these three tumordisease viruses were isolated and identified from the chicken bloods in the group of poisoning.The results are as follows:1Isolation and identification of the three disease virusThis study, with a combination of clinical symptoms and necropsy symptoms, initiallydetermined tumor diseases infection. And isolation and identification of viruses wereconducted. Virus isolation from the chickens blood, which were identified by indirectimmunofluorescence assay(IFA) with ALV-J, REV and MDV monoclonal antibodies,respectively. IFA results for MDV showed that monoclonal antibody BA4, H19have beenpositive results, the virulent strain was isolated MDV virus from H19monoclonal antibodyIFA positive result; the positive results of monoclonal antibody JE9showed that ALV-J wasisolated, at the same time. The positive rate of ALV p27antigen was65.5%(553/844) by ELISA, the results indicated the presence of ALV infection in Taishan Chai Chickens isserious; the positive results of monoclonal antibody11B118showed that REV was isolateddetected.The coinfection of MDV, ALV and REV are found by the isolation of the virus and theidentification of IFA, and the strain was named SD110503. The above IFA conclusion wasverified by the amplification of the oncogenes in the three tumor disease viruses,simultaneously. And the coinfection of different hypotypes of ALV were confirmed.2Sequence analysis of tumor-related genesIn this study,we analyzed the sequence results using a series of biological softwares.DNAstar shows that the MDV-meq, the pp24and the pp38of the isolate SD110503showed98.3%-99.0%,96.6%and98.1%-99.8%respectively, with that homology of the internationalreference strain. The ORF of Meq genes is1128pb, and the575th-577th bases are CACwhich is the character of the virulent virus. The710th-821th bases are the extra bases tocontrol with reference strain, but there is also a lack of some bases. The result indicated thisisolate is from the virulent virus. The sequence of81th base in pp24gene was C, indicatingthat this strain was originated from abroad, and it mostly came from CVI988. The320th basein pp38was G corresponding to the sequence of protein, and the107th amino acid wasarginine. The326th base was G, corresponding to the sequence of protein. The109th aminoacid was Glycine, which does not react with H19. So it is the vaccine strain. The resultsshowed that this mutant virus is virulent.The gene sequence analysis of ALV showed that the homology of env gene sequencesbetween the strain SD110503J and domestic and abroad ALV-J separation strains was84.1%-94.4%. The phylogenetic tree showed the evolutionary relationships betweenSD110503J and SD07LK01was recently. The homology of the amplified env gene betweenALV-A (SD110503A,SD110503A1) and the published ALV (A,B,C,D,E subtype) was70.6-88.4%. The result of the gene phylogenetic tree indicate that the genetic relationshipbetween the two strains and RSA-A was recent, and they both are ALV-A; the homology ofthe amplified env gene between the amplified ALV-C (SD110503C) strain and the publishedsequence was range from82.5to95.2%. The phylogenetic tree showed that the homology ofthis strain and the TW-3593strain was recent. And there could be a virus genes recombination. The above results showed that there are different subtypes of infection ALV inSD110503, and there may exist Genetic recombination between different subtypes.The analysis of REV env gene sequence showed that the homology of SD110503REV envgene amplified sequence with the published sequence REV were94.4-99.3%. Phylogenetictree showed that this strain has the closest relationship with HLJR0901.3Animal regression experimentAs the SD110503hybrid virus challenge test results showed, MDV，ALV and REV can beseparated from the blood of SPF that were from Challenged group, which can be confirmedpositive by IFA. It suggested that SD110503can be replicated in SPF chickens and spread,which is important for further research.更多还原