【作者】 陈静；

【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】 禽白血病病毒（Avian Leukosis Virus，ALV）为单股RNA病毒，归类于反转录病毒科反转录病毒属。AL在禽类中通常引起可传播的良性和恶性肿瘤。ALV造成的死亡率最低为1%-2%，最高可达20%。感染ALV造成的经济损失主要是免疫抑制带来的继发感染和产蛋下降等亚临床症状。根据病毒干扰模式、宿主类型、病毒中和作用可划分为10个亚型，其中能感染鸡的为A、B、C、D、E、J。Payne在1988年首次从英国肉鸡中分离得到J亚型ALV（ALV-J），主要引起髓样细胞瘤，各种品种鸡均易感，给全球养禽业带来巨大损失。由于种禽公司加强了对AL的净化工作，ALV-J的发病率有很大降低，但在免疫选择压和RNA病毒本身遗传不稳定性和多样性的特点的作用下，可能会促进病毒进一步突变、重组，从而导致宿主范围扩大、损失更为严重。本实验对分离自山东五大地方品种鸡ALV亚型进行分类鉴定，对分离得到的山东地方品种鸡的ALV-J分离株gp85基因进行分子特征分析。对与其它致鸡肿瘤病病毒共感染的ALV-J代表性毒株进行全基因组测序、分析。本研究发现，五大品种均感染了ALV-J，ALV-E的片段广泛存在，其中琅琊鸡、芦花鸡同时存在ALV-A的共感染；而全基因组序列分析发现，三个不同品种的肿瘤病鸡ALV-J分离株的基因组结构之间并无密切关系，ALV-J基因组结构中与肿瘤发生相关的关键基因或作用元件与致病性的关系需进一步研究。1.鉴定了山东五大地方品种鸡群中ALV的主要亚型，并对山东地方品种鸡ALV-J分离株gp85基因进行了分子特征分析收集来自山东的鲁西斗鸡、琅琊鸡的卵白，百日鸡、芦花鸡和寿光鸡的无菌抗凝血，经分离鉴定得到五大ALV分离株DJ146、LYJ15、BR170、LH091101、SG-1。经PCR扩增发现五个地方品种鸡均感染了ALV-J；均扩出内源性ALV片段，与不能产生传染性病毒粒子的ev-1，ev-3、ev-6毒株同源性最高，推测为鸡基因组所携带；同时还发现，琅琊鸡、芦花鸡存在A亚型ALV（ALV-A）的感染。针对ALV-J的普遍流行情况，挑选另与其它三株ALV-J有其它致鸡肿瘤病病毒共感染的分离株(SD1005、SD110503、BR119)，分析ALV-Jgp85基因的分子特征，探究山东地方品种ALV-Jgp85基因的变异趋势。分析发现8个山东地方品种鸡的gp85基因氨基酸同源性40.9%-97.4%。其中BR170、DJ46、LH091101、LYJ15、SG-1之间的同源达到98.1%-100%，与国外白羽肉鸡原型毒株的同源性为97.8%-98.3%，推测这五个山东地方品种鸡ALV-J分离株来源于同一宿主，即国外的白羽肉鸡。2.完成了三株与其它致鸡肿瘤病病毒共感染的J亚型禽白血病病毒（ALV-J）全基因序列的扩增并对其分子特点进行分析。实验选取了外源性ALV之间共感染的代表性毒株SD1005（ALV-A与ALV-J）；外源性与内源性ALV共感染的代表性毒株BR119（ALV-J与ALV-E）；ALV-J与其它致鸡肿瘤病病毒共感染的代表性毒株SD100503（ALV-J与REV、MDV）。参照秦爱建等发表的ALV-J引物，用高保真酶进行全基因组扩增，测序、分析发现全基因组序列分析发现SD110503、SD1005的E元件完整为146bp；SD1005在前导序列序列（leader sequence）序列有19bp（576-594）的插入；gp85基因有447bp的缺失。gp85基因负责识别宿主细胞膜上的特异性病毒受体，其结构的改变可能与病毒感染细胞有很大关联。BR119与国内早期肉鸡ALV-J分离株同源性最近。SD1005、BR119、SD110503来自三个不同品种的肿瘤病鸡ALV-J分离株的基因组结构之间并无密切关系，ALV-J基因组结构中与肿瘤发生相关的关键基因或作用元件与致病性的关系需进一步研究。3.发现了山东省地方品种泰山柴鸡中存在新的ALV重组株感染SD110503的变异株SD110503R为ALV-J与禽胶质瘤病毒（Fowl glioma-inducingvirus, FGV）、ALV-E重组病毒，其中env基因由FGV的gp85和ALV-E的gp37重组形成，多种病毒的共感染不仅增加了肿瘤发生率，同时也加剧了ALV的变异。更多还原

【Abstract】 Avian leukosis virus (ALV) is RNA virus which belongs to the retrovirus family Retroviridaegenus. It usually caused transmissible benign and malignant tumors. Mortality rate caused byALV is between1%and2%, even to20%. Immunosuppression induced secondary infectionsand egg drop have caused more ecnonmic losses than direct death. ALV can be divided into10subgroups based on the type of host, the virus neutralization. There are six subgroups, A,B, C, D, E and J, which can infected with chickens. Payne was first isolated subgroup J ALV (ALV-J) in the British broiler in1988. It mainly caused myeloid cell tumors, various strainsof chickens were susceptible to ALV-J, which has resulted in huge losses to the global poultry industry. Since the companies have strengthened purification of avian leukosis, the inciden-ce of ALV has decreased greatly. However, because of immune selection pressure and geneticinstability and diversity characteristics, ALV could results in mutation and recombination eve-n to expansion of host range, and much more economic losses.The study has identifid main epidemic subgroupies of five ALV strains isolated from local ch-icken breeds in Shandong Province; analyzed the molecular characteristics of gp85genes ofthe eight ALVstrains isolated from local chicken breeds in Shandong Province. The complet-e sequences were amplified and analyzed of the three co-infected strains which ALV-J infected with other tumor disease. The study found that the five groups of local chickens from Shandong Province were infected with ALV-J, ALV-E fragements existed in all lined of five localchickens, the co-infection of ALV-A and ALV-J was discovered in Langya chicken and Luhu-a chicken. The complete sequence analysis revealed that there is no close relationship betwe-en genome structures ALV-J isolates, the relationship between key genes or acting elements i-n ALV-J genome structure and tumorigenesis needs further study.1. Identification subtypes of five ALV strains isolated from local chicken breeds in ShandongProvince; the molecular characteristics of gp85genes of the eight ALV strains were analyzedThe five ALV strains were isolated and designated as DJ146, LYJ15, BR170, LH091101, SG-1through the egg albumen or sterile anticoagulant. Polymerase chain reaction (PCR)amplifica-tion found the five groups of local chickens from Shandong Province were infected with ALV-J; Endogenous ALV fragements were existed all lined of five local chickens, which has a great agreement with ev-1, ev-3,ev-6strains that cannot produce infectious virion. It is presumed that these fragments come from the chicken genome. Meanwhile, we also found that Langya chicken and Luhua chicken existed the co-infection of ALV-A and ALV-J.The homology of gp85genes at amino acid level of eight ALV strains (DJ146、LYJ15、BR170、LH091101、SG-1、SD1005、SD110503、BR119) was range from40.9%to97.4%.The homology among BR170, DJ46, LH091101, LYJ15, and SG-1is98.1%to100%.Comparing with prototype strain HPRS-103, the homology was ranged from97.8%to98.3%. It is presumed that five ALV-J strains of local chicken breeds possibily origined from the same host, foreign white feather broiler.2. Analysis the molecular characteristics of three co-infection strains of ALV-J and other tum-or diseasesIn this paper, representative strain between exogenous virus SD1005(ALV-J and ALV-A); co-infection between exogenous and endogenous virus BR119(ALV-J and ALV-E); ALV-J co-infected with other tumor diseases SD100503(ALV-J and REV, MDV) were selected. Thereference ALV-J complete genome primers published by Qin Aijian. PCR with high fidelity e-nzyme was carrid out. Sequences analysis found that SD110503and SD1005have completeE element146bp, respectively. SD1005isolate has19bp insertion in leader sequence and447bp deletion of gp85gene. Gp85gene is responsisble for identifying the host cell membrane.The change of structure to gp85gene may be associated with virus infected cells. There wasno close relation among ALV-J (SD1005、BR119、SD110503) genome structures from thethree different lines of chickens with tumors. The relationship between key genes or acting elements in ALV-J genome structure and tumorigenesis needs further study.3. A new recombinant ALV strain was discovered in Shandong local chicken breed TaishanChai chickenSD110503R is a variant of SD110503which is a recombinant virus among ALV-J and Fowl g-lioma-inducing virus (FGV), and ALV-E. FGV’ gp85and gp37’ALV-E composed of env ge-ne. Co-infection among multiple viruses not only induced the incidence rate of neoplasm, butalso increased variation of ALV.更多还原