【作者】 吴晓平；

【导师】 秦爱建；

【作者基本信息】 扬州大学 ， 预防兽医学， 2010， 博士

【摘要】 J亚群禽白血病病毒(avian leukosis virus subgroup J, ALV-J)首次分离于上个世纪80年代,系外源性ALV与宿主内源性序列EAV-HP的重组体。近年来,该病在我国养鸡业中呈流行趋势。由于诱发肿瘤、患鸡胴体废弃和引起免疫抑制,ALV-J给养禽业带来巨大经济损失和严重威胁。ALV-J在中国的一个重要的发病特点是致瘤宿主谱系的扩展,以往研究证实不易发生肿瘤的商品蛋鸡受到ALV-J感染后也能诱导较高的肿瘤发生率,同时研究证实中国的地方鸡种也可感染致瘤,表现出不同于国际上ALV-J感染的特殊性和复杂性。研究ALV-J的生物学特性及致瘤宿主谱扩展的机理有助于该病的控制与根除。本研究对ALV-J引起的商品蛋鸡血管瘤、髓细胞瘤的组织病理学变化进行了观察,对表现血管瘤、髓细胞瘤病的商品蛋鸡分离的ALV-J毒株进行了分离和鉴定,对不同鸡群的ALV-J前病毒基因组序列的变异规律进行了分析,对商品蛋鸡ALV-J受体进行了分离鉴定,为探索ALV-J致病机理提供了新材料。为弄清商品蛋鸡群中血管瘤的病毒感染和致瘤原因,本研究对送检的ALV-J疑似病例进行了剖检、病理学观察、免疫组化检测及ALV-J特异性RT-PCR检测,发现商品蛋鸡血管瘤型ALV-J病例在海兰褐送检样品中的比率为54.5%(6/11),在罗曼蛋鸡送检样品中比率为100%(2/2),白羽蛋鸡群中未发现感染病例。而感染的商品蛋鸡所表现出的主要肿瘤类型是单纯血管瘤以及血管瘤与髓样细胞瘤(ML)并存,各占44.4%(4/9)大体病变：体表及肝脏、脾脏、肾脏、肌胃等器官表面及切面可见大小不等的血管瘤,内含血液,部分病例内脏可见黄白色结节；组织学病变：脾脏、肝脏等器官可见典型的海绵状血管瘤,有或无清晰壁层,处于不同的发育阶段,部分病例的体表血管瘤壁层可见髓细胞浸润,部分病例在心脏、肝脏、脾脏或肾脏内出现数量不等的髓样细胞增生,表现为实体瘤或髓样细胞浸润。1个病例的肾脏中存在胞浆中含有嗜碱性颗粒的成髓细胞瘤。在血管瘤与ML并存的病例中,两种肿瘤的发育阶段存在差异,可能是相继发生。免疫组织化学检测发现：在肝脏、脾脏及其它组织内的髓细胞内、肝脏实质细胞、血管瘤壁层上皮细胞、脾脏网状细胞内可见阳性信号。因此,在褐色商品蛋鸡中发现血管瘤、髓细胞瘤型伴发多种肿瘤表型的J亚群禽白血病,在国内外尚属首次报道,显示出ALV-J在我国商品蛋鸡群中感染状况的特殊性和严重性。将病理学检测及ALV-J特异性RT-PCR初步诊断为商品蛋鸡血管瘤、髓细胞瘤型ALV-J感染的脾脏样品悬液分别接种单层DF1细胞以分离病毒,并以ALV-J特异性单抗JE9进行间接免疫荧光检测(IFA)对分离毒进行鉴定,结果证实从商品蛋鸡血管瘤、髓细胞瘤型J亚群禽白血病病例中分离到7株ALV-J,在初诊为ALV-J感染的病例中分离率为77.8%(7/9)为追溯病毒来源,对于所检测的6个血管瘤发病鸡场的共同父母代蛋鸡场进行ALV-J的ELISA检测,结果发现在此父母代蛋鸡场高日龄的鸡群中存在14%(36周龄)和31%(60周龄)ALV-J特异性抗体,证实在此临床表现正常的父母代蛋鸡场中存在ALV-J感染。父母代蛋鸡及商品蛋鸡感染ALV-J的临床表现不同,可能是由于遗传背景对于ALV-J感染应答的影响或者是垂直传播及水平传播的共同作用在蛋鸡繁殖育雏过程中放大了ALV-J的感染。本研究设计3对ALV-J特异性引物,以分离株感染的DF1细胞基因组作为模板,进行ALV-J前病毒DNA全基因序列进行分段PCR扩增,分析比较了2007-2009年的商品蛋鸡分离株NHH、JS09GY3、JS09GY6的全序列,JS09GY2、JS09GY5、HA08和HN的致瘤关键性序列,同时也对白羽肉鸡分离株SD9901、YZ9901、YZ9902、NM2002-1和JS-nt的全序列进行比较和遗传分析。结果表明：商品蛋鸡群分离毒株与肉鸡群分离毒株的全序列差异明显,在遗传进化树上分属两个大的分支；对LTR的分析可见,R区最保守,U3区最易变异,商品蛋鸡血管瘤型分离株NHH和JS09GY5的U3区域发生了不同的连续序列缺失。分析发现在商品蛋鸡血管瘤相关病例分离毒(NHH、JS09GY2、JS09GY3、JS09G5及JS09GY6)的U3区内含有几个独特的调控元件,包括C\EBP、c-Ets-1、TCF11,而其它一部分在肉鸡分离毒中较为保守的作用元件如GKLF和Lmo2在商品蛋鸡分离株中缺失,提示这些独特的调控元件的存在或者缺失可能与病毒的致肿瘤特性相关。序列分析还发现：PBS-Leader序列在所测的ALV-J毒株中高度保守,然而商品蛋鸡血管瘤型分离株JS09GY3与JS09GY6的PBS-Leader序列中有一段极为罕见的连续19bp的插入突变,这段序列只在RAV-1、RAV-2及RSV-SRB毒株中可见,说明这两株病毒可能是ALV-J与其它反转录病毒发生重组而产生,这种重组模式尚属首次报道；E元件在所有的中国肉鸡分离株及蛋鸡单纯ML病例分离株HA08均发生了几乎相同的大部分序列缺失,仅蛋鸡分离株HA08与肉鸡分离毒相同,说明蛋鸡中ALV-J可能部分来源于肉鸡,而商品蛋鸡分离毒NHH、JS09GY2、JS09GY5、JS09GY6和SD07LK1则完整保留E元件,值得注意的是商品蛋鸡分离毒JS09GY3的E元件中含有11bp的连续插入序列,这种E元件中的连续序列插入突变在ALV-J中尚属首次发现；不同年代分离株的序列分析还发现：non-functional TM序列逐渐缺失,说明这种变异可能与病毒进化相关；DR1的同源性与宿主类型表现出一定的相关性,蛋用型或肉用型宿主中分离株的DR1序列中往往发生各自相同的点突变。对ALV-J毒株的编码区的序列进行分析比对发现：pol基因保守性极强,而gag基因所编码的P10、P19和P15蛋白序列中各有一个较为明显的突变集中区,而且在某些位点上的突变与宿主类型有关。中国白羽肉鸡分离毒的gp85蛋白相互之间同源性很高,遗传距离较近；商品蛋鸡分离毒gp85之间却显示出进化的不平衡性：小部分毒株与白羽肉鸡分离毒遗传距离较近；部分毒株(特别是分离自临床单纯血管瘤肿瘤表型病例的毒株,如NHH)与黄羽肉鸡分离毒同源性较高,变异性较强；同时发现大部分的商品蛋鸡混合肿瘤表型病例分离毒株高度变异,与肉鸡分离毒的同源性较低,在遗传进化树中属于独立的分支。gp37保守性较高,对gp37胞浆区的调控区域预测发现env蛋白的转化潜力与分离宿主之间没有明显的相关性。为了检测商品蛋鸡细胞表面的ALV-J受体与DF1细胞表面ALV-J受体chNHE1是否存在差异,以及商品蛋鸡ALV-J亲嗜性组织细胞-心脏细胞与ALV-J抗性细胞-淋巴细胞表面的ALV-J受体的存在与否,本研究以PCR方法扩增来源于商品蛋鸡的分离毒JS09GY3的SU基因序列,包涵其信号肽序列,同时以RT-PCR方法从兔血细胞RNA中扩增IgG Fc片段的重链,并将两段序列以BamHI酶切位点进行连接,克隆入pcDNA3.1中,构建真核表达载体pcDNA3.1-SUJ-IgGFc;以磷酸钙转染法转染293细胞；以RT-PCR、免疫荧光、Western Blot、SDS-PAGE等方法检测和鉴定融合蛋白。结果表明,在293细胞内和培养上清中均得到融合蛋白SUJ-IgGFc的暂态表达,用竞争ELISA方法检测表达浓度,结果表明,在细胞表达上清中的融合蛋白浓度达到约155ng/ml。本研究从商品蛋鸡的脾脏和外周血中提取淋巴细胞,并取心脏心室部分组织,以膜蛋白提取试剂盒提取膜蛋白,以293细胞作为对照。以融合蛋白SUJ-IgGFc-Beads结合物为工具,以免疫共沉淀的方法从膜蛋白中捕获与ALV-JSU蛋白特异性结合的蛋白质,经过SDS-PAGE检测,结果发现在商品蛋鸡淋巴细胞膜蛋白和心脏细胞膜蛋白中捕获到的蛋白不同,其中在淋巴细胞膜蛋白获得约58KD和65KD的两个蛋白条带,而在心脏细胞膜蛋白中获得了与chNHE1分子量相符的蛋白和其它3个蛋白条带。这一有意义的发现初步揭示了不同细胞表面可与SUJ特异结合的蛋白可能存在不同,尚需要进一步的鉴定。1.本研究首次报道商品蛋鸡血管瘤、髓细胞瘤型ALV-J感染并进行了病毒分离鉴定。2.对分离自不同类型鸡群的ALV-J毒株的病毒cDNA全基因序列进行分段PCR扩增及序列测定,发现商品蛋鸡分离株与肉鸡分离株的全序列在遗传进化树上属于两大分支。3.ALV-J序列中与宿主类型相关的非编码区序列主要有U3、DR1和E元件：商品蛋鸡血管瘤型分离株的U3区中有独特的调控元件即C／EBP、c-Ets-1和TCF11,而缺失多数肉鸡分离毒所具有的GKLF和Lmo2结合位点；血管瘤、ML型ALV-J的PBS-Leader序列中有19bp的连续序列插入,是ALV-J与其它逆转录病毒的重组毒；DR1序列中发现有固定的几个位点发生与宿主类型相关的点突变；商品蛋鸡血管瘤型分离株的E元件较为保守,在血管瘤、ML型分离株JS09GY3中,发现了罕见的11bp的连续序列插入突变,在国内外尚属首次报道,而肉鸡分离毒的E元件则发生了大部分序列的缺失。4.gag序列中的P10、P15和P19蛋白序列中某些位点的氨基酸突变与宿主类型有相关关系；中国肉鸡分离毒的gp85归于同一个分支中,表现出高度的同源性；而商品蛋鸡血管瘤、ML型分离株的gp85蛋白在进化树中位于一个较为独立的分支。5.构建pcDNA3.1-SUJ-IgGFc真核表达载体并在293细胞中得到SUJ-IgGFc的表达,对融合蛋白进行了定量和定性分析。以SUJ-IgGFc作为配体,以免疫共沉淀的方法捕获商品蛋鸡淋巴细胞及心脏细胞膜蛋白中能与SUJ相结合的蛋白,结果通过SDS-PAGE检测发现：在同样的免疫共沉淀条件下,在淋巴细胞膜蛋白中获得约58KD和65KD的两个蛋白条带,而在心脏细胞膜蛋白中获得了与chNHE1分子量相符的蛋白和其它3个蛋白条带。这一有意义的发现初步揭示了不同细胞表面可与SUJ特异结合的蛋白可能存在不同,尚需要进一步的鉴定。更多还原

【Abstract】 Avian Leukosis Virus subgroup J (ALV-J) was firstly isolated in England in 1980s and was thought to be the recombinant of exogenous ALVs with host endogenous sequence EAV-HP. Until recently, ALV-J spreads in Chinese chicken flocks. ALV-J infection causes economic losses associated with mortality, delayed growth, and tumor development. This disease showed development in our country in recent years and spreaded its tumorigenesis host range from meat-type chickens to commercial layer chickens and Chinese local flocks. The ALV-J infection condition in China showed the difference from international ALV-J infection. The study on the ALV-J tumorigenesis host range spreading will be helpful to the control and eradication of ALV-J. This research of the histopathological change of hemangioma and myelocytoma induced by ALV-J in commercial layers was carried out. Viruses were isolated and identified. The proviral genome sequences difference in meat-type chickens and commercial layer chickens were compared and analyzed. The receptor on the ALV-J tropism cell and resistant cell surface of commercial layer chickens was isolated and identified. This study provided new information to explore the pathopoiesis mechanism of ALV-J.In order to explore the virus infection and tumorigenesis cause of hemangioma tumors in commercial layer flocks, the borderline cases in commercial layers were examined by autopsy, pathobiology observation, immunohistochemistry and RT-PCR specific for J subgroup. We found that the hemangioma cases in Hyline brown commercial samples reached 54.5%(6/11) and that in Roman chickens reached 100%(2/2). No infection cases were detected in white layer chickens. The main tumor types in commercial layers were simple hemangioma or the coexisting of hemangioma and myelocytoma (ML), and the rate in the infection cases were both 44.4%(4/9).The affection in general examination showed the hemangioma on bodysurface and internal organs including liver, spleen, kidney and stomachus muscularis. The yellow-white tumors were found in the internal organs of partial cases. The histology examination showed the typical angioma angiocavernoma in different developmental stages with or without clear parietal layer in spleen and liver. Partial hemangiomas on bodysurface showed the infiltrate of medullocells in the parietal layer. Myeloid cells proliferation were found in heart, liver, spleen and kidney. The tumors displayed solid tumor or infiltration. Myeloblastoma was detected in kidney of one case and the cells contained basophillic stipplings in endochylema. The developmental stages of the two tumors were different in the cases with hemangioma and ML, and it was suggested that the two tumors developed one after another. The immunohistochemistry examination indicated the positive signals of ALV-J in the medullocells in liver, spleen and other tissues. ALV-J antigens were also detected in the patenchymal cells of liver, cellula epithelialis of hemangioma and reticular cells in spleen. So we found the coexisting of hemangioma and ML accompany of multiple turners types induced by ALV-J in brown layers. This is the first report about the coexisting of the two tumors. This study showed the severity of ALV-J infection in China.Viruses were isolated from the spleen tissues of the hemangioma and ML cases dianosed by pathobiology examination and RT-PCR specific for ALV-J, respectively. The DF1 cells were inoculated with tissue homogenate and were maintained for two generations. The viruses in the inoculated cells were identified by IFA detecting the ALV-J gp85 protein. The results showed ALV-J were isolated from the hemangioma and ML cases of infected commercial layer chickens and the isolating rate in the infection cases was 77.8%.In order to ascend the resource of viruses, the same parental line flock of the commercial layer chickens were detected for ALV-J by ELISA. We found ALV-J infection in the old ages groups with the infection rate associated with ALV-J antibody about 14% and 31% in 36-week-old and 60-week-old groups. The asymptomatic parental line birds and the pathogenetic commercial line chickens maybe indicated the affect of variances of genetic background to ALV-J infection or the vertical and horizontal transmission amplified the infection in commercial groups.Three pairs of ALV-J specific primers were designed to amplify the full-length proviral DNA, using infected DF1 DNA as templete. The isolates were detected and compared including NHH, JS09GY2, JS09GY3, JS09GY5, JS09GY6, HA08 and HN from infected commercial layer chickens in 2007-2009. The full-length cDNA of SD9901, YZ9901, YZ9902, NM2002-1 and JS-nt from commercial meat-type chickens were also compared and genetic analyzed.The results showed that there were obviously difference between the isolates from commercial layer chickens and isolates from meat-type chickens, while the isoaltes from this two different host breeds clustered in the separate two groups. The R regions were the most conservative sequences in LTR and the U3 regions were most variable. We observed that in the U3 regions of NHH and JS09GY5 isolates from cases associated with hemangiomas showed different continous sequences deletions. Motif analysis revealed the special regulatory elements found in the isolates associated with hemangioma in commercial layer chickens including NHH, JS09GY2, JS09GY3, JS09G5 and JS09GY6. These presented special elements were C\EBP, c-Ets-1 and TCF11, while some elements including GKLF and Lmo2 conservatively presented in meat-type chickens strains were absent in most commercial layer isolates. The absence or presence of special binding sites in some isolates maybe exhibit association with the tumorigenesis characteristics. The PBS-Leader regions in the detected isolates were highly conservative. It is special that the acare continuous 19bp insertion was noted in the Leader region of JS09GY3 and JS09GY6 isolated from hemangioma and ML coexisting cases of commercial layer chickens. The insertion sequences were also found in RAV-1, RAV-2 and RSV-SRB, indicating the recombinate of ALV-J with other retroviruses. The recombinate mode was firstly reported here different from previous reports. The almost identical deletions of most sequences in the E elements of isolates from meat-type chickens and HA08 strain from layer chicken were noted. Only the HA08 strain was identical to meat-type chickens isolates indicated that it was possible partial viruses in infected layers came from infeted meat-type birds. The conservative E elements were found in the isolates from commercial layer chickens including NHH, JS09GY2, JS09GY5, JS09GY6 and SD07LK1. It was firstly reported that the E element of JS09GY3 associating with coexisting of hemangioma and ML in commercial layer chicken showed a special continous 11bp insertion. The non-functional TM showed the trend of deletion according to the isolating times. It indicated the mutations in non-functional TM exhibited relationship with the isolation time and showed weakly relation with the host breeds. The aligment of DR1 regions revealed the evident associativity between the sequences and host breeds. The identical point mutations in DR1 regions of the isolates from same host breeds frequently were noted.The pol gene of all the isolates examined showed high conservation. The P10, P19 and P15 proteins coded by gag gene exhibited one region showing some special mutations associated with host types respectively. The phylogenitic analysis of the gp85 protein showed that the isolates from meat-type chickens exhibited high identities but the isolates from layer chickens showed evolutive unbalancedness. Some of them were conservative to Chines meat-type chicken isolates, and some were relatively mutated. But the other including the isolates from hemangioma and ML cases of commercial layer chickens showed high variaty and located in one seprate group. The conservation of the gp37 proteins were high. By motif analysis of the gp37 proteins, it showed that there were no obviously relationship between the host breeds and the transform ability of env proteins.In order to reveal the difference of ALV-J receptor on the cell surface of DF1 cells and commercial layer chickens cells, We amplified the gp85 coding region including the signal peptide of the isolate JS09GY3 with PCR. Whereas, the IgG Fc heavy chain of rabbit were obtained with RT-PCR from the RNA of rabbit blood cells. The SU fragment and the IgG Fc heavy chain were ligated with BamHI restriction enzyme site and the fusion fragment was cloned into the eukaryotic expression vector pcDNA3.1. We transfected the pcDNA3.1-SUJ-IgGFc into the 293 cells and then detected the fusion protein with RT-PCR, immunofluorescence, western blot and SDS-PAGE. The results revealed that the transiently expressed SUJ-IgGFc were obtained from the 293 cells and the cell culture. The concentration of fusion protein in the cell culture reached 155ng/ml and the fusion protein was used to detect the receptor of ALV-J.The membrance proteins of commercial layer chickens’lymph cells resistant to ALV-J infection and the heart cells susceptible to ALV-J were extracted. The co-immunoprecipitation was carried out with the fusion protein binding with protein A/G beads as ligand. The proteins interactive with the SU proteins were detected by SDS-PAGE. The proteins obtained from the lymph cells and heart cells were different, and the protein with same molecular mass with chNHE1 was detected in the obtained proteins from heart cells while the lymph cells proteins showed two bands about 58KD and 65KD. This interesting result needs research deeply in future and needs to be confirmed by further identification.1. The coexisting of hemangiomas and ML in infected commercial layer chickens was firstly reported in our study and the isolates were obtained.2. The full-length genome analysis of ALV-J strains confirmed that the strains from the two different host breeds clustered in two separated groups.3. The genes associating with host breeds were mainly gp85, U3, DR1 and E element. U3 regions of the layer hemangiomas isolates showed the special binding sites including C/EBP, c-Ets-1 and TCF11 but lack the motifs GKLF and Lmo2 presented in most meat-type chickens isolates. Whereas, we noted a special continous 19bp insertion in the PBS-Leader regions of hemangioma and ML isolates JS09GY3 and JS09GY6, indicating the recombinants. We observed several points mutations corresponding to host breeds in DR1 regions. In the isolates from commercial layer chickens, the E elements were conservative but the isolates from meat-type chickens deleted most E element sequences. Interestingly, continous llbp insertion was found and firstly reported in the layer hemangioma and ML isolate JS09GY3.4. The P10, P19 and P15 proteins coded by gag gene exhibited some special mutations associated with host type. We found that the isolates from infected meat-type chickens clustered in one group. The isolates associating with hemangiomas and ML coexisting cases in commercial layer chickens clustered in a separate group. 5. We constructed the eukaryotic expression vector pcDNA3.1-SUJ-IgGFc, and the fusion protein expressed transiently. The SUJ-IgGFc was used as ligends to precipitate the receptor protein of ALV-J on the lymphocytes and heart cells surface using the method of co-immunoprecipitation. The precipitated proteins were detected by SDS-PAGE and stained. The proteins obtained from the lymph cells and heart cells were different, and the protein with same molecular mass with chNHE1 was detected in the obtained proteins from heart cells while the lymph cells proteins showed two bands about 58KD and 65KD. This interesting result needs research deeply in future更多还原