【作者】 赵冬敏；

【导师】 崔治中；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2010， 博士

【摘要】 禽白血病是由禽白血病病毒和禽肉瘤病病毒引起的禽类多种肿瘤性疾病的统称。禽白血病病毒是在鸡群中普遍存在的一群反转录病毒,该病毒可引起禽的多种具有传染性的良性肿瘤和恶性肿瘤。根据在不同遗传型鸡胚成纤维细胞上的宿主范围、与相同或不同亚群成员的干扰模式以及用病毒--血清中和试验鉴定的病毒囊膜抗原,鸡的禽白血病病毒可分为A、B、C、D、E和J共6个亚群。其中A、B、C、D主要引起经典的淋巴样白血病,而J亚群主要引发髓样细胞瘤。在过去十年中,中国已对J亚群ALV的流行状况有了大量报道,由于中国在过去几十年中从未对ALV采取过有效的净化措施,中国鸡群、尤其地方品系中存在其它亚型ALV感染的可能性很大。但对J亚群以外其它亚群ALV还很少有研究。1. B亚群ALV野毒株SDAU09C2的分离和鉴定本文通过接种DF-1细胞(C/E)系,从山东某地方品系芦花鸡的鸡群中分离到一株外源性白血病病毒(ALV)SDAU09C2。根据GenBank中已发表不同亚群序列设计合成7对连续、相互部分重叠的引物,完成了该外源性禽白血病病毒分离株SDAU09C2的前病毒全基因组核苷酸序列,并与A、B、C、D、E、J亚群参考毒株进行了同源性分析。分析结果表明,该分离毒株gp85氨基酸序列与ALV-B的四株参考株的同源性最高,92.5%-95.1%之间,而与其他亚群的同源性在78.9-89.9%之间,与J亚群参考株的同源性更是低至36.9%-39.5%,因此将此分离毒株划归为B亚群。将其gag、pol基因与其它各亚群参考毒株进行比较,发现该分离毒株与各亚群毒株的同源性均在95%以上,实际上这两个基因在各个亚群之间非常保守。gp37基因编码的氨基酸序列同源性比较发现,虽然SDAU09C2属于B亚群ALV,但是其gp37却与ALV-A的MAV-1具有极高的同源性,说明gp37的同源性与亚群没有明确相关性。SDAU09C2的R区域与其它各亚群的同源性在80.0%-100.0%之间。U5区域与各亚群参考株相应序列同源性比较发现,其与外源性A、B、C、D、J亚群的同源性在90.0%以上,与E亚群的同源性在85.9%-87.5%,说明R和U5序列与ALV的亚群没有相关性。将SDAU09C2的U3序列与其它各亚群U3序列比较发现,SDAU09C2的U3序列与ALV-A参考株MQNCSU株的同源性最高,为91.1%,与ALV-B参考株RSR B和MAV-2的同源性分别为84.4%和36.1%,这说明不同毒株之间U3序列的同源性与毒株所属亚群同样没有直接的相关性。通过分析调控元件发现,SDAU09C2的U3序列调控元件与MQNCSU没有差异,而与B亚群RSR B株相比,SDAU09C2多出1个增强子序列CAAT box和1个CArG box,但缺少了一个PRE box。2. SDAU09C2株对不同品种鸡的致病性比较本研究通过动物试验比较了SDAU09C2株对不同品种鸡的致病性。由于本实验室的前期试验中对J亚群分子化克隆毒株NX0101的致病性等已进行了深入研究,因此采用NX0101株作为对照毒株。两个毒株在三种鸡(白来航SPF鸡、HN蛋鸡、地方品系芦花鸡)中均采用鸡胚接种的方式。动物试验的比较分析结果如下:2.1两株ALV肿瘤发生率的比较自出壳20周后, SDAU09C2毒株和NX0101毒株开始诱发产生肿瘤。截止目前,两株病毒均在SPF鸡中诱发肿瘤,发生率分别为8.7%和14.3%,在HN蛋鸡中只有SDAU09C2引发肿瘤,发生率为5.6%,而两株病毒均未在芦花鸡中引发肿瘤。两株病毒均可在不同部位引发多种类型肿瘤,SDAU09C2在目前诱发的肿瘤中多表现为血管瘤,另外还有卵巢和肠系膜的肿瘤,说明该B亚群毒株具有一定的致病性;而NX0101则诱发包括血管瘤、纤维肉瘤及弥漫性肿瘤在内的不同部位的多种类型肿瘤。2.2两株ALV在不同品种鸡中病毒血症检出率的比较白来航SPF鸡和HN蛋鸡中,两株病毒均在1-3周之内即可检测到病毒血症,特别是白来航SPF鸡,在3周时9/10的鸡可检测到病毒血症。SDAU09C2在这两种鸡中的横向感染能力强于NX0101。地方品系芦花鸡中,SDAU09C2接种的鸡只在5周时检测到一过性病毒血症,且阳性比例不高(2/12)。横向感染组病毒血症检测表明,SDAU09C2在芦花鸡中的横向传播能力弱于其在白来航SPF鸡和HN蛋鸡中的横向传播能力。2.3两株ALV在不同品种鸡中泄殖腔棉拭子p27检出率的比较白来航SPF鸡和HN蛋鸡中,在5-21周时SDAU09C2接种鸡泄殖腔棉拭子p27阳性检出率高于NX0101接种组。SDAU09C2在这两种鸡中的横向感染能力强于NX0101。地方品系芦花鸡中,在5-21周时NX0101接种组泄殖腔棉拭子p27阳性检出率高于SDAU09C2接种组。两株病毒在芦花鸡中的横向传播能力均弱于白来航SPF鸡和HN蛋鸡。2.4两株ALV在不同品种鸡中对体重抑制作用的比较SDAU09C2和NX0101均可对3个品种鸡的增重产生抑制作用。在HN蛋鸡中,SDAU09C2株的对体重增重的抑制作用强于NX0101株。但是在白来航SPF鸡中,SDAU09C2的抑制作用则弱于NX0101株。在地方品系芦花鸡中,SDAU09C2对体重的抑制作用略强于NX0101,但两株病毒对体重的抑制效果均弱于在另外2种鸡中的抑制效果。2.5两株ALV在不同品种鸡中免疫抑制作用的比较对NDV,AIV-H5,AIV-H9灭活免疫后35d的HI抗体的滴度的比较表明,SDAU09C2株可在白来航SPF鸡及芦花鸡中对NDV的抗体反应显示一定的免疫抑制作用,而在HN蛋用型鸡则没有免疫抑制作用。该株病毒在三个品种的鸡对AIV-H5和AIV-H9的抗体反应则无影响。与SDAU09C2株不同,J亚群的NX0101株在三个品种鸡对三种病毒的抗体反应都不表现明显的免疫抑制作用。出壳后6周随机剖杀,观察病毒对免疫器官发育的抑制作用。在白来航SPF鸡中,两株病毒均对法氏囊的发育产生明显的抑制作用;在蛋鸡中,SDAU09C2对胸腺及法氏囊的发育均具有抑制作用,而NX0101则未对三种免疫器官的发育表现抑制作用;在芦花鸡中,NX0101对法氏囊的发育具有抑制作用,而SDAU09C2则未对三种免疫器官的发育产生影响。两株病毒在三个品种鸡中均未对脾脏的发育产生抑制作用。2.6两株ALV在不同品种鸡中其它检测指标的比较2.6.1特异性抗体的检测两株病毒均可在3种鸡接种组和横向感染组诱导产生抗体,但是在白来航SPF鸡和HN蛋鸡中抗体水平和检出率均高于芦花鸡。2.6.2对鸡蛋的相关检测目前只有蛋鸡试验组全部开产。鸡蛋的检测结果说明,两株病毒接种鸡所产蛋中可在蛋清中检测到p27抗原及卵黄抗体。SDAU09C2株两者的检出率均高于NX0101株。2.6.3血液常规指标的检测在连续的跟踪监测过程中,两株病毒可使血液中白细胞和淋巴细胞数轻微变化,与对照组相比,差异不显著。但是在第21周时,两株病毒均可使血液中白细胞和淋巴细胞总数显著上升或下降。动物试验的结果表明,SDAU09C2和NX0101在不同品种鸡中具有不同的感染能力和致病性。这种差异主要是由于每只鸡的个体差异造成的,另外,不同品种鸡的遗传背景也会产生一定地影响。更多还原

【Abstract】 Avian leukosis is a variety of tumor diseases caused by avian leukosis virus and avian sarcoma virus. Avian leuksis virus (ALV) is a member of the family Retroviridae. ALV infection of chickens is widespread and known to be capable of inducing a variety of infectious tumor diseases. Avian leukosis viruses, isolated from chickens, are classified based on their host range, cross-neutralization, and viral interference into six groups (A, B, C, D, E, and J). Subgroup A, B, C, D mainly induced classical lymphoid leukosis (LL), while subgroup J has been found to be associated primarily with myeloid leukosis (ML). In the past 10 years, ALV-J has been widely recognized and reported in many regions of China. In the past few decades, since there is no strict nationwide eradication program for ALV infection on chicken farms in China, it is very likely that Chinese chickens, especially the local breeds, may be infected by other subgroups of ALV. However, the related reports are relatively rare.1. Isolation and identification of subgroup B field strain SDAU09C2In this study, by inoculation blood samples in DF-1 (C/E) cell cultlure, an exogenous avian leukosis virus (ALV) strain SDAU09C2 was isolated from a breeder farm of Chinese native breed“Barred Plymouth Rock chicken”in Shandong province. To full proviral genome cloning, seven overlapping primers were designed according to reference strains published in GenBank. Identity of the sequence of SDAU09C2 was compared with other ALV reference strains of different subgroups. The results show that it had 92.5%-95.1% of amino acid identity in gp85 to four ALV-B reference strains, the gp85 identity to subgroups A, B, C, D, E was only in the range of 78.9%-89.9%. The identity to subgroup J was as low as 36.9 %-39.5%. So SDAU09C2 was divided into subgroup B.Comparison of gag、pol between the isolation and other reference strains showed that the sequence identity for gag and pol between SDAU09C2 and other subgroups was more than 95% on average. Actually, these two genes were very conservative between different subgroups.The analysis of amino acid encoding by gp37 showed that gp37 in SDAU09C2 has high identity to gp37 in ALV-A MAV-1 although SDAU09C2 belonged to subgroup B. The identity of gp37 among different subgroups has no clear correlation with its subgroup.The fragment R identity to subgroups A, B, C, D, E, J was in the range of 80.0%-100.0%. The U5 identity to exogenous subgroups A, B, C, D and J was more than 90.0%, while the identity to subgroup E was in the range of 85.9%-87.5%. The results indicated that R and U5 region had no relationship with subgroups.Its U3 had highest identity of 91% to ALV-A strain MQNCSU, much higher than 84.4% and 36.1% to ALV-B reference strains RSR B and MAV-2. It indicated that the identity of U3 among different subgroups has no clear correlation with its subgroup. Analysis of regulatory elements showed that none of the differences noted in the U3 sequences of SDAU09C2 involved any of the known regulatory elements present in the U3 region of MQNCSU. However, an enhancer CAAT box and a CArG box (CC(A/T)6GG) of U3 in SDAU09C2 is not present in the RSR B. In contrast, a PRE box of RSR B is not present in the SDAU09C2.2. Pathogenicity of SDAU09C2 in different chickensPathogenicity of SDAU09C2 was studied in three different breeds by animal experiments. Because the pathogenicity of NX0101 has been studied deeply in our lab, NX0101 was chosen as a control strain. In the experiments, two viruses were both inoculated in ovo in different chickens, including white Leghorn SPF chicken, HN line egg-type chicken and Barred Plymouth Rock chicken. The comparison analysis of animal experiments was the following:2.1 Rates of tumors in different chickensOn the 20th week after hatching, SDAU09C2 and NX0101 started inducing tumors. Up to now, both two viruses can induce tumor in white Leghorn SPF chicken, the rate was 8.7% and 14.3% respectively. While only SDAU09C2 induced tumor in HN egg-type chicken and the rate was 5.6%. Neither of two viruses can induce tumor in Barred Plymouth Rock chicken. Two viruses lead to a variety of tumors in different organs. SDAU09C2 mainly induced hemangioma, in addition to ovarian and mesenteric tumors. NX0101 can induce a variety of tumors in different parts, such as hemangioma, fibrosarcoma, diffuse tumor and so on.2.2 Viremia induced by two ALVs in different chickensIn white Leghorn SPF chicken and HN line egg-type chicken, both SDAU09C2 and NX0101 induced viremia during 1-3 wk of age in the most birds of white Leghorns SPF chickens and HN line egg-type chickens. Especially in white Leghorn SPF chicken, the rate of viremia can reach 9/10 on 3rd week. The SDAU09C2 demonstrated stronger horizontal transmission ability than NX0101. In the local breed Barred Plymouth Rock chicken, SDAU09C2 only induced transient viremia on the 5th week and the rate of viremia was low (2/12). In horizontal infection groups, horizontal infectivity of SDAU09C2 in Barred Plymouth Rock chicken was relative lower than in other 2 chicken breeds.2.3 Comarison of p27 in cloacal swabIn white Leghorn SPF chicken and HN line egg-type chicken, during the 5-21 week of age, SDAU09C2 induced higher positive detection rate of p27 in cloacal swab than NX0101. The SDAU09C2 demonstrated stronger horizontal transmission ability than NX0101. In Barred Plymouth Rock chicken, during the the 5-21 week of age, NX0101 induced higher positive detection rate of p27 in cloacal swab than SDAU09C2. The horizontal infection of two viruses in Barred Plymouth Rock chicken was lower than that in white Leghorn SPF chicken and HN line egg-type chicken.2.4 Inhibition on body weight by two ALVs in different chickensBoth strains induced growth retardation in all 3 chicken breeds, such growth inhibitory effects of SDAU09C2 were stronger than NX0101 in HN egg type chickens but smaller than NX0101 in white Leghorn SPF chickens. In local breed Barred Plymouth Rock chicken, growth inhibitory effects of SDAU09C2 were a little stronger than NX0101, and the inhibitory effects of two viruses were weaker than that of two viruses in other 2 breeds. 2.5 Inhibition on immunity by two ALVs in different chickens35d after vaccine, SDAU09C2 could inhibit antibody reactions to vaccination against Newcastle disease virus in white Leghorn SPF chicken and Barred Plymouth Rock chickens but not in HN egg type chickens. While, SDAU09C2 didn’t affect the antibody reactions to vaccination against H5- and H9- avian influenza viruses (AIV-H5 and AIV-H9) in 3 chicken breeds. It indicated that NX0101 did not show significant inhibitory effects on HI antibody titers to NDV, AIV-H5 and AIV-H9 after vaccination in 3 chicken breeds.6 weeks after hatched, chickens were killed randomly and the immune organs were obtained to determine the inhibition on the development of immune organs. In the White Leghorn SPF chickens, both viruses inhibited the development of bursa of fabricius. In HN egg-type chickens, SDAU09C2 inhibited the development of thymus and bursal of fabricius while NX0101 didn’t show inhibition on the development of three immune organs. In the Barred Plymouth Rock chickens, NX0101 inhibited the development of the bursa of fabricius, while SDAU09C2 didn’t show inhibition on the development of three immune organs.2.6 Other tests in different chickens2.6.1 Analysis of specific antibodyTwo viruses can induce antibodies both in inoculated groups and horizontal transimission groups in the 3 breeds. In white Leghorn SPF chicken and HN line egg-type chicken, antibody level and positive rate were higher than those in Barred Plymouth Rock chicken.2.6.2 Detection of eggsUp to now, only HN egg-type chicken started to lay eggs. The results showed that p27 antigen in albumen and yolk antibody can be detected in SDAU09C2 and NX0101 inoculated HN egg-type chicken groups. And the eggs contained yolk antibody when the hens have developed antibodies. the positive rates of SDAU09C2 were much higher than NX0101.2.6.3 Influences on white blood ball and lymphocytes in the bloodIn the continuous tracking, the two viruses allowed the number of white blood ball and lymphocytes changed a little, while compared with the control group, the difference was not significant. But on the 21wk, the two viruses could make the total number of white blood ball and lymphocytes either increased or decreased significantly.The results suggested that two viruses showed different infectivity and pathogenicity in different chicken breeds. This difference may be caused by the individual difference of each chicken between chickens. In addition, different genetic background of different breeds of chickens can also play roles in it.更多还原