【作者】 袁秀芳；

【导师】 童光志；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2004， 博士

【摘要】 为了阐明马传贫驴白细胞弱毒疫苗（DLA-EIAV）致弱及免疫保护的分子机制，给其它慢病毒的免疫预防提供借鉴，本实验室构建了DLA-EIAV感染性分子克隆（命名为pOK8266）并获得其衍生病毒（命名为vOK8226），同时对我国马传染性贫血病毒强弱毒株进行了序列分析。在此基础上，以pOK8226为骨架，构建了马传染性贫血病毒强／弱LTR嵌合毒感染性分子克隆（命名为pOKVltr），并获得了其衍生病毒（命名为vOKVltr）。本研究对vOK8226、vOKVltr和DLA-EIAV进行了动物接种试验，通过对临床和病理组织学变化、各结构蛋白抗体、感染马体内病毒载量的监测以及LTR及env基因在感染马体内的变异规律进行了研究。 将7匹EIAV阴性健康马分为4组，第1组（4#和5#马）接5ml×10^-5TCID₅₀嵌合毒（pOKVltr），第2组（6#和7#马）接种5ml×10⁵TCID₅₀克隆毒（vOK8226），第3组（8#）接种5ml×10⁵TCID₅₀疫苗毒（DLA-EIAV），同时设第4组（1#和2#马）作为非接种对照组。接种后第220天，除了2#马攻毒剂量为3ml×10^-4血清稀释度外，其它所有马均用3ml×10^-5血清稀释度EIAV强毒辽宁株（L-EIAV）进行攻击。攻毒前后对每匹马进行体温监测，发现在接种后，所有试验马未见体温升高现象，说明我们所用的毒株接种后对马是很安全的，用L-EIAV LTR置换vOK8226的LTR得到的嵌合病毒对马也没有表现出临床致病性；攻毒后，非接种对照组1#和2#马体温均出现典型的稽留热，并分别于攻毒后第134天和19天死亡，病理剖检发现死亡马呈现典型的马传贫的病理组织学变化。第1组和第3组（嵌合毒接种组和疫苗毒接种组）在攻毒后未见任何临床变化。证明嵌合毒和疫苗毒接种马得到了免疫保护。第2组（克隆毒接种组）中的6#马在攻毒后没有异常变化，而7#马却经历6次发热期，最终于攻毒后第185天死亡，但开始发热和死亡的时间均比对照组明显滞后，病理剖检发现发病死亡马具有典型的马传贫的病理组织学变化。在攻毒后第450天剖杀所有存活马，并进行病理组织学检查，结果表明所有免疫保护马均未见任何异常变化。 在试验过程中，我们利用ELISA方法对不同试验马攻毒前后血清中针对EIAV结构蛋白p15、p11、p26、p9以及gp45的抗体进行了检测。攻毒前各接种马抗p9、p11和p15抗体水平极低或检测不到，p26抗体上升后又逐渐下降，gp45抗体缓慢上升后并持续在较高水平。攻毒后发病马（1#，2#，7#）体内抗p9、p11、p15、p26和gp45抗体均显著升高。并持续至死亡，6#和8#马体内抗p15、p26和gp45抗体也有升高外。攻毒后，嵌合病毒免疫马（4#和5#）体内抗这五种结构蛋白抗体和攻毒前没有明显变化，表明攻毒后没有回忆反应，表明其免疫效果可能优于其它各接种组。抗gp45抗体变化在发病马和未发病马间无明显差异，提示gp45抗体变化与疾病进程没有直接的关系。 利用实时定量PCR技术对攻毒前后马外周血白细胞中EIAV前病毒DNA及马血浆中病毒RNA的载量进行了监测，发现感染马体内前病毒DNA载量与p15和p26抗体水平有一定的相关性，血浆中病毒RNA的载量与p9和p11抗体水平成正相关。发病马血浆病毒RNA超过10⁶拷贝／ml血浆，而免疫保护马血浆中病毒RNA载量较低(10^0-5拷贝／ml血浆)。免疫保护马在攻毒后3个月血浆病毒RNA降到很低水平，用实时定量PCR方法检测不到。但在整个试验过程中，在中国农业科学院博卜论文摘要各试验马外周血白细胞中均能检测到EIAV前病毒ONA，说明马传染性贫血病毒以前病毒形式长期潜伏存在。 由于EIAV基因组具有高度变异性，特别是LTR和env基因的变异可能与病毒的致病性及免疫保护性有着密切的关系。因此，本实验对攻毒前后马外周血白细胞中EIAV前病毒的LTR（包括U3和R区）和。nv（包括c4，V3，CS，V4，C6，VS在内的580个核营酸）基因进行了序列测定和分析，发现LTR和env在马体内存在3种序列类型，即强毒型（序列与L-EIAV一致）、弱毒型 （序列与OLA一EIAV一致）和中间型（L一EIAV和OLA一EIAV的混合序列）。对EIAV弱毒感染性分子克隆及其衍生毒免疫后攻毒马长时间跟踪，并采血样进行LTR和env基因序列分析，结果发现马体内同时存在强毒型和弱毒型的序列以及少量的强弱毒衍化的中间型序列。当强毒型LTR和env基因序列占绝对优势时，马表现临床发病。对LTR各基序分析表明，强毒型LTR在增强子高变区没有E一box基序，而在U3一R结合处有1个E一box基序，而弱毒LTR在增强子高变区有2个E一box基序，在U3一R结合处没有该基序。推测该基序的变化对病毒的致病性可能有一定的关系。对env序列分析发现，在攻毒后获得免疫保护的马体内存在着大量的带有强毒特征，但在其中发生移码突变和域出现终止密码的env基因序列，这些缺陷型前病毒的大量存在可能与免疫保护存在一定的关系。在我们所测的175个氨基酸中，强毒型env序列比弱毒型env序列多4个糖基化位点，而中间型位于二者之间，由于糖基化位点可以屏蔽抗原位点，使病毒逃避免疫系统的监视，因此糖基化的数目和位置对病毒致病性有着一定的影响。更多还原

【Abstract】 In order to investigate the molecular mechanism of the attenuation and protection of donkey leukocyte attenuated equine infectious anemia virus (DLA-EIAV), the complete genomes of DLA-EIAV and EIAV strain Liaoning (L-EIAV) proviruses were cloned and sequenced. The infectious molecular clone (pOK8266) of DLA-EIAV was construct and its derived virus was obtained (designated as vOK.8266), Based on pOK.8266, a chimeric infectious molecular clone (pOKVltr) was constructed by replaced the long terminal repeat (LTR) sequence of DLA-EIAV with the counterpart of L-EIAV. and its derived virus was generated (designated as vOKVltr).In this study, seven EIAV-negative horses were randomly divided into four groups, two horses (4# and 5#) in Group 1 were inoculated with vOKVltr, two horses (6# and 7#) in Group 2 with vOK8266. one horse in Group 3 with DLA-EIAV, and 2 horses (1# and 2#) in Group 4 were served unvaccinated control. Eight months post-inoculation, all horses were challenged with L-EIAV. Pre- and post-challenge, the body temperature of all horses was recorded. All horses showed no abnormal change pre-challenge, indicating that all viruses used were safe for horses, and the replacement of the LTR of the attenuated molecular clone by L-EIAV LTR didn’t result in virulence increasing. The horses in Group 4 experienced 1 (2#)to 4 (l#)episodes of fever and died 19 days and 134 days post-challenge, respectively. The horses in Groups 1 and 3 had no abnormal changes post-challenge, indicating that the chimeric virus and vaccine virus protected the horses from challenge. In Group 2, the horse 6# showed no clinical signs, but the horse 7# experienced 7 episodes of fever and died 185 days post-challenge. Dead horses (1#, 2# and 7#) showed typical El A pathological changes at autopsy, and survived horses (4#, 5#, 6# and 8#) were euthanized 450 days post-challenge, and displayed no pathological changes.The antibodies against the EIAV structural proteins pi5, pi 1, p26, p9 and gp45 were measured by ELISA based on the recombinant proteins expressed in E.coli. The antibody levels induced by p!5, pll and p9 proteins were very low or undetectable pre-challenge, antibody against p26 was increased soon after immunization and slowly come down later, antibody to gp45 was increased slowly and persistentat high level. After challenge, the titers of antibodies against the five structural proteins in horses 1 #, 2 #and 7# increased significantly and sustained till death, antibodies to pi5, p26 and gp45 were also increased in horses 6#and 8#. Antibodies level against the five structural proteins did not show significant change in horses 4# and 5# after challenge, the lower or no remembrance response might reflect good protection. The anti-gp45 titers had no obvious difference between diseased and protected horses, so the anti-gp45 antibody seems to have no relationship with the disease progress.The EIAV provirus DNA in PBMCs and the EIAV RNA in plasma of inoculated horses wereinvestigated by real-time PCR and RT-PCR. We found that the quantity of L-EIAV provirus DNA had positive relationship with the antibodies level of anti-pi 1 and p9, and the EIAV provirus DNA content were related to antibodies against p26. The virus loads exceeded 106copies/ml plasma in diseased horses, but presented low level in protected horses, and decreased to undetectable level three months post-challenge. But the provirus DNA in PBMCs from all horses can be detected, indicating that EIAV exists as latent provirus.The variation of EIAV LTR and env may associate with the pathogenesis. By sequencing ed the provirus LTR and env gene from PBMC, we found that there are three types of LTR and env gene sequences, representing virulent type (similar to L-EIAV), attenuated type (similar to DLA-EIAV) and intermediate type (between L-EIAV and DLA-EIAV). In diseased horses the virulent type of LTR and env was dominant, but all three types could be found in protected horses. Sequence analysis demonstrated that there are two E-box motifs in LTR highly variable region of at更多还原