【作者】 王雪峰；

【导师】 王凤龙；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2011， 博士

【摘要】 20世纪70年代,哈尔滨兽医研究所研制成功的马传染性贫血病毒(Equine Infectious Anemia Virus, EIAV)弱毒疫苗为控制马传染性贫血(Equine InfectiousAnemia,EIA)在我国的流行做出了重要的贡献。该研究成果荣获1982年国家发明一等奖。该疫苗克服了慢病毒疫苗的开发难点,能在被免疫动物体内产生坚强的保护性免疫,是迄今为止唯一广泛应用并证明确实有效的慢病毒疫苗。马传染性贫血弱毒疫苗的成功,其中必定蕴藏着慢病毒致弱或诱导保护性免疫的机制,在HIV疫苗开发屡受挫折的背景下,系统阐明EIAV疫苗的减毒和保护机理,为HIV等慢病毒疫苗的研究提供参考。EIAV弱毒疫苗是由自然野毒株经马体、驴体和体外培养的驴外周血白细胞及驴胎皮肤细胞长期传代培养而成。本文通过PCR和RT-PCR的方法分别扩增EIAV弱毒疫苗制备过程中不同代次病毒株的前病毒基因组序列、EIAV驴白细胞弱毒疫苗(EIAVDLV121)及其亲本毒株EIAVLN40感染马匹后5个月内病毒S2/gp90序列,并进行序列分析比较。研究结果表明：(1)EIAV在体外传代致弱过程中LTR、env和S2等区域是主要的变异区域：随着病毒在体外传代培养次数的增加,各病毒株与亲本毒株的遗传距离逐渐增加；伴随着病毒毒力的逐渐减弱,在基因组的各个区域出现了一系列稳定变异位点,包括Gag的100A/T、103T/S和484D/N的替换;Pol的16K/E、598K/R和619N/D的替换；gp90的46A/E、98G/R、103H/Y、189K/E、190E/K、193S/N、236D/-、237N/K、247E/K和321K/E的替换；Tat的7R/H的替换；S2的41T/I、51T/I和55Q/K的替换；Rev的74V/I的替换；LTR负调节区的细胞因子AP-1结合位点、增强子区E box基序、R区的转录起始位点和TAR的起始位点的变异。(2)EIAVLN40在体内进化过程中病毒分为两个大的分支,感染早期和无症状阶段的病毒处在同一分支,发病阶段的病毒和EIAVLN40处在进化树的另一分支;gp90的变异主要分布在8个变异区,V3、V4和V5区的糖基化位点的变化通常与发病状态有关,EIAVLN40和多数发病后的序列在这三个区域都具有糖基化位点191NSSN194、237NNTW240和280NDTS283；病毒进化过程中,S2有12%以上的氨基酸出现稳定的替换,这些变异的出现与疾病发展相关。(3)EIAVDLV121在体内进化过程中病毒gp90分成三个大的分支,各时间点分离的病毒gp90与EIAVLN40的平均差异在0.91%-6.49%之间,与EIAVDLV121的差异在3.88%-6.73%之间;病毒S2基因进化过程中分为两个分支,各时间点分离的病毒S2与EIAVLN40的平均差异在0.79%-8.83%之间,与EIAVDLV121的差异在.3.08%-8.14%之间。研究证实：在EIAV弱毒疫苗制备过程中,随着病毒毒力减弱在基因组的各个区域都出现了一系列稳定的变异：EIAVLN40在体内进化过程中gp90和S2的变异与疾病进程的发展相关；EIAVDLV121在体内低水平复制过程中,病毒基因会继续进化,特别是主要的抗原基因gp90的多样性得到进一步丰富,其中包括与强毒株相似的抗原成分。更多还原

【Abstract】 An attenuated equine infectious anemia virus (EIAV) vaccine was developed by scientists led by Rongxian Shen of Harbin Veterinary Research Institute in the 1970s. The pandemic of equine infectious anemia (EIA) in China, which caused the death of millions horses, was successfully controlled by the country-wide application of this vaccine from 1980-1990. A First-Class National Invention Prize was awarded for the development of this novel lentiviral vaccine in 1982. This vaccine is the first lentiviral vaccine, which elicits broad and effective immunity against the infection of pathogenic strains. The efficacy of this EIAV vaccine implicates a hidden mechanism that covers the attenuation of virulence and induction of protective immunity of lentiviruses. The development of vaccines for AIDS is still a global challenge. Due to the similarity between EIAV and HIV, decoding the mechanism of EIAV vaccine on inducing protective immunity will provide insight to the development of other lentiviral vaccines, such as HIV vaccine.The attenuated EIAV vaccine was developed through successive passages of a wild-type virulent strain firstly in horses and donkeys to enhance the virulence and immunogenicity. A consistently passaging of this virus in donkey peripheral blood mononuclear cells (PBMC) and in fetal donkey dermal (FDD) cell derived fibroblasts in vitro was performed thereafter to attenuate the virulence of the virus. To better understand the changes in characterizations of this vaccine during the above developing process, genomic evolution of the vaccine strain was examined in this study.Proviral genomes were prepared from the parental virulent strain (EIAVLN40), the fetal donkey dermal cell-adapted vaccine strain (EIAVFDDV13) and strains of representative intermediate attenuating steps, and were amplified by PCR. In addition, S2 and gp90 gene fragments were amplified from EIAVLN40 and EIAVDLV121 isolated from horses infected up to five months by RT-PCR. All these amplified fragments were sequenced and compared for sequence alterations.Results of sequence analysis demonstrated that:(1) Mutations generated during attenuation were mostly identified in LTR, env and S2. The genetic distances of these cell-adapted EIAV strains to the parental virulent strain were gradually enlarged with the increase of passages. There were multiple stable mutations in multiple genes during EIAV attenuation process in vitro, including substitutions of 100A/T,103T/S and 484D/N in Gag, 16K/E,598K/R and 619N/D in Pol,46A/E,98G/R,103H/Y,189K/E,190E/K 193S/N, 236D/-,237N/K,247E/K and 321K/E in gp90,7R/H in Tat,41T/I,51T/I and 55Q/K in S2, 74V/1 in Rev and change of transcription-factor AP-1 binding motif in the negative regulating element (NRE), transcription factor binding motif E-box within the enhancer, the transcription start position and the first residue of trans-activation responsive (TAR) element of LTR. (2) Phylogenetic analysis showed that the sequences of viruses isolated from EIAVLN40-infected horses were obviously split into two major branches. Viruses isolated in the early stage and in the asymptomatic stage of infection in all EIAVLN4o-infected horses clustered in to a branche different from EIAVLN40. Viruses isolated after disease onset remained in the same branch as EIAVLN40.The variations of gp90 were mostly concentrated in eight highly variable regions V1-V8. Changes in the glycosylation sites within V3, V4 and V5 regions were usually associated with the disease status. Specifically, glycosylation sites 191NSSN194.237NNTW240, and 280NDTS283 within these three regions were present in EIAVLN40 and most of the quasispecies isolated after, but not before. disease onset. During the evolution of EIAVLN40, over 12% substitutions of amino acid residues presented in S2. which associated with the disease status. (3) Phylogenetic analysis showed that the gp90 sequences of viruses isolated from EIAVDLV121-infected horses were obviously split into three major branches. The divergences of gp90 sequences isolated from EIAVDLV121-inoculated horses were 3.88%-6.73% compared with EIAVLN40, and were 0.91%-6.49% when compared with EIAVDLV121. The S2 sequences of viruses isolated from EIAVDLV121-infected horses were obviously split into two branches, the average differences between the S2 sequences from EIAVDLV121-inoculated horses and EIAVLN40 were from0.79%-8.83%, and were 3.08%-8.14% when compared with EIAVDLV121.Conclusion:A series of consensus substitutions in multiple genes of the attenuated EIAV vaccine strain were generated during attenuation process. The mutations in gp90 and S2 were associated with the disease status. The vaccine strain replicates in a relatively low level in inoculated horses, which enabled to the vaccine quasispecies to evolve continuous and to increase diversity of gp90.更多还原