CARDIOVIRAL POLY-C TRACTS AND VIRUS PATHOGENICITY

一系列含有孟加拉病毒和孟加拉病毒全长拷贝的cDNA载体 脑心肌炎病毒嵌合基因组已被开发出来 并以此为特征。这些分离株彼此不同，也不同于 野生型Mengo和EMC病毒序列仅为其5‘非 编码聚(C)区。来自这些结构的RNA转录本 是完全感染HeLa细胞的，由此产生的 基因工程子代病毒。然而，在接种小鼠后，自然的 心脏病毒的宿主，携带缩短版本的工程病毒 聚(C)道(如：C8、C12或C13UC10)表现出戏剧性 相对于原始野生型病毒或 相对于一种新设计的具有野生型-Mengo长度的子代病毒 聚(C)道(C50UC10)。衰减在106-1010倍中很明显 提高病毒的半数致死量。此外，接受亚致死剂量的动物 从任何较短的聚(C)菌株特征地发展为高水平 中和抗体和获得性终身保护性免疫 对抗野生型病毒的(致命)挑战。这就是我们的目标 探索和表征聚(C)-分子基础的建议 中介衰减，最终目标是利用这一点 现象和从中吸取的原则，为发展 新型有效的小核糖核酸病毒活疫苗。具体目标是：(1) 利用基因工程技术构建全长心脏病毒质粒谱 含有改变的聚(C)区、间断、病毒嵌合体 5‘非编码序列中的序列、重排和其他变体 区域；(2)测试这些结构对 转录本的感染性和后代病毒的活性 组织培养细胞系；(3)评价其致病作用 在小鼠中感染遗传工程后代病毒，以便 评估宿主保护反应、病毒病原学、复制、 清除和序列回复潜力；(4)制定和测试 在分子水平上解释小核糖核酸病毒5‘的作用的假设 限制性片段中的非编码序列，特别是聚(C)区 确定病毒与宿主的相互作用。细胞因子的潜在作用 诱导是增强野生型病毒的一个重要因素 将对感染进行检查。

A series of cDNA plasmids containing full-length copies of Mengovirus and MengoEMC (encephalomyocarditis) virus chimeric genomes have been developed and characterized. The isolates differ from each other and from the wildtype Mengo and EMC virus sequences only in the lengths of their 5' non- coding poly(C) tracts. RNA transcripts derived from these constructions are completely infectious to HeLa cells and the resulting genetically engineered progeny viruses. However, upon inoculation of mice, the natural host for cardioviruses, the engineered viruses carrying shortened versions of the poly(C) tract (eg: C8, C12 or C13UC10) exhibit dramatically attenuated pathogenicity relative to the original wildtype viruses or relative to a newly engineered progeny virus with a wildtype-Mengo length poly(C) tract (C50UC10). The attenuation is manifest in a 106-1010 fold increase in the virus LD50. Moreover, animals receiving sublethal doses from any of the short poly(C) strains characteristically develop high level of neutralizing antibodies and acquired lifelong protective immunity against (lethal) challenge with wildtype virus. It is the goal of this proposal to explore and characterize the molecular basis for poly(C)- mediated attenuation with the eventual objective of exploiting this phenomena and the principles to be learned from it, for the development of new and effective, live picornavirus vaccines. The specific aims are: (1) To genetically engineer a spectrum of full-length cardiovirus plasmids containing altered poly(c) tracts, discontinuities, viral chimeric sequences, rearrangements and other variations within the 5' non-coding regions; (2) To test the relative effects of such constructions on transcript infectivity and progeny virus viability in several relevant tissue culture cell lines; (3) To assess the pathogenic effects of infectious genetically engineered progeny viruses in mice, in order to evaluate the host protective responses, viral etiology, replication, clearance and sequence reversion potential; (4) To formulate and test hypotheses to explain at the molecular level, the role of picornaviral 5' non-coding sequences, and in particular poly(C) tracts, in the restrictive determination of virus-host interactions. The potential role of cytokine induction as a contributing element to the enhancement of wildtype viral infections will be examined.