The influence of host barriers on viral quasispecies diversity and pathogenesis

描述(申请人提供)：由于广泛的基因组变异，RNA病毒种群以复杂的突变种群存在，称为准种。准种的多样性使疫苗设计复杂化，促进了免疫逃逸，甚至产生了抗药性。对于脊髓灰质炎病毒，通过突变使其基因组多样化的能力是感染动物完全毒力所必需的。然而，在受感染的小鼠中，病毒多样性受到瓶颈的限制，这些瓶颈阻止准种从外围传播到中枢神经系统，从而潜在地限制了病毒的适合性。这种瓶颈效应可以解释为什么疫苗相关的脊髓灰质炎在人类中很少见，尽管在接种减毒的萨宾脊髓灰质炎病毒疫苗后，肠道中存在毒力病毒。这项建议的目的是以脊髓灰质炎病毒作为模型系统来研究RNA病毒瓶颈的机制，并确定这种瓶颈对病毒种群的影响。这一提议的中心假设是，物理障碍导致了瓶颈，而瓶颈病毒种群的进化能力有限，适应度降低。在目标1和目标2中，将使用一种新的基于杂交的准物种多样性分析来确定物理屏障对病毒注射或口服接种后瓶颈的贡献。在AIM 3中，将使用激光捕获显微解剖和活细胞纯化来识别肠道中的受感染细胞。在目标4中，病毒适合度和毒力阈值将在存在或不存在瓶颈的情况下进行测量。阐明这一毒力阈值可能对建立许多减毒活疫苗的合理设计很重要。 与公共卫生相关：像脊髓灰质炎病毒这样的RNA病毒具有难以置信的基因组多样性，这使疫苗设计变得复杂，并可能导致耐药性。然而，在受感染的宿主内被称为瓶颈的自然屏障会限制病毒的多样性，并可能增加减毒活疫苗的安全性。拟议的研究将确定哪些宿主因素导致脊髓灰质炎病毒的瓶颈，以及瓶颈对病毒毒力的影响。

DESCRIPTION (provided by applicant): Due to extensive genome variability, RNA virus populations exist as complex mutant populations called quasispecies. The diversity in the quasispecies complicates vaccine design, facilitates immune escape, and even confers drug resistance. For poliovirus, the ability to diversify its genome by mutagenesis is required for full virulence in infected animals. However, in infected mice, viral diversity is limited by bottlenecks that block quasispecies spread from the periphery to the CNS, thereby potentially limiting viral fitness. This bottleneck effect could explain why vaccine-associated poliomyelitis in humans is rare, despite the presence of virulent virus in the gut after vaccination with the attenuated Sabin poliovirus vaccine. The objective of this proposal is to use poliovirus as a model system to examine the mechanism of RNA virus bottlenecks, and to determine the effects of such bottlenecks on viral populations. The central hypothesis of this proposal is that physical barriers contribute to the bottleneck, and that the bottlenecked viral population has limited evolution capacity and reduced fitness. The contribution of physical barriers to the bottleneck following virus injection or oral inoculation will be determined in Aims 1 and 2 using a novel hybridization-based quasispecies diversity assay. Infected cells in the gut will be identified in Aim 3 using laser-capture microdissection and purification of live cells. In Aim 4, viral fitness and virulence thresholds will be measured in the presence or absence of the bottleneck. Elucidating this virulence threshold likely will be important for establishing the rational design of many live-attenuated viral vaccines. PUBLIC HEALTH RELEVANCE: RNA viruses such as poliovirus have incredible genome diversity, which complicates vaccine design, and can result in drug resistance. However, natural barriers called bottlenecks within an infected host can limit viral diversity, and possibly increase the safety of live-attenuated vaccines. The proposed research will determine which host factors contribute to the poliovirus bottleneck, and the effect of the bottleneck on the virulence of the virus.