The influence of host barriers on viral quasispecies diversity and pathogenesis

描述（由申请人提供）：由于广泛的基因组变异性，RNA病毒种群以复杂的突变体种群形式存在，称为准种。准种的多样性使疫苗设计变得复杂，促进免疫逃逸，甚至产生耐药性。对于脊髓灰质炎病毒来说，要在受感染的动物中具有完全毒力，需要通过诱变使其基因组多样化的能力。然而，在受感染的小鼠中，病毒多样性受到阻碍准种从外周传播到中枢神经系统的瓶颈的限制，从而可能限制病毒的适应性。这种瓶颈效应可以解释为什么尽管在接种减毒萨宾脊髓灰质炎病毒疫苗后肠道中存在剧毒病毒，但人类与疫苗相关的脊髓灰质炎却很少见。该提案的目的是使用脊髓灰质炎病毒作为模型系统来检查RNA病毒瓶颈的机制，并确定此类瓶颈对病毒种群的影响。该提案的中心假设是物理障碍导致了瓶颈，并且瓶颈病毒种群的进化能力有限且适应度降低。目标 1 和 2 中将使用基于杂交的新型准种多样性测定来确定物理屏障对病毒注射或口服接种后瓶颈的贡献。在目标 3 中，将使用激光捕获显微切割和活细胞纯化来识别肠道中受感染的细胞。在目标 4 中，将在存在或不存在瓶颈的情况下测量病毒适应性和毒力阈值。阐明这种毒力阈值可能对于建立许多减毒活病毒疫苗的合理设计非常重要。 公共卫生相关性：脊髓灰质炎病毒等 RNA 病毒具有令人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，受感染宿主内被称为瓶颈的自然屏障可能会限制病毒多样性，并可能提高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.