The influence of host barriers on viral quasispecies diversity and pathogenesis

描述（由申请方提供）：由于广泛的基因组变异性，RNA病毒群体作为称为准种的复杂突变群体存在。准种的多样性使疫苗设计复杂化，促进免疫逃逸，甚至赋予耐药性。对于脊髓灰质炎病毒，通过诱变使其基因组多样化的能力是在受感染动物中完全毒力所必需的。然而，在感染的小鼠中，病毒多样性受到瓶颈的限制，这些瓶颈阻止准种从外周扩散到CNS，从而潜在地限制了病毒适应性。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是罕见的，尽管在接种减毒Sabin脊髓灰质炎病毒疫苗后肠道中存在强毒病毒。该提案的目的是使用脊髓灰质炎病毒作为模型系统，以检查RNA病毒瓶颈的机制，并确定这种瓶颈对病毒种群的影响。该提议的核心假设是物理障碍导致了瓶颈，并且被阻断的病毒群体具有有限的进化能力和降低的适应性。在目的1和2中，将使用基于杂交的新型准种多样性试验，确定病毒注射或经口接种后物理屏障对瓶颈的贡献。在目标3中，将使用激光捕获显微切割和活细胞纯化来鉴定肠道中的感染细胞。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度和毒力阈值。阐明这一毒力阈值对于建立许多减毒活病毒疫苗的合理设计可能是重要的。 公共卫生相关性：RNA病毒（如脊髓灰质炎病毒）具有令人难以置信的基因组多样性，这使疫苗设计复杂化，并可能导致耐药性。然而，被感染宿主体内称为瓶颈的天然屏障可以限制病毒的多样性，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.