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病毒群体以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，甚至产生耐药性。对于脊髓灰质炎病毒，需要通过诱变使其基因组多样化的能力才能在受感染的动物中具有完全的毒力。然而，在受感染的小鼠中，病毒多样性受到瓶颈的限制，这些瓶颈阻止准物种从外周扩散到中枢神经系统，从而潜在地限制了病毒的适应性。这种瓶颈效应可以解释为什么尽管在接种减毒Sabin脊髓灰质炎病毒疫苗后肠道中存在毒力病毒，但人类疫苗相关的脊髓灰质炎却很少发生。本提案的目的是利用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，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试验来确定物理屏障对病毒注射或口服接种后的瓶颈的贡献。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。阐明这一毒力阈值可能对建立许多减毒活疫苗的合理设计非常重要。

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.