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.