A fundamental problem in virology is to understand the evolutionary dynamics of virus populations and the forces that drive them within an individual host. These intrahost dynamics determine the rate at which a virus will adapt to new hosts, develop new routes of transmission, or generate novel antigenic variants. The current paradigm is that, for RNA viruses, high mutation rates lead to increased genetic diversity, which allows for more rapid adaptation and enhanced virulence. However, most newly generated mutations are either lethal or detrimental to viral replication. These data suggest that mutational tolerance, or robustness, may have a greater impact on viral diversity than mutation rate. The mechanistic basis for the beneficial effect of diversity is also uncertain, since intrahost minorit variants are often present at an extremely low frequency. Thus, the importance of mutation rate, mutational robustness, and diversity to the virulence of acute RNA virus infections is unclear. The long-term goal of this research is to elucidate the fundamental mechanisms through which novel viral variants are generated and maintained within a host. The objective of this project is to define the evolutionary forces that shape the spectrum of viral mutants that arise in vivo and to determine their relevance to pathogenesis. The central hypothesis is that for many acute viral infections, the observed patterns of diversity are a consequence of selection for replicative speed and reproductive success as opposed to high mutation rate. This hypothesis has been formulated on the basis of preliminary data in the poliovirus system, which show that (i) high fidelity variants exhibit a growth defect, (ii) minority variants are rare in infected hosts, and (ii) mutational robustness determines a virus' diversity, fitness, and virulence. Building on these strong preliminary data, the hypothesis will be tested with three aims. (Aim 1) Define the trade-off between replicative speed and fidelity. Fast replicating variants will be derived by experimental evolution, and their replication kinetics will be correlated with mutation rates, fitness in vitro, and virulence in infected hosts. (Aim 2) Define the impact of mutational robustness on population diversity and evolvability. Poliovirus populations that vary in mutational tolerance will be derived by codon-based rational design. The relationship between robustness, diversity and adaptive capacity will be determined. (Aim 3) Determine the extent to which host selective pressure causes shifts in viral diversity. To successfully colonize and spread within a host, poliovirus must survive the innate immune response and transit several stringent bottlenecks. Deep sequencing and advanced modeling will be used to determine whether this process is accompanied by genetic changes in the population. The approach is innovative since new concepts, experimental tools, and analytic methodologies are employed to rigorously test assumptions about the causes of viral diversity and its consequences for intrahost evolution. The proposed research is significant because it will define the relevance of replicative speed, mutation rate, mutational robustness, and diversity to the pathogenesis of RNA viruses.