Intra-host GI.1 norovirus evolution is shaped by genetic drift and purifying selection

Human noroviruses are genetically diverse and over 30 different genotypes, mainly from genogroups GI and GII, are known to infect humans. Understanding of human norovirus diversity is still incomplete, and this large genetic diversity across and even within genotypes raises important questions about the origins of this diversity and how it is shaped within and between infected individuals. To gain insight into the origin of this genetic diversity, we analysed intra-host norovirus evolution from individuals experimentally infected with the prototype norovirus strain, Norwalk virus (GI.1[P1]). We investigated intra-host viral dynamics, the impact of antibody responses on intra-host viral evolution, inoculum-to-host viral evolution, and whether detected intra-host mutations have evidence of global (population-level) circulation. Notably, most intra-inoculum mutations detected in two GI.1 norovirus inoculum pools were not detected in individuals challenged with those inocula. Likewise, the majority of all intra-host mutations detected in challenged individuals were not detected in the inoculum and thus likely arose <i>de novo</i> within these individuals. Most of them were detected only once during shedding and have not been circulating at appreciable levels at the population level. Structural analyses confirmed that there was no significant difference in the distribution of intra-host mutations on the proteins among specimens with or without positive serum antibody responses. Our analyses suggest that stochastic processes, rather than host immune pressure, govern patterns of intra-host viral diversity in experimental, acute human norovirus infections. They further suggest that strong fitness constraints act to purify the majority of mutations during infection. Further studies that investigate structural and chemical constraints of this virus as well as fitness effects of mutations across the viral genome could help in our understanding of norovirus evolution.