Long-term experimental evolution of seasonal influenza A/H3N2 virus reveals diverse evolutionary potential

The repeatability of evolutionary outcomes is a fundamental question with implications for predicting the evolution pathogens like influenza viruses. Here, we tested whether seasonal A/H3N2 influenza virus evolution follows reproducible paths in a simplified setting where the natural cellular environment is maintained but adaptive immune selection is removed. We serially passaged replicate populations of an A/H3N2 virus for 1,200 generations across 12 independent differentiated human airway epithelium culture lines (three replicates of four donors) and performed deep sequencing to reconstruct the evolutionary dynamics. Rates of variant accumulation differed significantly among lines but not among donors, and parallel evolution was minimal. Populations generated polymorphisms not observed in the global human population, reversions to historical sequences, and changes at positions held near-invariant globally since 1968. These findings suggest A/H3N2 evolutionary trajectories are shaped less by intrinsic functional constraint than by selective context and help explain why predicting influenza virus evolution remains difficult.