Synchronous co-infection drives lineage representation in influenza A virus populations.

RNA viruses evolve through replication errors that generate diversity but can also trigger cellular antiviral responses, creating an inherent tension between adaptation and host control. Influenza A virus biology adds to this complexity through genome segmentation, which enables reassortment but also creates infection heterogeneity through incomplete genome expression and packaging defects, and through superinfection exclusion, which restricts complementation. Together, these constraints would be expected to limit the consistent expansion of individual variants, yet the influenza A virus continues to adapt in nature, suggesting that specific infection contexts may allow disproportionate lineage amplification. To identify such contexts, we paired in vitro single-cell transcriptomics with viral lineage tracking to link infected cellular environments to viral output dynamics. We find that lineage enrichment predominantly occurs during synchronous coinfection, when multiple viral genomes occupy the same cell, and viral output is increased. These findings identify a timing-dependent condition under which lineage representation can shift, and predict that traits increasing burst output or the likelihood of synchronous coinfection may disproportionately shape population composition.