Longitudinal analysis of influenza A virus deletion-containing viral genomes reveals key determinants of co-evolutionary dynamics and interference.

Influenza A virus generates extensive genetic diversity during replication, including abundant deletion-containing viral genomes (DelVGs) that compete directly with wild-type (WT) genomes. To investigate how DelVGs and WT genomes interact and evolve within mixed populations, we performed high-MOI serial passaging and next-generation sequencing to track DelVG emergence over time. Early passages produced a highly diverse DelVG repertoire, followed by the rise of a small set of PB2-derived DelVGs that persisted at high frequency. We identified a single-nucleotide mutation that enhanced DelVG replication and interference capacity while being detrimental in the WT background. These data illustrate how DelVGs act as dynamic genomic elements shaped by selection and capable of influencing within-population viral dynamics.