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

The substantial genetic diversity generated during influenza A virus replication facilitates both evasion of pre-existing host immunity and cross-species emergence. A major contributor to this diversity is the ubiquitous production of deletion-containing viral genomes (DelVGs) - viral RNAs with large internal deletions that arise during replication. DelVGs directly compete with wild-type (WT) genomes, and their accumulation has been implicated in modulating disease severity. However, the specific functional and genetic interactions between DelVGs and WT genomes remain poorly understood. To examine how DelVGs and WT genomes may co-evolve in mixed populations, we serially passaged influenza A virus under sustained high multiplicity-of-infection (MOI) conditions and used next-generation sequencing to build a longitudinal profile of DelVG emergence and dynamics. Early passages yielded a highly diverse repertoire of DelVGs across multiple segments, followed by a sharp contraction in overall diversity and the rise of one to two PB2-derived DelVGs that persisted at sustained high frequency. We identified a single-nucleotide substitution within PB2-derived DelVGs that significantly enhanced their replication and interference, whereas the same mutation proved lethal in the WT background. Collectively, these findings indicate that DelVGs are not mere byproducts of replication but dynamic genomic elements shaped by selection; their capacity to acquire adaptive mutations and outcompete WT genomes underscores their active role in shaping within-population viral dynamics.