Host genotype and sex shape influenza evolution and defective viral genomes.

Viral evolution during early pandemic waves favors mutations that enhance replication and transmission over antigenic escape. Host genotype and sex strongly shape this early adaptation, yet their individual and combined effects remain unclear. Here, we experimentally adapt influenza A virus to male and female BALB/c and C57BL/6 mice, generating 28 independent lineages, and employ a rolling sphere approach to identify recurrent selection hotspots in three-dimensional protein structures. In BALB/c mice, adaptation favors nonsynonymous substitutions linked to increased virulence, including hemagglutinin variants that become fixed exclusively in female lineages. We also observe sex-dependent selection acting on a viral protein interface, as substitutions disrupting a key NS1 dimerization motif converge on a single residue in female-adapted viruses, but are dispersed across the same interface in male-adapted viruses. Conversely, adaptation to C57BL/6 results in fewer substitutions but altered defective viral genome formation, leading to reduced cytopathic effect and attenuated virulence. These findings provide in vivo evidence that host genetic background alone can modulate defective viral genome formation and demonstrate that specific genotype-sex combinations impose selective pressures that favor mutations increasing virulence across hosts.