Influenza virus evolution and defective genome formation are shaped by host genotype and sex

Host-specific factors, including genetic background and sex, shape viral adaptation and influence virulence, yet their interactions and quantitative effects remain poorly understood. Additionally, multiple infections, where a host is infected with viruses from more than one source, are hypothesized to enhance viral diversity and increase virulence, but their impact in vertebrate hosts remains largely unexplored.

We experimentally adapted influenza A virus (IAV) to male and female BALB/c and C57BL/6 mice under single and multiple infection conditions. Using a novel three-dimensional mapping approach, we identified genotype- and sex-specific selection hotspots that drive viral adaptation at multiple scales, from localized substitutions to broader structural changes. Our findings reveal that host genotype plays a dominant role in shaping viral evolution, with sex-dependent selection patterns observed in certain contexts. In BALB/c-adapted viruses, selection favored mutations at a specific site of a protein interface in females, whereas male-adapted lineages exhibited a more diffuse distribution of mutations across the same region. We further demonstrate that host genotype influences the formation of defective viral genomes (DVGs), with C57BL/6-adapted viruses accumulating significantly more and longer deletions, leading to reduced cytopathic effect and altered virulence trajectories.

Multiple infections accelerated viral adaptation, increasing replication and mortality in a host-dependent manner. Adaptation to BALB/c hosts selected for high-virulence variants that maintained pathogenicity across diverse host backgrounds, whereas C57BL/6-adapted viruses exhibited attenuated virulence in novel hosts. These findings highlight the role of host genotype and sex in shaping viral evolution, reveal a previously unrecognized host-specific effect on DVG formation, and provide insights into how multiple infections drive the emergence of virulence-associated variants.