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

## TL;DR

This study shows how mouse host genotype and sex influence influenza virus evolution, including virulence and defective genome formation.

## Contribution

The study experimentally demonstrates sex- and genotype-dependent viral adaptation and defective genome formation in mice.

## Key findings

- Female-adapted influenza viruses show substitutions in a key NS1 dimerization motif, while male-adapted viruses show dispersed changes.
- Adaptation to C57BL/6 mice promotes defective viral genome formation, reducing virulence and cytopathic effects.
- Host genotype alone modulates defective viral genome formation, offering new insights into host–pathogen interactions.

## Abstract

Viral evolution during initial 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. We experimentally adapted influenza A virus to male and female BALB/c and C57BL/6 mice, generating 28 independent lineages, and employed a novel “rolling sphere” approach to identify mutational hotspots in three-dimensional protein structures. In BALB/c mice, adaptation favored nonsynonymous substitutions linked to increased virulence, including a hemagglutinin variant exclusively fixed in female lineages. It also revealed the first demonstration of sex-dependent selection shaping a viral protein interface. In female-adapted viruses, substitutions disrupting a key NS1 dimerization motif converged on a single residue, while in male-adapted viruses, they were dispersed across the same interface. Conversely, adaptation to C57BL/6 resulted in fewer substitutions but promoted defective viral genome formation, leading to reduced cytopathic effect and attenuated virulence. This provides the first
in vivo
evidence that host genotype alone can modulate defective viral genome formation. Our results offer critical insights into host–pathogen interactions and reveal that selective pressures imposed by specific genotype–sex combinations can increase virulence across host genotypes, enabling new epidemiological modeling and disease control strategies.