DNA methylation-mediated suppression of endocytosis confers resistance to duck hepatitis A virus type 3

Host DNA methylation plays a crucial role in antiviral defense, yet its impact on viral susceptibility remains unclear. Using ducklings resistant (R) or susceptible (S) to duck hepatitis A virus type 3 (DHAV-3), we integrated whole-genome bisulfite and oxidative bisulfite sequencing with transcriptomics. We observed a distinct intragenic 5-methylcytosine (5mC) signature between the two lines, with endocytic pathway genes being hypomethylated in susceptible, while hypermethylated in resistant ducklings. Critically, the genebody 5mC level displayed a dominant negative correlation with gene expression, and functionally determined viral entry efficiency. Following infection, the methylation program further coordinated post-entry host responses, including immune, fatty acid metabolism, and autophagy pathways. This genebody methylation signature defines a regulatory framework governing DHAV-3 susceptibility, offering insights for antiviral strategies and disease-resistance breeding.IMPORTANCEDuck hepatitis A virus type 3 (DHAV-3) causes severe mortality in ducklings, leading to substantial economic losses in poultry production. Understanding why some ducks resist infection while others succumb is critical for developing sustainable control strategies. Here, we discovered that a chemical "tag" on DNA-called methylation-acts as a natural switch that determines susceptibility. Resistant ducks carry higher methylation on genes controlling viral entry, effectively closing the door to the virus. Susceptible ducks lack this protective methylation, allowing viral access. Experimentally manipulating this switch directly altered infection efficiency, proving methylation is a functional controller of disease outcome. Our findings reveal that epigenetic variation, independent of genetic sequence, shapes host vulnerability. This opens new possibilities for breeding virus-resistant poultry through epigenetic markers, reducing reliance on vaccines and antiviral drugs. The work also provides a framework for understanding how animals defend against viral pathogens at the molecular level.