We first determined that DHAV-1 infection induces autophagy in duck embryo fibroblasts (DEFs) and found that autophagy induction was dependent on the integrity of viral proteins by infecting DEFs with UV- or heat-inactivated DHAV-1. Then, in experiments using the autophagy inducer rapamycin (RAPA) and the autophagy inhibitor chloroquine (CQ), autophagy inhibition was found to reduce DHAV-1 genome copies and viral titers in and out of cells. These results suggest that autophagy activated by DHAV-1 infection in DEF affects DHAV-1 proliferation and extracellular release. Next, we screened the autophagy-inducing effects of DHAV-1 structural proteins VP0, VP3 and VP1 and found that all DHAV-1 structural proteins could induce autophagy in DEF, but not full autophagic flux. Finally, we found that VP1 protein promoted the protein expression of PI3KC3 and Beclin1 by immunoblotting experiments, and VP1 interacted with PI3KC3 by co-immunoprecipitation experiments; in addition, the autophagy inhibitor 3-methyladenine (3-MA) led to the reduction of PI3KC3 protein Thus, VP1 protein-induced autophagy in DEF was inhibited. Taken together, the DHAV-1 structural protein VP1 regulates the PI3KC3 complex by interacting with PI3KC3 to induce autophagy in DEF.Related research was published in Veterinary Research under the title "The DHAV-1 protein VP1 interacts with PI3KC3 to induce autophagy through the PI3KC3 complex", doi: 10.1186/s13567-022-01081-6.