Autophagic extracellular vesicles (AEVs) are distinct from exosomes and play crucial roles in viral infections.

Emerging evidence indicates important interconnections between autophagy and the secretion of small extracellular vesicles. However, our understanding of these secretory vesicles remains incomplete. Here, we identify a subtype of small extracellular vesicles, termed autophagic extracellular vesicles (AEVs), which are distinct from exosomes. Extracellular AEVs characterized by a size less than 100 nm exhibit increased secretion when the autophagy response is induced. Amphisomes, the hybrid organelles, are essential for the secretion of these types of vesicles. Further exploration reveals that autophagic cargos, certain ESCRT Ⅲ components and the Rab13 serve as distinctive markers for distinguishing AEVs from exosomes. Moreover, we find that the biogenesis of AEVs functionally requires components of the ESCRT Ⅲ complex and the GTPase Rab27a. Finally, we confirm that the enteroviral particles or genomes can be encapsulated into AEVs and subsequently infect receptor-negative cells with high efficiency. This model represents a prominent pattern of EV-mediated virus transmission.