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Sialic Acid-Containing Glycolipids Extend the Receptor Repertoire of Enterovirus-D68 Enterovirus D68 (EV-D68) emerged as a pathogen of increasing health concern globally, particularly due to its association with outbreaks of severe respiratory diseases and acute flaccid myelitis (AFM) in children. Knowledge regarding the tissue tropism and pathogenesis of EV-D68 within the respiratory tract and central nervous system remains limited, primarily due to an incomplete understanding of the host factors that facilitate the entry of EV-D68 into host cells. Several cellular receptors involved in EV-D68 infections have been identified, including ICAM-5, sialylated glycoproteins, and heparan sulfate (HS). Here, we investigate the receptor requirement of a panel of EV-D68 strains covering all clades, focusing on HS and sialosides utilizing glycan arrays. We found that all EV-D68 strains binding to HS harbor a cell culture adaptive substitution in the structural protein VP1 at position 271, which changes the amino acid into a positively charged one. Glycan array analyses revealed that EV-D68 strains prefer α2,6-linked sialic acids presented on N-glycans, α2,8-linked sialic acids on gangliosides, or both. Inhibition of glycolipid biosynthesis or multivalent glycolipid mimics confirmed that ganglioside structures serve as entry receptors for certain EV-D68 strains. Lastly, we examined whether EV-D68 strains that bind to HS or glycolipids require different uncoating mechanisms. Bafilomycin A1 minimally affected the cell entry of HS-binding EV-D68 strains B2/039 and B2/947, and the ganglioside preferring B1/2013 and other viruses were strongly inhibited. Together, we identified that EV-D68 strains can use disialoglycolipids as novel receptors and that different EV-D68 strains show a promiscuous sialic acid binding repertoire. https://pubs.acs.org/doi/10.1021/acsinfecdis.5c00063