Enterovirus 71 (EV71) is the major causative pathogen of human hand, foot, and mouth disease (hHFMD) and has evolved to use various cellular receptors for infection. However, the relationship between receptor preference and EV71 virulence has not been fully revealed. By using reverse genetics, we identified that a single E98K mutation in VP1 is responsible for rapid viral replication in vitro. The E98K mutation enhanced binding of EV71-GZCII to cells in a heparan sulfate (HS)-dependent manner,

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... nd it attenuated the virulence of EV71-GZCII in BALB/c mice, indicating that the HS-binding property is negatively associated with viral virulence. HS is widely expressed in vascular endothelial cells in different mouse tissues, and weak colocalization of HS with scavenger receptor B2 (SCARB2) was detected. The cGZCII-98K virus bound more efficiently to mouse tissue homogenates, and the cGZCII-98K virus titers in mouse tissues and blood were much lower than the cGZCII virus titers. Together, these findings suggest that the enhanced adsorption of the cGZCII-98K virus, which likely occurs through HS, is unable to support the efficient replication of EV71 in vivo. Our study confirmed the role of HS-binding sites in EV71 infection and highlighted the importance of the HS receptor in EV71 pathogenesis.