Ephrin B2 and Ephrin B3 are receptors for a novel putative henipavirus with zoonotic potential

Next-generation sequencing has accelerated the discovery of novel putative viruses in wildlife reservoirs, while identifying those with zoonotic potential remains challenging. In this study, we report the identification and characterization of Ailong virus, a novel putative henipavirus from previous bat metagenomes in China that utilizes human ephrin B2 (EFNB2) and EFNB3 as functional receptors. Using an integrated approach combining phylogenetic analysis, pseudotyped virus entry assays, antibody blockade assays, and structural modeling, we demonstrate that Ailong virus glycoprotein binds human EFNB2 and EFNB3 with high specificity, mediating pseudovirus entry into both human neuronal and respiratory epithelial cells. Structural analysis revealed the Ailong virus glycoprotein-EFNB2 interface closely resembling that of Nipah virus (NiV), with conservation of all critical receptor-binding residues. Moreover, AiV encodes an exceptionally large phosphoprotein, 1,033 amino acids in length, which is larger than any other known phosphoprotein in the subfamily Paramyxoviridae. Given its receptor usage, structural similarities to NiV, and efficient entry in human airway epithelia, Ailong virus is believed to pose a spillover risk.