(A) Binding of EBOV GP (WT and mutant V141A) to soluble NPC1 domain C proteins derived from African pteropodids measured by an ELISA. RaNPC1, Egyptian rousette; EbNPC1, Büttikofer’s epauletted fruit bat; HmNPC1, Hammer-headed fruit bat; EhNPC1, African straw-colored fruit bat. (B) Infection of African straw-colored fruit bat cells with VSV pseudotypes bearing EBOV GP (WT or V141A). Means ± SD (n = 3–4) from a representative experiment are shown in each panel. Means for VSV-EBOV GP WT vs V141A infection were compared by unpaired two-tailed Student’s t-test with Welch’s correction (**p < 0.01). (C) Surface-shaded representation of a single GP1-GP2 monomer (PDB ID: 3CSY (Lee, et al., 2008) highlighting key residues in the NPC1-binding site (yellow) and residue 141 (red). GP1, blue. GP2, grey. (D) Alignments of GP1 sequences from a panel of filoviruses. V141, orange; A141, white text on blue shading; other residues divergent from consensus sequence, black text on green shading. (E) Infection of African pteropodid cells with VSV pseudotypes bearing SUDV GP (WT or A141V). Means ± SD (n = 4) from two biological replicates are shown. Means for VSV-SUDV GP WT vs A141V infection on each cell line were compared by unpaired two-tailed Student’s t-test with Welch’s correction (*p < 0.05, **p < 0.01, ****p < 0.0001).