One of the potent neutralizing monoclonal antibodies, E16, inhibited infection of genetically diverse WNV lineage I strains that were isolated from mosquitoes, birds and horses in New York. E16 neutralized all WNV strains with PRNT 50 values of 4-18 ng and PRNT 90 values of 53-297 ng (Supplementary Table 2 online) . Notably, Fab fragments of E16 inhibited WNV (PRNT 50 , 23 ng), suggesting that neutralization does not require bivalent E protein binding. E16 potently blocked infection with strain 956, the original lineage II strain isolated in 1937 (ref. 24 ), yet was virus specific, as it neither recognized nor neutralized other flaviviruses including distantly related dengue and yellow fever viruses (data not shown) and closely related Japanese and St. Louis encephalitis viruses (Supplementary Table 2 Figure 1 Mapping of monoclonal antibodies to DIII with yeast. (a) Surface display of WNV E protein on yeast. A fusion protein is composed of the ectodomain or DIII of WNV E protein and the yeast Aga2 protein, which becomes attached to the Aga1 protein on the yeast cell wall. Yeast were transformed with the vector alone (pYD1; top row), the entire WNV E ectodomain (amino acids 1-415; middle row), or DIII alone (amino acids 296-415; bottom row). 24 h after induction, yeast were stained with the indicated monoclonal antibodies (negative control, anti-SARS ORF7a) and processed by flow cytometry. Data for a representative neutralizing (E16) and non-neutralizing (E18) antibody are shown. (b) Flow cytometric enrichment for DIII-expressing yeast variants that lose binding of E24. After each round, an increased percentage of DIII expressing yeast are recognized by the polyclonal WNV E-specific antibody but not by E24. After the final round, DIII-expressing variants (boxed region) were harvested. a b Figure 2 Fine epitope mapping of DIII neutralizing and non-neutralizing monoclonal antibodies. (a) Flow cytometry profiles for immunoreactivity by nonneutralizing (E2), weakly neutralizing (E1) and strongly neutralizing (E34) monoclonal antibodies with yeast expressing wild-type and variant DIII. The red, yellow and green arrows point to mutations that abolish yeast surface binding of individual monoclonal antibodies and correspond to distinct regions of DIII (see b). (b) Mapping of neutralizing and non-neutralizing monoclonal antibodies on the surface of WNV DIII. A molecular surface representation is depicted based on the nuclear magnetic resonance structure of WNV DIII 16 . The indicated amino acid residues associated with binding of neutralizing, weakly neutralizing and non-neutralizing monoclonal antibodies are shown in red, yellow and green, respectively.