We used error-prone PCR mutagenesis of DIII of WNV E protein and yeast surface expression to map antibody contact residues in a highthroughput manner. We performed individual screens to identify DIII mutants that lost binding selectively to strongly neutralizing (E16, E24 and E34), weakly neutralizing (E1), and non-neutralizing (E2 and E22) monoclonal antibodies. To eliminate mutants that abolished surface expression of DIII, yeast were stained sequentially with an Alexa Fluor 647-conjugated individual monoclonal antibodies and an Alexa Fluor 488-conjugated oligoclonal antibody derived from a pool of individual monoclonal antibodies. After cell sorting, we identified yeast that selectively lost expression of an individual monoclonal antibody epitope but retained surface expression of DIII (Fig. 1b) . Multiple independent yeast that lost binding of individual monoclonal antibodies were subjected to plasmid recovery and sequencing. Monoclonal antibodies that localized to DIII and strongly neutralized WNV had reduced binding when residues S306, K307, T330 or T332 were altered ( Fig. 2a and Table 1 ). These are located on adjacent loops and form a contiguous patch 16 on the solvent-exposed surface at the lateral tip of the DIII (Fig. 2b) . Only two other mutations caused considerable loss of binding of any of the ten neutralizing monoclonal antibodies tested: K310E or P315R reduced binding only of E49. No two neutralizing monoclonal antibodies had identical loss-of-binding patterns. For example, S306L reduced binding of E16, E27, E40, E43 and E49 but not E24, E33, E34, E47 and E58. K307R abolished binding of E34, E40, E43, E47, E49 and E58 but affected E16, E24, E27 and E33 less strongly. In contrast, K307E decreased binding of all neutralizing monoclonal antibodies yet did not affect non-neutralizing or poorly neutralizing monoclonal antibodies. Changes in residues T330 and T332 also abolished binding of neutralizing but not non-neutralizing monoclonal antibodies. T330I or T332M strongly reduced binding of all neutralizing monoclonal antibodies with the exception of E27, and T332V or T332A weakened binding of only E24, E27, E33 and E58.