Six monoclonal antibodies that recognized DIII were either poorly neutralizing or non-neutralizing, and none engaged the dominant neutralizing epitope defined by S306, K307, T330 or T332. E2 and E9 were abolished or reduced by mutation of D381 and H396, and binding of E22 was weakened by a change in P315. D381 and H396 are proximal to one another but physically distinct from the four residues that affect binding of neutralizing monoclonal antibodies (Fig. 2b) . None of the mutations identified by loss-of-binding sorts for E2 or E22 had any effect on two other non-neutralizing monoclonal Individual WNV-specific monoclonal antibodies (25 µg/ml) were mixed with yeast that displayed wild-type or mutant DIII on their surface. After washing, and incubation with an Alexa-647 goat-anti mouse IgG secondary antibody, yeast were analyzed for antibody binding by flow cytometry. The two values represent the percentage of yeast that were positive for DIII expression with a given monoclonal antibody, and in parentheses, the mean linear fluorescence intensity of the positive cells. Yeast were analyzed at 24 h after induction with galactose, which gives a baseline surface expression of DIII of 60-80% positive cells (Fig. 1a) . Bold values indicate an almost complete (>80%) loss of binding, whereas underlined values show a marked (50-79%) reduction in either the percentage or the mean fluorescence intensity of the positive cells. Results are representative of at least two independent experiments for each antibody and DIII variant.