For each individual antibody, we screened the yeast library of DIII mutants according to the following protocol. To identify yeast that selectively lost binding to a given monoclonal antibody epitope, the library was initially stained with an Alexa Fluor 647-conjugated WNV-specific monoclonal antibody for 30 min at 4 °C. To control for the surface expression of DIII, after washing, yeast were subsequently stained for 30 min at 4 °C with an Alexa Fluor 488-conjugated oligoclonal antibody that was derived from a pool of individual monoclonal antibodies (E1, E2, E9, E16, E24 and E34). After immunostaining, we subjected yeast to flow cytometry and identified the population that was single monoclonal antibody negative but pooled oligoclonal antibody positive. The yeast cells were sorted at an event rate of ∼4,000 cells/s and this population (monoclonal antibody-negative and oligoclonal antibody-positive) was enriched after three rounds of sorting. After the final enrichment sort, we plated yeast and selected individual colonies and tested them for binding to individual monoclonal antibodies. For individual clones that had lost only the desired monoclonal antibody epitope, the DIII-pYD1 plasmid was recovered using the Zymoprep Yeast Miniprep kit (Zymo Research). The plasmid was then transformed into DH5α cells, purified using the Qiaprep Spin Miniprep kit (Qiagen) and sequenced.