Library construction and screening. We mutated DIII of the WNV E protein using an error-prone PCR protocol 25 that included Mn 2+ and Mg 2+ at concentrations of 0.3 and 2.0 mM respectively. Subsequently, the cDNA library was ligated into pYD1 and transformed into XL2-blue ultracompetent cells (Strategene). The colonies were pooled and the plasmid DNA was recovered using the Qiagen HiSpeed Maxi kit.