Purified WNV E protein expression. WNV E protein ectodomain was generated using a baculovirus expression system according to previously described methods for related flaviviruses 38 . The last 45 nucleotides of prM (endogenous signal sequence) and the first 1,290 nucleotides of WNV E protein from the New York 1999 strain 39 were fused downstream of the polyhedrin promoter and upstream of a histidine repeat in a baculovirus shuttle vector (pFastBac, Invitrogen) by PCR using a high-fidelity Taq polymerase (Platinum Taq, Invitrogen). Three days after baculovirus infection of Hi-5 insect cells at a multiplicity of infection (MOI) of 1, supernatants were harvested, filtered, buffer-exchanged and purified by nickelaffinity chromatography according to the manufacturer's instructions. The purified WNV E ectodomain lacks the C-terminal 71 amino acids that are associated with the membrane proximal, transmembrane and cytoplasmic domains.