Recombinant polypeptide containing the 260-466 amino acid sequence of West Nile virus (WNV) strain LEIV-Vlg99-27889-human glycoprotein E (gpE, E260-466) was constructed. Immunochemical similarity between the E260-466 and gpE of WNV was proven by enzyme immunoassay (EIA), immunoblot, competitive EIA, hemagglutination inhibition, and neutralization tests using polyclonal and monoclonal antibodies against the viral gpE and recombinant E260-466. Polypeptide E260-466 induced formation of virus neutralizing and cross-reactive antibodies that were interactive with various epitopes of this recombinant protein. It is shown by evaluation of the interaction of E260-466 with one of the proposed cell receptors of WNV that average E260-466-alpha V beta 3 integrin-specific interaction force measured using atomic force spectroscopy was 80 and 140 pN for single and double interactions, correspondingly. Taken together with previously described interaction between laminin-binding protein (LBP) and WNV gpE domain II, it is proposed that WNV gpE can interact specifically with two cellular proteins (LBP and alpha V beta 3 integrin) during virus entry.