Flavivirus membranes fuse with cellular membranes by low pH-induced mechanisms. Although Japanese encephalitis virus (JEV) has similar mechanisms, fusion induced under neutral conditions has been observed. We report herein polykaryocyte formation using Vero cells infected with recombinant vaccinia viruses, vP829 expressing JEV premembrane (prM) and envelope (E), or vP555 expressing prM, E, and the nonstructural proteins NS1 and NS2A. Polykaryocytes were detected under pH 7.0 as early as 9 h after infection. Higher fusion indices were shown with vP555 than vP829. A monoclonal antibody to E suppressed vP829/vP555-induced polykaryocyte formation. Polykaryocytes were also formed under alkaline conditions (pH 8.0).