Abstract Human infections with the Japanese encephalitis virus (JEV) are a leading cause of viral encephalitis. An unprecedented outbreak of JEV genotype 4 was recently reported in Australia, with an isolate (JEV NSW/22 ) obtained from a stillborn piglet brain. Herein we conduct a thorough characterization of JEV NSW/22 in three different mouse strains and in human cortical brain organoids (hBOs), and determined the ability of JEV NSW/22 to be neutralized by sera from humans vaccinated with IMOJEV. JEV NSW/22 was less virulent than JEV FU (genotype 2) and JEV Nakayama (genotype 3) in C57BL/6J mice and in interferon regulatory factor 7 deficient ( Irf7 −/− ) mice, with infection of wild-type and knockout murine embryonic fibroblasts indicating JEV NSW/22 is more sensitive to type I interferon responses. Irf7 −/− mice provide a new model for JEV NSW/22 , showing higher viremia levels compared to C57BL/6J mice, and allowing for lethal neuroinvasive infection. All JEV strains were universally lethal in Ifnar −/− mice by day 3, with histological signs of brain hemorrhage, but no other lesions. There were no indications of brain infection in Ifnar −/− mice, with viral protein detected in blood vessels, but not neurons. All JEV isolates showed robust cytopathic infection of human cortical brain organoids, albeit lower for JEV NSW/22 . IMOJEV vaccination in humans induced antibodies capable of neutralizing JEV NSW/22 , although, for all JEV strains, cross-neutralization titers declined with increasing divergence from IMOJEV in the envelope amino acid sequences. Overall, our study establishes JEV NSW/22 mouse and hBO models of infection, allowing for possible lethal neuroinvasive infection in mice that was rarer than for other JEV genotypes. JEV vaccination regimens may afford protection against this newly emerged JEV genotype 4 strain, although neutralizing antibody responses are sub-optimal.