An attenuated Australian strain of West Nile virus (WNV), Kunjin (KUN), shares ~ 98% amino acid homology with the pathogenic New York 99 NY99 strain (NY99). To investigate the viral factors involved in NY99 virulence we generated an infectious cDNA clone of the WNV NY99 4132 isolate from which virus was recovered and was shown to be indistinguishable from the parental isolate. We then introduced the regions of the NY99 non-structural (NS) proteins and/or untranslated regions (UTRs) into the KUN backbone. Chimeric KUN viruses containing NY99 5′UTR and the parts of NS coding region were more virulent in mice than parental KUN virus. Chimeric NY99 viruses, containing KUN NS2A protein with alanine 30 to proline substitution were significantly less cytopathic in cells and less virulent in mice. Our results identify the 5′UTR and NS proteins as WNV virulence determinants and confirm a role for the NS2A in WNV cytopathicity and virulence.