Abstract Background: West Nile virus (WNV) is a neurotropic flavivirus transmitted through mosquito bites and whose reservoir hosts are wild birds. Equids and humans are incidental dead end hosts and can develop severe neurological symptoms. Despite enhanced reporting of WNV outbreaks in Europe since 2008, much remains to be explored about the virulence level and determinants of WNV strains circulating in Europe. Of note, recently expanding lineage 2 virus has gained a proline residue at position 249 in the non structural 3 (NS3) protein and has caused the worst epidemics ever experienced in central and Southern Europe. Interestingly, Brault et al. (2007) identified NS3249P as being crucial for the virulence of WNV in American crows. Methods & Materials: An infectious clone, based on WNV lineage 1 IS-98-ST1, a highly neuroinvasive strain, harbouring NS3249P, was constructed (Bahuon et al., 2012) and a NS3P249Tmutant was generated by directed mutagenesis. We aimed at deciphering the properties of recombinant viral particles in vitro and in vivo, in mammalian and bird models (Dridi et al., 2013). Results: In Vero cells, virus with a NS3249T protein proved to replicate at a slower rate than the parental NS3249P virus. When injected intraperitoneally in adult female Balbc/J mice, parental virus was found to be highly virulent (LD50 < 1 pfu), while only 4 out of 20 animals infected with the NS3249T virus succumbed, regardless of the initial infecting dose (1-103pfu). Mice infected with NS3249T virus experienced milder clinical and virological outcomes, characterized by delayed weight loss and decreased viremia 4 days pi (1.4x103 vs 3.2x104 viral copies/mL blood). Birds, one-day old chicks and young corvids (Corvus corone), also indicated that NS3249T virus was attenuated for model and susceptible European birds. In particular, in young crows, 16.7% (1/6) animals died after subcutaneous infection with NS3249T virus whereas a 100% lethality (7/7) was observed with NS3249P virus. Conclusion: The presence of a proline residue at position 249 in NS3 appears as a primary determinant for WNV virulence in wild birds, as well as in mammals and could be a genetic factor accounting for enhanced reporting of WNV neuro-invasive cases in humans in Europe.