A single positively selected West Nile viral mutation confers increased virogenesis in American crows

West Nile virus ( WNV), first recognized in North America in 1999, has been responsible for the largest arboviral epiornitic and epidemic of human encephalitis in recorded history. Despite the well- described epidemiological patterns of WNV in North America, the basis for the emergence of WNV-associated avian pathology, particularly in the American crow ( AMCR) sentinel species, and the large scale of the North American epidemic and epiornitic is uncertain. We report here that the introduction of a T249P amino acid substitution in the NS3 helicase ( found in North American WNV) in a lowvirulence strain was sufficient to generate a phenotype highly virulent to AMCRs. Furthermore, comparative sequence analyses of full- length WNV genomes demonstrated that the same site ( NS3- 249) was subject to adaptive evolution. These phenotypic and evolutionary results provide compelling evidence for the positive selection of a mutation encoding increased viremia potential and virulence in the AMCR sentinel bird species.