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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 low-virulence 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.

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Figure 1: Genetic relatedness and geographic distribution of WNV genotypes (a) Maximum likelihood phylogenetic tree of 21 complete genomes of WNV.
Figure 2: Virulence of recombinant WNVs in AMCRs.


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We thank C. Cope of the Kansas Department of Wildlife Resources (Wichita, Kansas) and T. Janousek for their assistance with crow trapping and K. Bird, R. McLean and L. Clark of the US Department of Agriculture Wildlife Health Center (Fort Collins, Colorado) for assistance with avian housing. We thank W. Reisen and C. Barker for their assistance with statistical analyses. Trapping of AMCRs was performed under US Fish and Wildlife Scientific Collecting Permit number MB-032526, and experimental inoculations were performed under Centers for Disease Control and Prevention IACUC protocol numbers 02-26-012-MSA and 03-13-013-MSA, CSU IACUC approval 02-058A and UC Davis IACUC protocol number 11279. Funding for these studies was provided by the US Centers for Disease Control and Prevention (CI000235), the US National Institutes of Health (AI061822) and the Pacific Southwest Regional Center for Excellence (U54 AI065359). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

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This study was designed by A.C.B., R.M.K., A.M.P. and B.R.M. Mutant constructions were produced by C.Y.-H.H., R.M.K., S.A.L. and W.N.R. Avian infections were performed by R.A.B. and N.A.P. Viral titrations were performed by S.A.L., N.A.P., W.N.R. with assistance from A.M.P. Positive selection analyses were performed by E.C.H.

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Correspondence to Aaron C Brault.

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Brault, A., Huang, CH., Langevin, S. et al. A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nat Genet 39, 1162–1166 (2007).

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