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

Abstract

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.

Figure 1

Genetic relatedness and geographic distribution of WNV genotypes (a) Maximum likelihood phylogenetic tree of 21 complete genomes of WNV. Viruses are grouped according to the four described lineages of WNV, and the substituted amino acid at each NS3-249 site is indicated (and color coded). The most parsimonious reconstruction of the amino acid residue at internal nodes is also shown. The tree is midpoint rooted, and branch lengths are drawn to scale to indicate the number of substitutions per site. For details on the strains used in this tree, including isolate name, infected organism and GenBank accession number, see Methods. The asterisks represent nodes with bootstrap support values >95%. (b) Geographic distribution of WNVs in Africa, Europe and Asia (light blue). Colored dots indicate NS3-249 genotypic identity.

Figure 2

Virulence of recombinant WNVs in AMCRs. (a) Viremia in AMCRs infected with WN/IC-P991 (n = 8), WN/IC-KEN (n = 16), WN/IC-P991-NS3-P249T (n = 8) and WN/IC-KEN-NS3-T249P (n = 16) viruses. AMCRs were bled daily, and viral titers were determined by plaque titration on Vero cells with a detection limit of 1.6 log₁₀ PFU/ml serum. Error bars represent s.d. (b) Survivorship of AMCRs inoculated with 1,500 PFU of WN/IC-P991 (n = 8) and WN/IC-KEN (n = 16) viruses as well WN/IC-P991-NS3-P249T (n = 8) and WN/IC-KEN-NS3-T249P (n = 16) viruses. Parental viruses are designated by solid lines and symbols, and viral point mutants are designated by dashed lines and open symbols. Red lines and symbols represent viruses with the NS3-T249P substitution, and blue lines and symbols represent viruses with NS3-Thr249. Diamonds represent the WN/IC-P991 backbone, and circles represent the WN/IC-KEN backbone.