Date Published: June 27, 2014
Publisher: Public Library of Science
Author(s): Stanley A. Langevin, Richard A. Bowen, William K. Reisen, Christy C. Andrade, Wanichaya N. Ramey, Payal D. Maharaj, Michael Anishchenko, Joan L. Kenney, Nisha K. Duggal, Hannah Romo, Aloke Kumar Bera, Todd A. Sanders, Angela Bosco-Lauth, Janet L. Smith, Richard Kuhn, Aaron C. Brault, Tian Wang.
A single helicase amino acid substitution, NS3-T249P, has been shown to increase viremia magnitude/mortality in American crows (AMCRs) following West Nile virus (WNV) infection. Lineage/intra-lineage geographic variants exhibit consistent amino acid polymorphisms at this locus; however, the majority of WNV isolates associated with recent outbreaks reported worldwide have a proline at the NS3-249 residue. In order to evaluate the impact of NS3-249 variants on avian and mammalian virulence, multiple amino acid substitutions were engineered into a WNV infectious cDNA (NY99; NS3-249P) and the resulting viruses inoculated into AMCRs, house sparrows (HOSPs) and mice. Differential viremia profiles were observed between mutant viruses in the two bird species; however, the NS3-249P virus produced the highest mean peak viral loads in both avian models. In contrast, this avian modulating virulence determinant had no effect on LD50 or the neurovirulence phenotype in the murine model. Recombinant helicase proteins demonstrated variable helicase and ATPase activities; however, differences did not correlate with avian or murine viremia phenotypes. These in vitro and in vivo data indicate that avian-specific phenotypes are modulated by critical viral-host protein interactions involving the NS3-249 residue that directly influence transmission efficiency and therefore the magnitude of WNV epizootics in nature.
Since 1999, WNV has expanded its geographic range to include both continents of the Western hemisphere and the Caribbean islands, with recent WNV activity documented on every major continent except Antarctica –. One hallmark feature associated with epizootic transmission events in North America has been avian mortality, predominantly in corvid species such as the American crow (AMCR; Corvus brachyrhynchos) –. Consequently, WNV dead bird surveillance programs have allowed, for the first time, a detailed molecular characterization of different geographic isolates as WNV expanded across vast ecological habitats in North America , . Results of these studies have shown that WNV is genetically stable with maximum genetic variation of 0.2% at the amino acid level among isolates made during its trans-continental spread between 1999–2004 . During this same time, WNV was responsible for large epizootics resulting in marked declines in highly susceptible avian populations–.
The recent expansion of WNV throughout the Western hemisphere and its re-emergence worldwide has highlighted questions concerning the introduction of specific WNV genotypic mutations that have enhanced avian transmission capacity leading to larger WNV epizootics. Naïve avian populations, moderately competent vector mosquito species, and WNV phenotypic adaptations to augment vector  and host competence  are possible driving forces that led to the expeditious spread of WNV across the continental U.S. in less than 5 years. Previous studies have identified a critical genetic determinant within the helicase domain of the NS3 protein (NS3-249) that modulates WNV pathogenesis in the AMCRs , . Introduction of an NS3-P249T substitution in a North American East Coast lineage 1a genotype backbone (NY99) restricted peripheral viral replication and dramatically increased the survivorship in AMCRs . Selection modeling coupled with the phenotypic evidence that this site modulates avian replication provided further support that replicative capacity in an avian host could be an instrumental feature for the emergence of different WNV lineages. A comprehensive study involving sequence alignments of 146 WNV NS3 proteins demonstrated this protein to be highly conserved among WNVs with a maximum divergence of 10% at the amino acid level. Highly conserved sequence motifs within the NS3 helicase flank the NS3-249 position (NS3-235-243 and NS3-256-282) and the NS3-256-282 motif comprises a portion of the DEAD/H binding domain involved in phosphorylation of protein kinase C (Fig. 2a) . Interestingly, amino acid alignments of WNV demonstrate that a series of hydrophobic residues (NS3-243-254) within the NS3 helicase that are highly conserved among WNVs, with variation being observed only at the NS3-249 residue (Fig. 2a). The NS3-249 residue is positioned on the terminus of the hydrophobic loop and would be in a strategic orientation for direct interaction with alternative viral or host proteins (Fig. 2b). Furthermore, this positioning would allow for any number of amino acids to be present at this loci and, as such, explains the potential for the variety of lineage-specific amino acid identities observed at this site.