Abstract
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.
Highlights
Since 1999, West Nile virus (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 [1,2,3,4,5,6,7,8]
Sequence analyses In order to assess the extent of intra- and inter-lineage incorporation of the NS3-249 proline mutation, Bayesian (Fig. 1a) and Maximum likelihood (Fig. 1b) phylogenies of WNV strains from lineages 1–4 were constructed and the amino acid identity at position NS3-249 for individual isolates color-coded on the resulting phylogeny
Results demonstrated the threonine to occupy the ancestral position at basal nodes and within this phylogeny, an NS3-249 mutation from a Thr to Pro, Ala, His or Asn has been demonstrated on at least twelve independent occasions. Eight of these mutations led to a NS3249P at this position, with seven of these NS3T249P mutations having occurred within lineage 1a strains, and one having occurred in a lineage 2 strain (Greece 2010) (Fig. 2a)[23]
Summary
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 [1,2,3,4,5,6,7,8]. 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 [12,13]. 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 [13]. Kunjin viruses (lineage 1b) have an alanine (Ala), lineage 2 WNVs are associated with histidine (His) at this locus [20] and lineage 3 [21] exhibit an asparagine (Asn), whereas many lineage 1 and the single representative lineage 4 WNV strain [21,22] have a Thr at NS3-
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