Molecular epidemiology of West Nile virus in North America
Charles Todd Davis
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The introduction of West Nile virus (WNV) into the U.S. during the summer of 1999 led to the largest epidemic of arboviral encephalitis ever recorded in the Western Hemisphere during 2002. Over the course of six years, the distribution of WNV has expanded to include each of the contiguous U.S. states and seven Canadian provinces, as well as Mexico, several Caribbean Islands, and Colombia. In order to understand how the virus has evolved since its emergence in North America, this dissertation investigates the genetic and phenotypic variation among WNV isolates collected in various regions of North America during different transmission seasons. The overall objectives of this dissertation were to study the extent to which WNV has evolved since its emergence in North America and to better understand the relationship between viral evolution and phenotypic variation in an emerging viral population. The first aim of this project was to compare nucleotide and deduced amino acid sequences of WNV isolates collected in North America during 2002, 2003, and 2004 to those collected during earlier years. Sequence comparisons of WNV isolates collected throughout North America during different years have identified nucleotide/amino acid substitutions that reveal the emergence of genetically divergent variants of WNV in recent years and support the hypothesis that microevolution of WNV will continue from year to year and as the distribution of the virus expands. In addition, these studies have identified a dominant genotype of North American WNV that has displaced all other known genotypes throughout North America. This research also led to the discovery of several genetic variants with altered phenotypes. Thus, the second aim of this project investigated the phenotypic characteristics of WNV variants collected in 2003 and 2004 by evaluating their small plaque and temperature-sensitive phenotypes, their multiplication kinetics in cell culture, and attenuation in a mouse model. Finally, reverse genetic techniques were used in a third aim in order to precisely identify the mutations responsible for the observed phenotypic changes in WNV and to illustrate novel molecular mechanisms of attenuation of WNV. The results from these aims demonstrate that as WNV has accumulated mutations in its genome, phenotypic variants have emerged with significantly different biological properties when compared to progenitor virus isolates that initiated the epidemic in North America. Also, this study has identified novel molecular determinants of attenuation in WNV that provide valuable insight into the multigenic components of pathogenicity for WNV and possibly other related flaviviruses.