Genotypic and Molecular Determinants in the Continued Evolution of West Nile Virus in the United States
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Following the introduction of West Nile virus (WNV) into New York in 1999, the virus continues to evolve in the U.S. with the emergence, extinction, and co-circulation of multiple, distinct U.S. genotypes. Despite annual local, regional, or national epidemics (varying in size by year), limited progress has been made dissecting how and when viral outbreaks occur; therefore, the objective of this dissertation was to investigate both genotypic and molecular determinants involved in continued evolution of WNV by relating in silico and in vitro differences in the molecular epidemiology, population dynamics, and phenotypic properties of both natural and infectious clone-derived viruses. Phylogenetic analysis revealed bi-directional virus transmission (i.e., gene-flow) on the U.S.-Mexican border despite limited prior isolation or evidence of WNV circulation in northern Mexico; in addition, paired geospatial and phylogenetic comparisons identified the emergence of four distinct genetic clusters (Groups 7-10) of novel 2010-2012 WNV isolates in both Houston (Harris County) and Dallas/Fort Worth, Texas concurrent with the regional 2012 Texas WNV epidemic—implicating recent emergence and circulation of virus isolates with novel genotypic signatures. In silico application of 2002-2012 Houston, Texas isolates implicated the Harris Co. paradigm as a novel, surrogate model for national WNV surveillance and evolution in the U.S. Incorporation of nineteen 2002-2012 Harris Co. isolates from this paradigm in the Illumina HiSeq1000 next generation sequencing (also known as “deep” sequencing) platform identified both host- and genotypic-dependent trends in the diversity and evolution of regional WNV populations; additional, paired phenotypic studies revealed a relationship between an attenuated neuroinvasive mouse phenotype and low genetic diversity in natural WNV quasispecies structure. These in silico results and prior applications of the Harris Co. paradigm identified several natural mutations encoded in the NS4B protein—a protein that has been linked to interferon (IFN)-α/β antagonism. In vitro implementation of engineered NY99 infectious clone (NY99ic)-derived viruses—encoding natural or in vitro-selected NS4B substitutions—in the IFN-competent human A549 in vitro cell line revealed both residue- and domain-dependent dysregulation of host innate antiviral responses. Overall, the studies in this dissertation support continued evolution of WNV with evidence of genetic and phenotypic changes over time.