Deep sequencing of WNV isolates collected in Harris County, TX 2013-2016

Harris County, Texas experienced its largest outbreak of West Nile (WNV) disease in 2014. Characterization WNV isolates from the outbreak using next generation sequencing approaches, phylogeny, and animal studies revealed a novel genotype characterized by selection of an amino acid substitution, NS2A-R188K. Retrospective analysis of Genbank showed this genotype was first identified in New York and Connecticut in 2008 and then in Texas subsequently. Thus, we have termed this the Northeast 2008 (NE/WN08) genotype. Emergence of this genotype in Texas likely involved the displacement of the SW/WN03 genotype. Quasispecies diversity revealed significant variation occurred among viral isolates, but no phenotypic changes were observed in terms of temperature sensitivity and mouse virulence, suggesting that despite WNV being in North America for 15 years there are strong mutational robustness among naturally occurring WNV isolates. Examination of Genbank showed that convergent evolution of NS2A-R188K was also taking place worldwide. Mosquito pools and dead birds in Harris County, TX were collected by the Harris County Public Health, Mosquito & Vector Control Division (HCPHMVCD) in Houston, TX, USA. The brain homogenate of twenty nine WNV-positive birds and a single WNV-positive mosquito pool was passaged once or twice in Vero cells, and virus was collected from the supernatant of infected Vero cells when CPE was apparent. WNV RNA was extracted using the QIAamp Viral RNA Mini kit according to the manufacturer’s recommendations. Libraries were generated with a TruSeq RNA v2 kit (Illumina) and samples were sequenced on an Illumina HiSeq 1500 platform.