West Nile virus (WNV) was first identified in North America in New York City in 1999.1 By 2002, WNV was detected in 44 states, including Texas.2 Epidemic levels of WNV human cases occurred in the United States in 2002 and 2003, and WNV has been endemic from 2004 to 2011 with a fairly stable annual incidence in humans.3–6 West Nile virus is maintained in an enzootic cycle between mosquitoes (predominantly Culex spp.) and passerine birds, with annual outbreaks in humans typically peaking in August.6 Previous studies have estimated that ∼20% of humans infected with WNV develop West Nile fever (WNF), and < 1% develop West Nile neuroinvasive disease (WNND).7

The largest outbreak of human WNF and WNND cases in the United States since 2003 occurred during 2012. As of December 11, 2012, 48 states and the District of Columbia reported a total of 5,387 confirmed or probable human WNF and WNND cases to the Centers for Disease Control and Prevention (CDC) through ArboNET. Approximately one-third of all United States WNF and WNND cases were reported from Texas. Within Texas, the incidence of WNV disease varied between counties. For example, the incidence of WNF and WNND was 16.1 cases/100,000 population in Dallas County and 3.6 cases/100,000 population in Montgomery County (Figure 1A and B). The reason for the increased incidence of WNV disease in 2012 is unknown, although climate, ecological changes in WNV vectors, and viral genetics have been suggested.8

In a manner similar to other arboviruses, WNV evolution is predominantly subject to purifying selection,9,10 and limited phylogeographic structure has been identified for WNV isolates10–12; few adaptive changes have been identified during its evolutionary history. Notable examples are a valine to alanine mutation at position 159 of the envelope protein that has been reported to infer fitness benefits in mosquitoes13,14 and has swept across the United States,15,16 and a threonine to proline mutation at position 249 in NS3 that has fitness benefits in crows and has been identified to have evolved under the effects of positive selection.17 As dead-end hosts, humans are unlikely to affect the evolution of WNV on a large scale; however, mutations arising in bird and mosquito hosts could stochastically affect viral replication and/or pathogenesis in humans. Additionally, altered transmissibility between mosquitoes and avian hosts could be associated with increased amplification and transmission to humans.

To determine whether the 2012 WNV outbreak may have been driven by recent WNV evolution, we assessed the phylogenetic relationship between WNV isolates from Texas in 2012 and isolates from North America in previous years. We also measured the genetic diversity and divergence of WNV isolates from Dallas and Montgomery Counties with variable disease incidence rates in Texas. Together, these data indicate that WNV evolution in 2012 was similar to previous years and is unlikely to explain the increased incidence in humans.

In this study, we investigated the evolution of WNV isolates made in Texas during the largest outbreak in recent United States history. To determine whether WNV genetics may have contributed to the increased incidence of human WNV disease in 2012, we sequenced full-length WNV genomes from mosquitoes collected in Texas during the 2012 epidemic. We found that the mutations in the 2012 Texas isolates were largely driven by genetic drift and viral population structure molded by purifying selection in a manner similar to previous years. Furthermore, despite differences in WNF and WNND incidence between Dallas and Montgomery Counties, the WNV isolates from these areas were phylogenetically indistinguishable. Together, these data indicate that a major role for WNV genetic determinants in the 2012 outbreak was unlikely.

The 2012 Texas WNV isolates clustered in two distinct clades, with the most closely related isolates from Connecticut in 2008 and New York in 2009 (Figure 2). This indicates that the 2012 WNV epidemic was derived from two WNV strains endemic to the United States; however, there may have been two recent reintroductions of WNV into Texas from the East coast. This is characteristic of the long-range movements of WNV across North America.11 However, there is likely short-distance viral gene flow between Dallas and Montgomery Counties because WNV isolates from these areas were phylogenetically similar. Even though WNF and WNND incidence in Dallas County was five times higher than in Montgomery County (Figure 1), similar WNV strains were found in both locations. This shows that a geographically isolated virulent strain of WNV did not cause the 2012 outbreak in Dallas. Thus, differences in WNV genetics are unlikely to explain the difference in WNF and WNND incidence between Dallas and Montgomery Counties.

Rapid evolution is a characteristic of genomes involved in antagonistic virus-host interactions28; therefore, identifying sites evolving under positive selection can pinpoint mutations with fitness consequences. In this study, we identified two residues that are rapidly evolving in the United States lineage of WNV: NS2A₅₂ (Table 3) and NS5₃₁₄. The NS5₃₁₄ has previously been shown to have evolved under positive selection, making it a tempting candidate for an adaptive change29; however, NS5₃₁₄ only showed variation in Montgomery County isolates and was therefore unlikely to have affected the 2012 outbreak. In a previous study of WNV pathogenesis in American crows, an alanine-to-threonine mutation at NS2A₅₂ was shown to be one of 11 mutations potentially involved in the increased virulence in American crows of WNV NY99 compared with a Kenyan strain of WNV.30 However, most United States WNV strains since 1999 have contained a threonine at NS2A₅₂, suggesting that this mutation is either unrelated to the 2012 outbreak or requires additional mutations to affect fitness. Of the 17 additional non-synonymous mutations in the 2012 Dallas isolates, one site was identified to be subject to purifying selection (Table 3), suggesting this mutation may be deleterious to viral fitness. The remaining 16 mutations, although not rapidly evolving or having a known functional consequence, may elicit an adaptive phenotype such as increased transmission efficiency in mosquitoes; however, vector competence studies would be required to fully assess this question.

Although WNV sequences from humans infected during the 2012 outbreak were not available for analyses, the mosquito isolates that were assayed should encompass the diversity of the strains circulating during the summer of 2012. Genetic diversity of WNV is maintained in mosquitoes,31,32 and studies of human and mosquito WNV isolates have not shown phylogenetic separation by host species.11 In addition, the level of divergence between the 2012 Texas isolates and other United States isolates was comparable to estimates from previous years in Texas and other geographic regions of the United States.9,10,33–35 Interestingly, significantly higher genetic diversity within the Clade II Dallas population compared with the Clade II Montgomery population was observed (Table 2), although the sampling area and timeframe of isolate collection were not matched between populations. The Dallas isolates were collected over 8 weeks from multiple cities, whereas the Montgomery isolates were collected over a 7-week span from one city (Table 1 and Figure 1). Thus, our population genetic diversity estimates could be skewed by mosquito sampling.

In addition to viral genetics, ecological factors have been shown to increase viral transmission in mosquitoes and birds. For example, temperature affects the WNV extrinsic incubation period of mosquitoes, such that increased temperature increases the rate of WNV transmission by mosquitoes.13,36 Previous outbreaks of WNV in 2002–2004 were associated with higher than average temperatures,36,37 and the average annual temperature in Texas in 2012 was 2° warmer than the average annual temperature in 2002–2011 (NOAA National Climatic Data Center). Furthermore, the average monthly temperature during the peak of the 2012 outbreak (June through September, Figure 1) was 1° to 6° warmer in Dallas than in Houston (NOAA National Climatic Data Center), which may help to explain the higher viral diversity in Dallas County mosquito isolates or the greater rates of WNV transmission in Dallas County. In addition, changes in mosquito or avian populations may have contributed to altered viral transmission rates. Based on the results presented here, rather than genetic changes in WNV leading to increased transmission efficiency to humans, it is more likely that a combination of other environmental and ecological factors could explain the magnitude of the 2012 WNV outbreak.