WNV Diversity is Maintained in Mosquitoes Figure 1. Percent nucleotide diversity by time and tissue. The percent nucleotide diversity was determined for each sample and ploted by either days post infection (7 dpi (A), 14 dpi (B), & 21 dpi (C)) or by tissue type (midguts (D), legs (E), & saliva (F)). Dotted lines connect the means for each sample set. P-values were determined by ANOVA using Tukey’s multiple comparison post test. Letters above sample sets represent statistically significant groupings (p-value,0.05). Figures without letters denote that samples were not significantly different from one another. doi:10.1371/journal.pone.0024466.g001 analysis between the genetic diversity metrics and log transformed viral genome equivalents. The Pearson correlation analysis revealed that percent nucleotide diversity is significantly inversely correlated to viral genome equivalents (p = 0.003; Pearson r2 = 20.2747) (Figure 3A). Similarly, viral genome equivalents are inversely correlated to the proportion of unique viral variants (p = 0.0205; Pearson r2 = 0.1772) (Figure 3B). Frequency and Migration Analysis Analysis of the frequency and location of viral variants revealed that 78 of 883 sequences sampled were unique. These variants were found in all three tissue types and at all three time points. There were 16 variants unique to the ‘input’ mosquitoes, 6 were found in all four categories (input, midgut, legs, saliva), 19 were unique to saliva, 15 unique to legs, and 9 unique to midguts, and the remaining variants were found in multiple tissues. The 14 most common variants were then plotted to display their relative proportion in each mosquito sample (Figure 4). By analyzing the data by this approach we were able to track individual variants from ‘input’ through infection (midguts), dissemination (legs), and transmission (saliva). The ‘input’ set is a combination of all three 0 hpi mosquitoes and as expected represents a complex population of multiple variants. Generally, the midgut populations, at all three time points, are composed of only a few variants with no one variant PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24466