WNV Diversity is Maintained in Mosquitoes Figure 2. Proportion unique viral variants by time and tissue. The proportion of unique viral variants was determined for each sample and plotted 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 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.g002 dominating in all samples. Likewise, the WNV populations recovered from the legs had, in general, low intrahost variability, but with no overrepresentation of any one variant between mosquitoes. Interestingly, there was an expansion in the total number of variants identified in the saliva compared to the midguts and legs. These findings are supported by the proportion of unique viral variants analysis (Figure 2). Further, many of the variants that were present in the input samples and subsequently undetected in the midguts and legs were recovered from the saliva (Figure 4). Included in this data was a variant that contained a single nucleotide deletion at nucleotide 2194 in the E-glycoprotein. This deletion mutant was found in the legs or saliva of three different mosquitoes at 7 and 14 dpi (black colored sections of Figures 4A & 4B). Migration analyses were performed in order to more closely look for evidence of genetic bottlenecks and test for tissue compartmen- talization (Figure 5). A hypothetical tree was generated to represent what would be expected if strong genetic bottlenecks were influencing WNV populations during the EIP (Figure 5A). Under this scenario, a single ‘input’ variant initiates infection in the midgut. Subsequently, a single midgut variant establishes an infection in the hemolymph from which a single variant invades the salivary glands and is transmitted. However, this is not what we observed. Three PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24466