WNV Diversity is Maintained in Mosquitoes Figure 3. Viral genome equivalents and genetic diversity are inversely correlated. Black = ‘input’, Orange = midguts, Green = legs, and Purple = saliva. (A) Log transformed genome equivalents for each sample plotted against percent nucleotide diversity, n = 30, p = 0.003, Pearson r2 = 0.2747. (B) Log transformed genome equivalents for each sample plotted against the proportion of unique viral variants, n = 30, p = 0.0205, Pearson r2 = 0.1772. doi:10.1371/journal.pone.0024466.g003 representative mosquitoes, one from each time point, are shown and the remaining six trees are provided in a supplement (Figure S3). The migration analysis indicates some evidence for compartmen- talization based on the p-values for ordered character states as estimated from 10,000 randomly sampled trees. A cluster of closely related variants isolated from legs was identified in our represen- tative mosquito at 7 dpi (Figure 5B p,0.0001). Similarly, clusters of saliva variants were identified at 14 dpi and 21 dpi (p,0.0001 and p,0.0001, respectively) (Figures 5C & 5D). In vivo Competition Assay To test for the presence of genetic bottlenecks using a less genetically complex virus population, mosquitoes were fed on chicks infected with a known mixture of marked-reference and wild-type WNV (Text S1). The mean proportion of wild-type to reference virus in chick viremia was 0.73 (n = 4, SEM 0.011) (Figure S4). Subsequent analysis of the mosquito samples revealed that there was no change in the proportion of wild-type to reference WNV in any mosquito tissue compared to chick viremia (bodies 0.8 SEM 0.031, legs 0.73 SEM 0.07, and saliva 0.75 SEM 0.087). Figure 4. Frequency and location of unique viral variants. There were 883 clones sequenced, of which 78 sequences were unique. The frequency of the fourteen most common viral variants was mapped back to each sample. The column labeled input combines the data from all three 0 hours post infection mosquitoes. Samples are broken down by days post infection (7 dpi (A), 14 dpi (B), & 21 dpi (C)). Each time point includes three mosquitoes (denoted mosquitoes 1–3) and further broken down by tissue (M (midguts), L (legs), & S (saliva)). The white sections of the histograms represent the remaining 64 uncommon variants and the black sections represent a single nucleotide deletion mutant found in multiple samples. doi:10.1371/journal.pone.0024466.g004 Discussion Population bottlenecks during transmission may profoundly influence the evolution of arboviruses by stochastically reducing population variation, thereby selecting random genomes that may be less fit than the overall population. Currently, it is unclear PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24466