proportion of unique viral variants in midguts are significantly lower than in the ‘input’ population (Figures 1D & 2D). Surprisingly, however, the genetic diversity of peripheral WNV was not significantly different from virus in the bloodmeal, with saliva-associated WNV (i.e. the WNV that would be transmitted by a feeding mosquito) tending to be the most genetically diverse of the three tissue types sampled (Figures 1 & 2). The mechanism(s) that lead to increased genetic diversity outside of the midgut are not clear. The presence of a ‘leaky midgut’ may explain this discrepency between the percent nucleotide diversity in legs and saliva compared to that of the midguts [24,25]. However, we found little evidence of WNV bypassing the midgut and directly infecting secondary tissues (Figure S2). In addition, we observed a significant inverse correlation between viral genome equivalents and the genetic diversity metrics (Figure 3). Taken together, these findings suggest that although WNV populations appear to be restricted in the midguts, and to a lesser extent in hemolymph, the genetic diversity of transmitted WNV was similar to that of the ingested virus population, and that variables other than tissue of origin determine viral genetic diversity in mosquitoes. It may be that WNV accumulates mutations during the course of mosquito infection: relaxation of purifying selection on WNV sequences has been associated with mosquito infection [15]. To assess this possibility, we compared the frequency and location of viral variants present in our mosquito tissue samples to those of the ‘input’ population. Not surprisingly, the majority of the viral variants identified in the midguts were also present in the ‘input’ population (Figure 4). Interestingly, however, variants found in legs and saliva were also represented in our ‘input’ dataset, without being present in the mosquito midguts. These findings support our quantitative analysis of genetic diversity, in these tissues, and indicate that the increased variation observed in peripheral WNV populations was more attributable to genetic diversity in the ‘input’ WNV population than to the generation of novel mutants during mosquito infection. Additional evidence supporting the infection of a single cell by multiple WNV variants was obtained through examination of a defective WNV sequence in our dataset. Specifically, we identified a single nucleotide deletion mutant that was found in multiple mosquito samples, including peripheral compartments, but not in the ‘input’ (Figure 4). Although it is possible that these mutants arose independently, it seems more likely that an ancestral mutant was present but undetected in the M24 population and was maintained in mosquitoes by complementa- tion. Numerous studies have observed complementation of defective Flavivirus genomes in cell passage experiments [26,27]. Typically, these studies have found large, ,2 kb, in-frame deletions at the 59-end of the genome in the structural genes. Interestingly, one study found long-term transmission of a defective DENV-1 virus with a premature stop codon in the E gene [21]. This data suggests that defective WNV particles can infect mosquitoes, propagate through complementation and ultimately be transmitted (Figure 4; mosquito 3 saliva 7 dpi). This implies that multiple WNV virions may frequently infect a single midgut cell, providing a mechanism by which WNV genetic diversity may be maintained in mosquitoes despite limitations in the number of susceptible midgut cells [22,23]. Finally, we performed a migration analysis to formally test for the presence of bottlenecks and compartmentalization. We detected compartmentalization in legs and saliva, but found no evidence of genetic bottlenecks (Figure 5). In this analysis, if genetic bottlenecks exist, viral variants from the tissue samples would originate from a single ‘input’ variant as demonstrated in our hypothetical tree. Rather, variants identified in the saliva were WNV Diversity is Maintained in Mosquitoes found to originate from multiple ‘input’ variants indicating the ability of numerous ‘input’ variants to overcome multiple mosquito barriers to infection (i.e. midgut infection, midgut escape, and salivary gland escape barriers). The artificial nature of this experimental system may explain the discrepancies between our tests. Mosquitoes were offered a bloodmeal containing WNV M24 which contains an approximately 10 fold increase in the genetic diversity compared to natural WNV populations [14,19]. This approach was implemented as a means to more easily track variation in our populations. It may be that the perceived bottlenecks were artificial due to saturating the system. As a more realistic approach to testing for bottlenecks we performed an in vivo competition assay in which infectious clone-derived wild-type WNV was competed against a marked reference virus [19]. It was observed that the proportion of marked refernce virus to wild-type WNV remained unchanged from ‘input’ to bodies, legs or saliva (Figure S4). Together, these data suggest that genetic bottlenecks do not significantly influence WNV populations during the EIP in Cx. p. quinquefasciatus. Our genetic approach to transmission bottlenecks provides an intersting contrast to previous studies of bottlenecks in arbovirus transmission cycles [22,23]. Using virus-like particles to track binding and internalization, one study demonstrated that WNV infects only a few midgut epithelial cells during infection of Cx. quinquefasciatus [23]. Similar results were found during VEEV infection of Aedes taeniorhynchus [22]. By virtue of the small number of infected cells it was concluded that arbovirus populations may be stochastically reduced at the point of infection. Our genetics studies of WNV do not support this obsevation. Notably, these conclusions are not necessarily mutually exclusive: It may be that the small proportion of susceptible midgut cells are infected with more than one virus particle or that an undetectable level of infection occurred in a higher proportion of cells. In fact, a high frequency of dual infections were observed in the VEEV-Aedes system [22]. Essentially, only a few susceptible midgut cells may be needed to propagate a diverse arbovirus population. Our observation of a deletion mutant persisting, apparently through complementation, during mosquito infection supports this possi- bility. The literature regarding the role of bottlenecks in natural transmission cycles of RNA viruses is currently ambiguous. Bottlenecks are seemingly unimportant for Cauliflower mosaic virus in plants, but may exist for other RNA viruses [28–31]. Numerous factors may contribute to this discrepency such as virus species, single vs two-host systems, mode of transmission and/or site of inoculation. Further, environmental or host genetic factors may influence differences between individual hosts within a given population and likely explain the high variablity observed between individual mosquitoes in this experiment [32]. Never- theless, our data establish that transmitted WNV populations are at least as diverse as those of the imbibed population and therefore suggests that genetic bottlenecks are unlikely to significantly influence WNV population biology during horizon- tal transmission. Materials and Methods Virus and Mosquito Infections The highly genetically diverse WNV population, WNV M24, used for these studies has been previously described [19]. Briefly, 24 WNV isolates from naturally infected mosquitoes and birds were passaged once on C6/36 Aedes albopictus cells [14]. Titers were determined by plaque assay on Vero cells and mixed at a 1:1:1… ratio. This mixture was amplified once on C6/36 cells at PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24466