an MOI of 0.1 and the resultant population titered and genetically characterized. To infect mosquitoes, WNV M24 was mixed 1:1 with defibrinated goose blood and offered to adult female Culex pipiens quinquefasciatus 7–8 days post emergence. The virus titer in the bloodmeal was 66106 pfu/ml. Fully engorged females were separated from the remaining unfed mosquitoes and housed in an environmental chamber (27uC, 16:8 L:D photoperiod) for the remainder of the experiment. Sample Collection and Virus Detection To quantify viral genetic diversity of the ‘input’ virus population, three fully engorged mosquitoes were placed in RNA extraction buffer immediately after feeding and homoge- nized using a mixer mill. Viral genetic diversity was quantified as described below. At 7, 14, and 21 days post-infection paired tissues samples (midguts, legs, and saliva) were collected from 20–25 mosquitoes. To ensure that contaminating WNV from the hemoceol was not introduced into our midgut samples, dissected midguts were washed three times in PBS before placing the samples in RNA extraction buffer. Dissecting forceps were flame sterilized between dissections to avoid cross contamination between samples. Total RNA was extracted from mosquito hemolymph and tissues using the RNeasy Mini Protect kit (Qiagen, Valencia, CA) and screened for the presence of WNV RNA by one-step RT-PCR using the Superscript III kit with platinum Taq (Invitrogen, Carlsbad, CA). WNV specific primers used in this study spanned a 934 nt. region corresponding to the E-NS1 junction (1971 nt–2928 nt). Three mosquitoes with detectable WNV RNA in all three tissue types were selected from each time point for further analyses. Quantification of Viral Genetic Diversity Viral genetic diversity was determined according to methods previously described [14]. Briefly, cDNA was generated from 5 ml of total RNA using the High Fidelity Reverse Transcription kit (Stratagene, Cedar Creek, TX) according to the manufacturers specifications and WNV specific primers, WNV 1971 F and WNV 2928 R. Subsequently, the cDNA served as template for high fidelity Pfu Ultra polymerase amplification (Stratagene). Amplicons were PCR purified and cloned into the pCR Script Amp (+) vector (Stratagene). Between 21–30 individual clones from each of the samples were sequenced using the M13F, M13R, WNV 2369 F, and WNV 2768 R primers. DNAStar’s SeqMan module (DNAStar Inc., Madison, WI) was used for sequence alignment and analysis of genetic diversity. Only clones with two-fold sequencing coverage were considered complete. As a means to estimate genetic diversity, consensus sequences for each sample were determined and individual clones within that sample were then compared to the specimen-specific consensus sequence. The percent nucleotide diversity (total number of mutations from all clones within a sample divided by the total number of nulcoetides sequenced per sample) and the proportion of unique viral variants (the number of unique clones differing from the consensus divided by the total number of clones sequenced per sample) were calculated and used as indicators of genetic diversity. Quantification of Viral Genome Equivalents WNV genome equivalents were determined by quantitative-RT- PCR (Q-RT-PCR). As a standard control for this assay a ,2 kb fragment from the WNV E gene was amplified using the WNV 1031 F and WNV3430 R primers. The resultant amplicon was cloned into the pCR2.1-TOPO vector (Invitrogen) downstream of the T7 promoter. The recombinant vector was linearized with Kpn WNV Diversity is Maintained in Mosquitoes I, purified and used as template for in vitro transcription using the T7 Megascript kit according the manufacturer’s instructions (Ambion, Austin, TX). The resultant RNA was quantified and aliquoted in serial ten-fold dilutions. Using a probe specific for the E gene, the WNV 1160 F and WNV 1229 R primers, and the TaqMan H One- Step RT-PCR Master Mix Reagent (Applied Biosystems, Foster City, CA) viral RNA copy numbers were determined [33]. Samples were run on the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Frequency and Migration Analysis The presence of genetic bottlenecks and/or compartmentaliza- tion was further assessed by migration analyses. To determine the frequency and location of viral variants, sequences from each sample were aligned in DNAStar’s SeqMan module, exported as FASTA files and duplicates removed using BioEdit [34]. Alignments were generated for each mosquito and tested for recombination using the Genetic Algorithm for Recombination detection program implemented on the datamonkey.org website [35]. Evidence of recombination was not detected, so the alignments were used to perform a migration analysis. To test the hypothesis of panmixis versus the alternative that there are distinct WNV sub-populations within different mosquito tissues, we used the Slatkin-Maddison test for gene flow in MacClade version 4 (Sinauer Associates, Sunderland, MA) [36]. Tissue of origin was assigned to each taxon in a one-character data matrix. ‘Input’ sequences from freshly-fed mosquitoes were included as an estimate of the population of variants present in the infectious bloodmeal. In total there were four character states (input, midgut, legs, and saliva). The Slatkin-Maddison test was performed independently for each mosquito. This analysis was performed on Bayesian phylogenies, generated with MrBayes 3.1.2 [37]. These were run with a general time reversible (GTR) model with invariable rates with substitution rates following a gamma plus invariants distribution. Two Markov Chains Monte Carlo (MCMC) tree searches of 5 million generations each were run in parallel with sampling one in every 250 trees. 50% majority-rule consensus trees are shown based on the last 19,000 trees. Briefly, the phylogenetic tree resulting from the nucleotide data was loaded into MacClade and the most parsimonious reconstruction of this ancestral character inferred with the Fitch algorithm [38] in order to estimate the minimum number of steps required to explain the distribution of tissue states on the tree of interest. We then generated 10,000 random trees by random joining and splitting of the input tree and compared the number of steps on our input tree to those calculated in the random trees, as described previously for HIV-1, using ordered tissue states [39]. Statistical Analysis Statistical analyses were completed in Microsoft Excel and GraphPad Prism. A one-way analysis of variance (ANOVA) with the Tukey’s multiple comparison post-test with a significance level of a = 0.05 was used for analysis of the percent nucleotide diversity and proportion of unique viral variants. A Pearson correlation analyses was perfomed on log transformed viral genome equivalents versus percent nucleotide diversity and proportion of unique viral variants. Figures were generated in GraphPad. Supporting Information Figure S1 WNV genome equivalents per tissue sample. WNV genome equivalents were determined by Q-RT-PCR for each sample characterized. (TIF) PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24466