Computational protocol: West Nile Virus Genetic Diversity is Maintained during Transmission by Culex pipiens quinquefasciatus Mosquitoes

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Protocol publication

[…] The presence of genetic bottlenecks and/or compartmentalization 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 . Alignments were generated for each mosquito and tested for recombination using the Genetic Algorithm for Recombination detection program implemented on the datamonkey.org website . Evidence of recombination was not detected, so the alignments were used to perform a migration analysis. To test the null 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) . 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 . 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 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 . […]

Pipeline specifications

Software tools BioEdit, GARD, Datamonkey, MacClade, MrBayes
Applications Phylogenetics, Population genetic analysis
Organisms West Nile virus, Culex quinquefasciatus
Diseases HIV Infections