Computational protocol: Spatial and Temporal Phylogeny of Border Disease Virus in Pyrenean Chamois (Rupicapra p. pyrenaica)

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Protocol publication

[…] Viral RNA was extracted from chamois spleen tissues using a commercial RNA extraction kit (Nucleospin Viral RNA Isolation, Macherey Nagel, Düren, Germany). Reverse transcription and PCR assays targeting 5’UTR region of pestivirus were performed using 324 and 326 pan-pestivirus primers [] following previously described protocols []. For each sample, amplicons of the expected size were purified and sequenced using forward and reverse primers by cycle sequencing using a Big Dye Terminator version 3.1 kit and an ABI PRISM 3130xl sequencing device (Applied Biosystems, CA, USA).All sequences from chamois and domestic hosts were aligned with BDV reference strains, retrieved from GenBank and representative of BDV phylogenetic groups according to [], using CLUSTALW (integrated within the Bio-Edit sequence editor, freely available at http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogeny was preliminary estimated by the neighbor-joining algorithm (NJ) and the maximum likelihood (ML) method. [...] The best-fitting nucleotide substitution model was estimated by means of jModeltest [], and selected a TrN model [] with gamma-distributed rates among sites.The phylogenetic tree, model parameters, evolutionary rates and population growth were co-estimated using a Bayesian Markov chain Monte Carlo (MCMC) method implemented in the BEAST v.1.74 package [].Statistical support for specific clades was obtained by calculating the posterior probability of each monophyletic clade. As coalescent priors, we compared four simple parametric demographic models (constant population size, and exponential, expansion and logistic population growth) and a piecewise-constant model, the Bayesian skyline plot (BSP) under both a strict and a relaxed (uncorrelated log-normal) clock [].Two independent MCMC chains were run for 50 million generations with sampling every 5,000th generation, and were combined using the LogCombiner 1.74 included in the BEAST package. Convergence was assessed on the basis of the effective sampling size (ESS) after a 10% burn-in using Tracer software version 1.5 (http://tree.bio.ed.ac.uk/software/tracer/). Only ESS’s of ≥ 200 were accepted.Uncertainty in the estimates was indicated by 95% highest posterior density (95% HPD) intervals, and the best-fitting models were selected using a Bayes factor (BF with using marginal likelihoods) implemented in BEAST [].In accordance with [],only values of 2lnBF ≥ 6 were considered significant.The trees were summarised in a target tree using the Tree Annotator program included in the BEAST package, choosing the tree with the maximum product of posterior probabilities (maximum clade credibility) after a 10% burn-in.The time of the most recent common ancestor (tMRCA) estimates were expressed as mean and 95% HPD years before the most recent sampling dates, corresponding to 2011 in this study. [...] The continuous-time Markov Chain (MCC) process over discrete sampling locations implemented in BEAST [] was used for the geographical analysis, implementing the Bayesian Stochastic Search Variable Selection (BSSVS) model which allows the diffusion rates to be zero with a positive prior probability. Comparison of the posterior and prior probabilities that the individual rates would be zero provided a formal BF for testing the significance of the linkages between locations: rates with a BF of >3 were considered well supported and formed the migration pathway. This analysis was performed for Pyrenean chamois BDV dataset (n = 50) assigned to 7 distinct geographic groups, on the basis of the sampling location, corresponding to: Alt Pallars (APA n = 7), Alta Ribagorça (ARI n = 4), Andorra (AND n = 4), Aran (ARA n = 14), Cadí (CAD n = 13), Cerdanya-Alt Urgell (CER n = 6) Freser-Setcases (FRE n = 2) (). In preliminary analysis, Ariege strains (n = 2) significantly clustered in Cerdanya-Alt Urgell area and therefore they have been assigned to this area due to limited number of sequences available. Centroids were used as reference coordinates to build discrete geographic groups. The Maximum Credibility Tree (MCC) final tree was visualised using FigTree version 1.4 (available at http://tree.bio.ed.ac.uk/software). The significant migration rates were analysed and visualised using SPREAD, which is available at http://www.kuleuven.be/aidslab/phylogeography/SPREAD.html. [...] With the aim to infer intraspecies reticulate evolutionary relationships among samples analysed, a Median Joining Network [] was constructed using PopArt (Population Analysis– http://popart.otago.ac.nz.) based on sequences available in . […]

Pipeline specifications

Software tools Clustal W, BioEdit, jModelTest, BEAST, FigTree, PopART
Applications Phylogenetics, Population genetic analysis
Organisms Ovis aries, Border disease virus, Rupicapra pyrenaica, Capra hircus, Rupicapra rupicapra
Diseases Border Disease, Infection, Virus Diseases