Computational protocol: Southernmost Asia Is the Source of Japanese Encephalitis Virus (Genotype 1) Diversity from which the Viruses Disperse and Evolve throughout Asia

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Protocol publication

[…] The most recent study showed that in evolutionary terms, the G1 JEV genotype is the youngest of five genotypes and is the dominant genotype circulating in Asia . For studying the dispersing patterns of G1 JEV, totally 656 E sequences of JEV, with information regarding the isolation time and place, were downloaded from GenBank (as of May 1, 2012). ClustalX version 2.0.9 was used to generate sequence alignments of 678 E gene sequences (including 22 newly contributed sequences). The dataset was screened for recombination using RDP3 (Recombination Detection Program3) and GARD (genetic algorithm for recombination detection) , . No recombination events were identified (data not shown). Subsequently, in order to differentiate G1 from the other four genotypes, the Neighbor-joining method in Mega Version 5.05 was applied for phylogenetic analysis. Finally, 359 E sequences of the G1 JEV genotype were obtained and comprised a sequence database for phylogenetic analysis (). [...] The sequence database constructed above was analyzed using Bayesian Markov chain Monte Carlo (MCMC) method. The GTR+I+G was selected as the optimal nucleotide substitution model by MrModelTest . The nucleotide substitution rate and divergence time of the most recent common ancestor (TMRCA) were estimated using the relaxed (uncorrelated lognormal) molecular clock model under a coalescent model of constant population size in the BEAST software package . Demographic histories were inferred by Bayesian skyline reconstruction. The analysis was run through 600,000,000 generations to ensure sufficient mixing. Convergence of parameters was checked using TRACER and was indicated as effective sample size (ESS>200), and the maximum clade credibility (MCC) tree was built using TreeAnnotator with 10% burn-in (http://beast.bio.ed.ac.uk/). Statistical uncertainty was expressed for nodal support by 95% confidence intervals of the highest posterior density (HPD).In order to infer the history of geographical dispersion of JEV, the Bayesian stochastic search variable selection (BSSVS) was used to provide evidence for statistically supported diffusion between state variables under BEAST v1.7.5 . This method estimates the most probable state at each node in the MCC trees, allowing us to reconstruct ancestral positions for ancestral viral lineages along the tree. For phylogeographic reconstructions, each region was coded as a discrete trait. BSSVS output and surfaces representing uncertainty for continuous diffusion processes were formatted as KML using the SPREAD utility . Determination of each locality was coordinated and performed using Google Earth v.6.2.2. Mapinfo was finally used to display the dispersal pattern of JEV based on the phylogeographic analysis. […]

Pipeline specifications

Software tools Clustal W, RDP4, MEGA, MrModelTest, BEAST
Application Phylogenetics
Organisms Japanese encephalitis virus
Diseases Encephalitis