2.3 Phylogenetic and evolutionary analyses
Eleven full-length genome sequences derived from this study were
analyzed and compared with the dataset of 188 full-length genome
sequences of CPV-2, originally isolated from domestic dogs during 1979
to 2017, available in the GenBank database (Supplementary Table S4). The
substitution rate of the full-length genome sequences and each
individual structural and nonstructural gene coding region was estimated
as the number of nucleotide substitutions per site per year (nt/s/y),
implemented in the Bayesian Evolutionary Analysis Sampling Trees (BEAST)
version 1.8.4 software (http://tree.bio.ed.ac.uk/) . The
rates were calculated based on the two main phylogenetic clades of the
CPV-2 strains discovered in Asia (Asian clade) and the CPV-2 strains
reported in Europe and America (Western clade). The best-fit model of
substitution (HKI + I + G4) was determined using the model section
function in the MEGA 7.0 software
(https://www.megasoftware.net/).
The coalescent Bayesian skyline was applied under exponential
relaxed-clock models (Drummond & Rambaut, 2007; Li et al., 2017). The
Markov Chain Monte Carlo (MCMC) algorithm was run for 200-million
generations and logged every 10,000 states. The convergence of all
parameters was checked by observing that the effective sample size (ESS)
> 200 with TRACER version 1.6.1. The maximum clade
credibility (MCC) tree was obtained from the posterior distribution of
trees using TreeAnnotator version 1.8 after discarding 10% of steps as
burn-in. The phylogenetic tree with timeline estimated divergences,
posterior probability (PP) and 95% highest probability density (HPD)
values were generated using FigTree version 1.4.2.