2.5 Selective pressure analysis
To determine whether the high rate of nucleotide substitutions might result in the rapid adaptation of the CPV-2, selective pressure tests were assessed on the individual NS and VP genes. Non-neutral selection of nucleotide substitutions was calculated using the ratio between nonsynonymous (dN) and synonymous (dS) substitutions, as assessed by phylogenetic reconstruction using the maximum likelihood (ML) model with general reversible nucleotide substitution, available on the Datamonkey web server (http://www.datamonkey.org). Non-neutral selection was implemented using different models for pervasive individual site measurement, including the single-likelihood ancestor counting (SLAC) and fixed-effects likelihood (FEL) on the HyPhy software package. A p -value of 0.1 was set as significant in all the described methods. The Bayes factor was set at 50 to estimate the rate of dN and dS within each individual codon. Positive selection (adaptive molecular evolution), neutral mutations, and negative selection (purifying selection) were defined as dN/dS > 1, dN/dS = 1, and dN/dS < 1, respectively.