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.