Calibration of multiple poliovirus molecular clocks covering an extended evolutionary range.

We have calibrated five different molecular clocks for circulating poliovirus based upon the rates of fixation of total substitutions (K(t)), synonymous substitutions (K(s)), synonymous transitions (A(s)), synonymous transversions (B(s)), and nonsynonymous substitutions (K(a)) into the P1/capsid region (2,643 nucleotides). Rates were determined over a 10-year period by analysis of sequences of 31 wild poliovirus type 1 isolates representing a well-defined phylogeny derived from a common imported ancestor. Similar rates were obtained by linear regression, the maximum likelihood/single-rate dated-tip method, and Bayesian inference. The very rapid K(t) [(1.03 +/- 0.10) x 10(-2) substitutions/site/year] and K(s) [(1.00 +/- 0.08) x 10(-2)] clocks were driven primarily by the A(s) clock [(0.96 +/- 0.09) x 10(-2)], the B(s) clock was approximately 10-fold slower [(0.10 +/- 0.03) x 10(-2)], and the more stochastic K(a) clock was approximately 30-fold slower [(0.03 +/- 0.01) x 10(-2)]. Nonsynonymous substitutions at all P1/capsid sites, including the neutralizing antigenic sites, appeared to be constrained by purifying selection. Simulation of the evolution of third-codon positions suggested that saturation of synonymous transitions would be evident at 10 years and complete at approximately 65 years of independent transmission. Saturation of synonymous transversions was predicted to be minimal at 20 years and incomplete at 100 years. The rapid evolution of the K(t), K(s), and A(s) clocks can be used to estimate the dates of divergence of closely related viruses, whereas the slower B(s) and K(a) clocks may be used to explore deeper evolutionary relationships within and across poliovirus genotypes.