The values of A, U, C, G and C+G were compared with the values of A 3 , C 3 , G 3 , U 3 , (G+C) 3 , respectively. An interesting and complex correlation was observed. In detail, the (C+G) 3 have highly significant correlations with A, U, C, G and C+G, respectively, indicating C+G may reflect interaction between mutation pressure and natural selection. However, the A have no correlation with A 3 , G 3 and C 3 , and U have no correlation with A 3 (Table 4 ). Both cases suggested that the nucleotide constraint possibly influence synonymous codon usage of polioviruses. In addition, the correlation between the Axis 1 (calculated by PCA) and the values of A, C, G, U, A 3 , C 3 , G 3 , U 3 , (G+C), (G+C) 3 of each strain was also analyzed. The significant correlation was found between nucleotide compositions and synonymous codon usage to some extent excluding Axis 1 and the value of A ( Table 4 ). The analysis revealed that most of the codon usage bias among ORFs of polioviruses strains was directly related to the base composition. Finally, the ENC-plot [ENC plotted against (G+C) 3 %] was used as a part of general strategy to investigate patterns of synonymous codon usage and all of the spots lie below the expected curve (Figure 1 ). These imply that the codon bias can be explained mainly by an uneven base composition, in other words, by mutation pressure rather than natural selection. Effect of other potential factors on codon usage