Many human RNA viruses show extraordinarily stringent selective constraints on protein evolution.

How negative selection, positive selection, and population size contribute to the large variation in nucleotide substitution rates among RNA viruses remains unclear. Here, we studied the ratios of nonsynonymous-to-synonymous substitution rates (d N/d S) in protein-coding genes of human RNA and DNA viruses and mammals. Among the 21 RNA viruses studied, 18 showed a genome-average d N/d S from 0.01 to 0.10, indicating that over 90% of nonsynonymous mutations are eliminated by negative selection. Only HIV-1 showed a d N/d S (0.31) higher than that (0.22) in mammalian genes. By comparing the d N/d S values among genes in the same genome and among species or strains, we found that both positive selection and population size play significant roles in the d N/d S variation among genes and species. Indeed, even in flaviviruses and picornaviruses, which showed the lowest ratios among the 21 species studied, positive selection appears to have contributed significantly to d N/d S We found the view that positive selection occurs much more frequently in influenza A subtype H3N2 than subtype H1N1 holds only for the hemagglutinin and neuraminidase genes, but not for other genes. Moreover, we found no support for the view that vector-borne RNA viruses have lower d N/d S ratios than non-vector-borne viruses. In addition, we found a correlation between d N and d S, implying a correlation between d N and the mutation rate. Interestingly, only 2 of the 8 DNA viruses studied showed a d N/d S < 0.10, while 4 showed a d N/d S > 0.22. These observations increase our understanding of the mechanisms of RNA virus evolution.