The codon usage analysis is the most essential feature that plays a major role in biological evolution. In this research, a series of analyses on ORF of 114 EV71strains was conducted. Firstly, nucleotide A was highly frequently presented in the genome ORF of EV71, on the contrary, which preferred T at the third position of codon (Table S1). Secondly, the relatively low ENc values (ENc = 56.696 > 40 in average) of all EV71 strains indicated low bias. In comparison with ENc value of other known RNA virus, such as polioviruses (HPV), hepadnaviruses, human immunodeficiency virus (HIV), Zika virus, West Nile virus (ENC = 53.754, 44.45, 51.9, 52.72, 53.81 respectively) [19–22], a widely recognized explanation was that low bias was optimal for intracellular replication and immune evasion. Finally, it was discovered in this research that CAI values of all EV71 strains were relatively high, which was optimal for adaptation to the host and intracellular translation.
Analyses on the PCA and evolution tree reveals that the EV71 viruses that were popular in China before 2008 mainly belonged to C4 serotype, so it was more stable at that time. However, after 2008, a huge fluctuation on the codon usage pattern of virus was found, which is consistent with large scale outbreaks in Anhui, Guangxi, Jiangxi and other province includes 2008 years and later. This suggests that due to the population surge during the grant outbreak, EV71 faced selective and mutational pressure that led to diversity of its codon usage pattern. The diversity of codon usage pattern has changed some of the genetic characteristics of the EV71 virus, which will bring new pressure to the prevention and treatment of the virus in the future. And all of the above are similar to our previous researches . On the other hand, it is discovered in principal factor analysis that the two isolates extracted from Taiwan in 1998 were greatly different from those in mainland China during the same period, which belonged to C2 serotype as verified by evolution tree analysis. It is proved for the first time through codon usage analysis that there outbreak of C2 isolates in China before 2000.
In this study, our analysis reveals that codon usage bias in EV71 is low, and mutation pressure is the main factor that affects codon usage variation in evolution of the virus. Other factors, including base composition, genotype, geography and host may also significantly influence codon usage bias. Our study aslo provides a basic understanding of the codon usage patterns of EV71 and the roles played by mutation pressure and natural selection. In addition, we studied the variation trend of codon usage pattern of EV71 in China, especially the great differences in codon usage pattern of the China isolates before and after 2008. But a more comprehensive analysis is needed to reveal more information about codon usage bias variation within EV71 viruses.