Molecular Evolution of Protein Sequences and Codon Usage in Monkeypox Viruses

Abstract

The monkeypox virus (mpox virus, MPXV) epidemic in 2022 has posed a significant public health risk. Yet, the evolutionary principles of MPXV remain largely unknown. Here, we examined the evolutionary patterns of protein sequences and codon usage in MPXV. We first demonstrated the signal of positive selection in OPG027, specifically in the Clade I lineage of MPXV. Subsequently, we discovered accelerated protein sequence evolution over time in the variants responsible for the 2022 outbreak. Furthermore, we showed strong epistasis between amino acid substitutions located in different genes. The codon adaptation index (CAI) analysis revealed that MPXV genes tended to use more non-preferred codons compared to human genes, and the CAI decreased over time and diverged between clades, with Clade I > IIa and IIb-A > IIb-B. While the decrease in fatality rate among the three groups aligned with the CAI pattern, it remains unclear whether this correlation was coincidental or if the deoptimization of codon usage in MPXV led to a reduction in fatality rates. This study sheds new light on the mechanisms that govern the evolution of MPXV in human populations.

Keywords: OPG027; Accelerated evolution; Codon usage bias; Mpox virus; Positive selection.

Figure 1

Positive selection on OPG027 during the evolution of MPXV A. Phylogenetic tree of MPXV strains from different clades. The phylogenetic tree was constructed using the maximum likelihood method based on whole-genome alignments of MPXV. B. The amino acid substitutions (in red) and synonymous SNPs (in blue) of OPG027 in the evolutionary process of MPXV. The divergent time of Clades IIb-A and IIb-B was retrieved from Nextstrain (https://nextstrain.org/), and the other divergent time was obtained from data reported by Babkin and his colleagues [59]. C. The distribution of the median dN/dS ratio (ω) between two sequences of each gene from different MPXV clades. The red arrow indicates the ω value of OPG027. It is noteworthy that only one nonsynonymous mutation was observed in the comparisons between IIa and IIb-A strains, with a dS value of 0 and a dN value of 0.0035 for OPG027. YA, years ago; dN, nonsynonymous substitutions per nonsynonymous site; dS, synonymous substitutions per synonymous site; SNP, single nucleotide polymorphism; MPXV, monkeypox virus; VARV, variola virus; VACV, vaccinia virus.

Figure 2

Accumulation of synonymous and nonsynonymous substitutions over time in Clade IIb-B genomes during the 2022 outbreak A total of 756 MPXV genomes with precise collection dates were analyzed. The Y-axis represents the number of synonymous (A) or nonsynonymous (B) substitutions in a strain collected on a specific day relative to the reference genome (NCBI: NC_063383). Spearman’s correlation coefficient (rho) between the number of substitutions in a strain and time is presented. Additionally, linear regression analyses were performed for the number of substitutions in a strain over time (with May 7th, 2022 assumed as day 1).

Figure 3

LD for the 15 linked SNP groups (r² ≥ 0.8) found in Clade IIb-B of MPXV The Y-axis illustrates the intergenic SNPs or amino acid changes resulting from SNPs within a given gene. Both the A82405C and A82406T mutations resulted in the Q427P change (CAA>CCT) in OPG105. Colored blocks represent SNPs, while gray areas indicate no mutation at that position within a lineage. The number of genome sequences containing the SNPs demonstrating linkage is provided for each lineage, divided by the total number of genome sequences within that lineage (these numbers are presented in parentheses). In total, data from 1726 genomes in Clade IIb-B are presented. LD, linkage disequilibrium.

Figure 4

Comparisons of CAI values and fatality rates among different MPXV groups A. A comparative distribution of CAI values for human genes and concatenated coding sequences from the reference MPXV genome (NCBI: NC_063383). B. Significant differences in CAI values among three distinct MPXV variant groups, classified based on their collection dates (1968–2008, 2017–2021, and 2022). C. Significant differences in CAI values for MPXV variant strains belonging to different clades and lineages. D. Fatality rates of different MPXV clades. The fatality rates for MPXV Clade I and Clades IIa and IIb-A were obtained from a previous study [8]. The fatality rate of Clade IIb-B (0.17%) was estimated in this study. The dots on the graph represent the estimated fatality rates, while the bars indicate the 95% CIs. ***, P < 0.001 (Wilcoxon rank sum test); CAI, codon adaptation index; CI, confidence interval.