Insights into the Evolution and Host Adaptation of the Monkeypox Virus from a Codon Usage Perspective: Focus on the Ongoing 2022 Outbreak

Abstract

The exceptionally widespread outbreak of human monkeypox, an emerging zoonosis caused by the monkeypox virus (MPXV), with more than 69,000 confirmed cases in 100 non-endemic countries since 2022, is a major public health concern. Codon usage patterns reflect genetic variation and adaptation to new hosts and ecological niches. However, detailed analyses of codon usage bias in MPXV based on large-scale genomic data, especially for strains responsible for the 2022 outbreak, are lacking. In this study, we analyzed codon usage in MPXV and its relationship with host adaptation. We confirmed the ongoing outbreak of MPXVs belonging to the West Africa (WA) lineage by principal component analysis based on their codon usage patterns. The 2022 outbreak strains had a relatively low codon usage bias. Codon usage of MPXVs was shaped by mutation and natural selection; however, different from past strains, codon usage in the 2022 outbreak strains was predominantly determined by mutation pressure. Additionally, as revealed by the codon adaptation index (CAI), relative codon deoptimization index (RCDI), and similarity index (SiD) analyses, the codon usage patterns of MPXVs were also affected by their hosts. In particular, the 2022 outbreak strains showed slightly but significantly greater adaptation to many primates, including humans, and were subjected to stronger selection pressure induced by hosts. Our results suggest that MPXVs contributing to the 2022 outbreak have unique evolutionary features, emphasizing the importance of sustained monitoring of their transmission and evolution.

Keywords: codon usage bias; evolution; host adaptation; monkeypox virus; mutation pressure; natural selection.

Figure 1

Maximum-likelihood phylogenetic tree of 161 MPXV strains based on full-length genomes. Viruses’ labels are filled according to their major lineage classification. The West Africa (WA) and Central Africa (CA) lineages are represented in blue and purple, respectively. Each 2022 outbreak strain is marked by an orange star. Numbers above the branches are ultrafast bootstrap support (%) and SH-aLRT support (%). For both metrics, only support values above 70% are displayed. The scale bar represents the expected substitutions per site. The circular color blocks at the periphery of the tree are distributed from inside to outside indicating the host, collection year, and country of the isolates, respectively. The country names are indicated using ISO alpha-3 codes.

Figure 2

Boxplots of the GC, GC1s, GC2s, and GC3s values of the coding sequences of MPXV from different clades. The strains that caused 2022 worldwide monkeypox outbreaks are noted as “WA-Outbreak2022”. The isolates from previous outbreaks in the WA lineage are noted as “WA-Others”. Benjamini–Hochberg (BH)-corrected Dunn’s test was used for comparisons between groups. All differences with p < 0.05 are indicated. * p < 0.01; ** p < 0.01; **** p < 0.0001.

Figure 3

Violin plot of the ENC values of the MPXVs from different lineages. Benjamini–Hochberg (BH)-corrected Dunn’s test was performed to infer the significance of differences between groups. All differences with p < 0.05 are indicated. **** p < 0.0001.

Figure 4

PCA of MPXV based on RSCU values for 59 synonymous codons. The ellipses show the 95% confidence interval. Each dot represents an MPXV strain. WA-Outbreak2022, WA-Others, and CA clades are depicted in orange, blue, and purple, respectively. The shape of the dot represents the host categories: human (circle), non-human primates (NHP, triangle), rodents (rectangular), and Soricidae (crisscross).

Figure 5

ENC-plot and neutrality analysis. ENC-plot of the coding sequences of the MPXV (A) and partially enlarged view (B). Neutrality plot analysis of all MPXV strains (C) and individual lineages (D). The continuous black line represents the expected ENC values. The interpretation of the color and shape are the same as described in Figure 4.

Figure 6

Spearman’s correlation analysis of the nucleotide composition, ENC, and the first two principal components (Dim.1 and Dim.2) of the PCA for MPXV coding sequences. Dark red and blue indicate a positive and negative correlation, respectively. Deeper colors indicate a higher correlation. Only significant correlations are displayed (p < 0.05).

Figure 7

(A) CAI, (B) RCDI, and (C) SiD analyses of codon usage for MPXV and its potential hosts. Benjamini–Hochberg (BH)-corrected Dunn’s test was used for comparisons between groups. All differences with p < 0.05 are indicated. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. The colors of labels on the horizontal axis correspond to the reference genome: humans (red), NHP (blue), and rodents (purple).