Objective To analyze the whole genome sequences and the population evolution characteristics of monkeypox viruses from four cases in Guizhou province from 2023 to 2024.

Methods  Samples were collected from four monkeypox cases in Guizhou province, and the whole genome sequences of the viruses were obtained by amplicon sequencing. High-quality genome sequences of monkeypox viruses in Asia from 2022 to 2024 were screened and downloaded from the GISAID database to construct a population dataset containing 173 sequences. This dataset was used for divergence time estimation, phylogenetic analysis, and mutation site identification.

Results  In the population dataset, the number of sequences was the largest in 2023 (126), followed by that in 2022 (33) and 2024 (14). It was estimated that the time to the most recent common ancestor (TMRCA) of monkeypox viruses emerged in 2013, with an evolutionary rate of 3.77×10⁻⁵ subs/site/year. From 2022 to 2024, the diversity of monkeypox virus lineages declined, with the dominant lineage changing from IIb.A.2 to IIb.C.1. China had the most diverse monkeypox virus lineages, including IIb.C.1, IIb.B.1.3, IIb.B.1.20, IIb.B.1, IIb.B.1.5, and IIb.A.1. The phylogenetic analysis revealed that the prevalent strains in 2022 were mainly related to lineage A of clade IIb. In 2023, IIb.C.1 became the dominant lineage, and it further evolved to be dominated by sublineage IIb.C.1.1 with the emergence of IIb.B.1. Due to the limited data volume of the 2024 samples, no significant new clusters were formed. The viruses from the four cases in Guizhou province all belonged to the clade IIb.C.1. Nine to 20 mutation sites were identified in each sample. Among them, N3R_R84K was the common mutation site of the four cases, and amino acid mutations and back mutations were observed at this site.

Conclusions Monkeypox viruses present variations and diversity in China, and its evolutionary dynamics show obvious time dependence. The monkeypox viruses from all the four cases in Guizhou province from 2023 to 2024 belonged to the lineage IIb.C.1 and all carried the key mutation site N3R_R84K. Continuous monitoring of the viral genome should be carried out, with close attention paid to the sources, transmission chains, and variations of the virus.