Evolutionary and Modification Features of Two Monkeypox Virus Strains: Insights from Integrated Genomic and Epigenomic Analyses

Since 2022, global outbreaks of monkeypox virus (MPXV) have been repeatedly designated by the World Health Organization (WHO) as a public health emergency of international concern (PHEIC), underscoring the urgent need to elucidate the multidimensional mechanisms underlying viral evolution and transmission. Current understanding remains largely focused on genomic variation, while the critical role of epigenetic regulation has been considerably overlooked. To address this gap, this study integrates high-throughput evolutionary genomic analysis with whole-genome DNA methylation profiling. Using parallel Illumina and Nanopore sequencing platforms, we comprehensively characterized two clinically derived MPXV isolates collected locally. The results revealed that both isolates belonged to the C.1.1 ancestral lineage, diverging into distinct clades (E.3 and E.4, respectively, supporting the presence of at least two independent viral introduction events into the region, each followed by limited local transmission. They had accrued a considerable number of single-nucleotide polymorphisms (SNPs), with APOBEC3-associated substitutions constituting 84.8% and 77.6% of all observed mutations. Furthermore, both 5-hydroxymethylcytosine (5hmC) and N6-methyladenine (6mA) modifications were identified and found to be preferentially enriched within the inverted terminal repeats (ITRs) regions of MPXV genome in both viral strains; moreover, the E.4 lineage viral strain exhibits a markedly more intricate and compositionally diversified modification landscape, a pattern that indicates appreciable epigenetic heterogeneity among MPXV lineages. Our study furnishes a multi-omics framework that presents a systematic evolutionary feature of two clinical MPXV isolates and their genomic DNA 5hmC and 6mA modification topologies, and enhances our understanding of MPXV viral adaptation and diversification.