Genomic Characterization and Molecular Evolutionary Analysis of Monkeypox Virus in Qinghai, China, 2025

Abstract Monkeypox virus (MPXV) lineage E.4 has emerged as the predominant strain in Asia, however, genomic data from northwestern China remains scarce. In this study, we performed comprehensive genomic, phylodynamic, and structural analyses of three newly identified MPXV isolates obtained from confirmed cases in Qinghai Province, China, between June and September 2025. Whole-genome sequencing, optimally performed on vesicular fluid owing to peak viral loads—confirmed that all isolates belong to Clade IIb, lineage E.4, and form a tight monophyletic cluster with contemporary strains from Japan. Bayesian phylodynamic reconstruction yielded a mean evolutionary rate of \(\:4.186\times\:{10}^{-5}\) substitutions per site per year and revealed a rapid population expansion between June and September 2025, coinciding with the regional rise of the E.4 lineage. Notably, mutational profiling uncovered a distinctive “core conserved, terminal differentiated” pattern. Although all isolates shared 11 signature mutations, substantial sample-specific heterogeneity—including eight unique mutations restricted to a single isolate—strongly suggest multiple independent introductions into Qinghai through a cryptic transmission network, rather than a single localized outbreak. In silico predictions further identified OPG102:T150I and OPG092:E155K as highly deleterious mutations that severely destabilize protein structure (ΔΔG = − 0.64 and − 0.13 kcal/mol, respectively). We hypothesize that these structural perturbations represent adaptive fitness trade-offs that may prioritize conformational flexibility or immune evasion. Collectively, these findings underscore the critical need for high-resolution genomic surveillance, integrated with structural bioinformatics, to decipher the ongoing microevolution of MPXV.