Monkeypox virus (MPXV) has garnered global attention in recent years. MPXV is a DNA virus that is categorized as belonging to the Orthopoxvirus, exhibiting close genetic and structural similarities to variola virus (VARV) and vaccinia virus (VACV). Despite the World Health Organization (WHO) announcing in 2023 that the global outbreak of monkeypox (mpox) is no longer a public health emergency, the new occurrence reported in most regions shows the urgency of the development of medicines for mpox. In this review, we reviewed a huge amount of literature on mpox, summarizing its development, scientific findings, and trustworthy data when it comes to viral structure, binding targets, treatments, and intervention strategies. Because no antiviral agents have been formally authorized by the US Food and Drug Administration (FDA) or the WHO for the targeted treatment of mpox, coupled with the limited effectiveness demonstrated by current antiviral medications in clinical evaluations, the progressive emergence of drug resistance, and the ongoing threat posed by novel viral variants, the advancement of new antiviral therapeutics for mpox has emerged as an urgent priority within the domain of global public health. The purpose is to explore measures, therapy techniques, and the medication development process for mpox, including vaccines, antibodies, chemicals, natural drug monomers, traditional Chinese medicines (TCM), and Chinese herbal formulations. Also, it clearly differentiates the progress of approved drugs, preclinical candidate compounds, and computer-predicted molecules in the development of therapeutics for mpox. It is beneficial to comprehend MPXV's research basis, improve the understanding of preventive measures and treatment methods, and promote medical and healthcare services.

Monkeypox virus (MPXV) is a zoonotic virus endemic to Africa that has recently re-emerged, becoming the first orthopoxvirus with sustained human-to-human transmission since smallpox eradication. The 2022 global epidemic of MPXV was caused by Clade IIb virus (lineage B.1) that derived from the lineage A endemic to West Africa through APOBEC3 microevolution[1-3]. Here, comparison of lineage B.1 virus with its closest earlier A.1 ancestor reveals that despite only 46 nucleotide differences, lineage B.1 has attenuating phenotypic changes. Both viruses replicated equivalently in cell culture, but B.1 had defects in long-range spread, which mapped to APOBEC3 mutation E353K in OPG057 (F13L). MPXV B.1 was also defective in innate immune control, inducing higher IFNβ and innate immune signalling gene expression, and showing reduced capacity to suppress interleukin-1β-mediated responses, which mapped to APOBEC3 mutation R48C in OPG047 (F3L). Finally, MPXV B.1 had reduced virulence in a mouse model of MPXV infection relative to the ancestral A.1 strain. Our work demonstrates that the 2022 global outbreak MPXV was intrinsically attenuated, with attenuating mutations that are absent in concurrent, more severe clade IIb outbreaks. Our data also reveal that APOBEC3 editing is a mechanism of MPXV phenotypic variation, potentially contributing to clade evolution and competition.

Since 2022, human monkeypox virus (hMPXV) has emerged in non-endemic regions, including Thailand. However, the genomic dynamics and clinical correlates of local transmission remain incompletely defined. Whole-genome sequencing was performed on hMPXV from 16 patients in Thailand (2023-2024) using targeted amplicon NGS. Phylogenetic analyses integrated global reference sequences. Mutational profiles, specifically non-synonymous substitutions and APOBEC3-associated signatures, were analyzed in relation to clinical data. Phylogenetic reconstruction identified three temporal phases. Early 2022 cases (clade IIb lineages A and B) were interspersed with global sequences, consistent with multiple introductions. In contrast, 2023-2024 cases were dominated by lineage C.1. All 16 genomes belonged to C.1 (one C.1.1), and formed a distinct mid-2023 cluster, designated C.1/Thai/Cluster, supporting sustained local transmission. APOBEC3-associated mutations were pervasive across the C.1 lineage overall, including within C.1/Thai/Cluster, without evidence of significant enrichment specific to this cluster. The cohort comprised exclusively male patients (81% HIV-positive, MSM), with predominantly genital painful lesions and a median recovery time of 23 days. No significant associations were detected between viral genetic variation and clinical outcomes. Mpox transmission in Thailand evolved from multiple introductions to sustained C.1-dominated local spread, underscoring the importance of continued genomic surveillance.

Monkeypox virus (MPXV) infection induces robust humoral immune responses; however, the kinetics of IgG avidity maturation following natural infection remain poorly characterized. Since IgG avidity reflects antibody affinity maturation over time, its evaluation may provide additional information for the interpretation of MPXV serology and infection staging. This study longitudinally assessed MPXV-specific IgG avidity in patients with confirmed mpox infection up to one year after symptom onset.

In 2024, Monkeypox virus (MPXV) clade I has triggered outbreaks in several countries world-wide. MPXV clade I demonstrates enhanced virulence and transmissibility, with a case fatality rate reaching 10%. In response, we have developed a one-pot detection assay specifically targeting MPXV clade I, combining recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system. The assay can be completed within 40 min and achieved a 95% limit of detection (LOD95) of 27.16 copies/μL. No cross-reactivity was observed with MPXV clade II or other tested viral templates, including Vaccinia virus (Tiantan strain). A preliminary room-temperature evaluation showed that the assay retained detectable performance at 25°C, supporting its potential use in equipment-limited settings. The assay also showed good intra-assay and inter-assay repeatability for recombinant plasmid templates, with all coefficient of variation (CV) values below 10%. In simulated clinical samples, the RPA-CRISPR/Cas12a assay detected more low-concentration plasmid-spiked samples than quantitative polymerase chain reaction (qPCR). These results indicate that the established assay is specific, sensitive, repeatable, and easy to perform, providing a practical tool for field-based screening and decentralized detection of MPXV clade I.

The global outbreak and ongoing spread of the monkeypox virus (MPXV) have highlighted the urgent need for effective antiviral therapeutics. Here, we integrated artificial intelligence-based protein structure prediction, large-scale virtual screening, and experimental validation to identify preliminary hit compounds targeting MPXV. Using AlphaFold2, we predicted high-accuracy structures for seven essential MPXV proteins, including three polymerase-related and four surface proteins. Molecular docking of these targets against 6405 drugs from the ZINC15 world-approved subset generated a docking score dataset of 44,835 drug-protein pairs, from which numerous high-scoring compounds were identified. Focusing on A35R, we selected 26 compounds for experimental validation using surface plasmon resonance (SPR). Three compounds, including cepharanthine, eltrombopag, and simeprevir, exhibited measurable A35R‑associated binding signals with equilibrium dissociation constants (KD) in the micromolar range. Molecular dynamics (MD) simulations and molecular mechanics generalized Born surface area were employed for stability analysis and relative energetic assessment. Notably, all three have been previously reported to target other MPXV proteins, reinforcing their potential for repurposing. This work establishes AI‑driven structure prediction as a useful tool for identifying preliminary binding compounds. However, no antiviral activity has been demonstrated for these compounds; therefore, the three hit compounds warrant further optimization and biological evaluation. Our integrated approach provides a framework for rapid drug screening against emerging viral threats.

Highly pathogenic airborne microorganisms pose a significant public health threat, particularly because their small particle size can support prolonged suspension and long-range transport. Here, we present an integrated platform that combines a composite bioaerosol collection system with a three-stage enrichment mechanism and recombinase polymerase amplification-CRISPR/Cas12a (RPA-CRISPR/Cas12a) detection. The platform incorporates model-based constant-temperature control, stacked optical filters, and multichannel fluorescence imaging. The temperature-control model accurately described the thermal response of the reaction module, while the optical and imaging subsystems supported stable fluorescence readout. Using aerosolized monkeypox virus (MPXV) pseudovirus as a controlled test target, the platform detected target nucleic acid in collection liquid down to 10 copies/μL under the tested conditions. These results support the feasibility of integrating aerosol enrichment with isothermal molecular detection in a compact prototype. The main contributions lie in innovations and the engineering integration of bioaerosol collection and enrichment, thermal control of biochemical reactions in molecular detection, and precision fluorescence detection techniques for molecular detection. Regarding molecular detection methods, the biochemical reaction parameters were experimentally optimized rather than introducing new biochemical reaction mechanisms. Validation with authentic clinical or environmental viral aerosols remains necessary before diagnostic or field-use claims can be made.

Poxviruses are double-stranded DNA viruses with large genomes. Among them, monkeypox virus (MPXV) has been responsible for two recent public health emergencies as declared by the World Health Organization[1]. The MPXV polymerase comprises three subunits-a catalytic subunit (F8) and a heterodimeric processivity factor (A22 and E4). The viral polymerase must coordinate activities with the hexameric helicase-primase (E5) to initiate replication of the viral genome[2]. Although structures of MPXV E5 (refs. [3,4]) and the polymerase[5-7] in isolation are available, how they assemble into a functional replisome remains unclear. In isolation, E5 is in an autoinhibited conformation and has very weak helicase activity[3,4], and the mechanism for helicase activation is unclear. Here we used cryo-electron microscopy to determine the structures of DNA-bound MPXV replisomes comprising the polymerase holoenzyme (F8, A22 and E4) and the E5 helicase hexamer. We show that, during replisome assembly, E5 undergoes large-scale conformational changes that allow two of its primase domains to interact with the polymerase F8 thumb and A22 subunit. Biochemical assays and single-molecule experiments reveal that this E5 conformational change is coupled to helicase activation and enhances primase activity. Taken together, these findings identify fundamental mechanisms governing coordinated helicase and polymerase activities during DNA replication for an important class of viral pathogens.

Mpox virus (MPXV) poses an increasing global health threat, as underscored by two World Health Organization declarations of Public Health Emergencies of International Concern, particularly after the emergence of a novel Clade Ib strain that exhibited high human-to-human transmissibility in the Democratic Republic of the Congo. However, the treatment options for MPXV infection remain extremely limited. To address this unmet need, we established an integrated platform combining single-cell transcriptomics and deep learning-based structural prediction to discover effective human monoclonal antibodies against MPXV. By integrating computational prediction with experimental validation, we identified five neutralizing antibodies targeting the following distinct viral forms: BA345, MA42, and MA49, which engage the extracellular enveloped virus-associated A35R glycoprotein; BAL31, which binds intracellular mature virus (IMV) protein A29L; and HB05, which targets IMV antigen H3L. Importantly, the elite monoclonal antibody BA345 conferred effective protection against MPXV and vaccinia virus both in vitro and in vivo. Combined in silico structure prediction and X-ray crystallography revealed a highly conserved epitope shared across orthopoxviruses. Surface plasmon resonance measurements revealed nanomolar equilibrium dissociation constants of BA345 for A35R homologs, corroborating its cross-reactive, broad-spectrum neutralizing activity against orthopoxviruses. Moreover, the BA345/BAL31 and MA49/BAL31 antibody cocktails developed in this study conferred robust therapeutic protection in MPXV-infected animals, substantially reducing disease severity and viral load. Our findings not only establish a practical paradigm for antibody discovery through the integration of deep learning-driven structure prediction with single-cell multiomics but also inform next-generation biodefense countermeasures against MPXV and related orthopoxviruses.

Since the first confirmed case in 1970, the monkeypox virus (MPXV) has emerged as a significant threat to global public health. The World Health Organization (WHO) has declared it a Public Health Emergency of International Concern (PHEIC) on two occasions. Despite decades of research, only tecovirimat has been approved by the European Medicines Agency (EMA) for the treatment of MPXV infection. The genome and structure are similar between MPXV and other orthopoxviruses (OPXVs), suggesting that the strategies used for other OPXVs may be applicable to MPXV. This review systematically summarizes the genome, structure, and critical stages in the life cycle of OPXVs, especially MPXV. A variety of antiviral agents against MPXV and other OPXVs are discussed according to their distinct mechanisms of action: 1) blocking viral entry and fusion, 2) inhibiting DNA replication and processing, 3) disrupting transcription and mRNA processing, 4) preventing virion assembly, maturation and release, 5) modulating immune responses, and 6) mechanism unknown. Overall, this article provides a systematic review of current research progress on potential therapeutic targets and agent for MPXV, aiming to offer innovative insights and strategies for the development of effective therapeutic agents against mpox.