31-Jul-2025
Monkeypox virus (MPXV), a DNA virus endemic to Central and West Africa, caused an unexpected multi-country outbreak in 2022, dominated by a less virulent but more transmissible lineage. While this lineage generally caused milder disease, it spread widely in non-endemic regions. In this study, supported by ISIDORe, this outbreak strain was compared with two previously studied Clade II isolates: USA 2003 and WRAIR 7-61, using multi-omics and cellular assays to investigate viral adaptation.
To support their multi-omics approach, the team collaborated with the Instruct Facility, the proteomics Unit at the University of Helsinki to conduct the proteomic and phospho-proteomic analyses of MPXV-infected cell lysates, enabling high-resolution mapping of both viral protein expression and host cell phosphorylation responses. These comparisons help clarify whether the Clade IIb strain's enhanced spread is linked to changes in viral growth, host protein modulation, or immune evasion mechanisms.
Using mouse embryonic fibroblasts (MEFs), the researchers conducted plaque assays and measured intracellular and extracellular viral titers over time. They found that 2022 MPXV replicates more slowly than the other two strains, forming significantly smaller plaques and producing lower titers. In contrast, USA 2003 demonstrated the most aggressive phenotype, forming large comet-shaped plaques characteristic of high extracellular virus release. This suggests that despite lower replication, the 2022 virus may have developed alternative mechanisms for spread.
Figure 1. Proteomic analysis displays differential protein expression between 2022 MPXV previous Clade II strains. (A) Volcano Plot representing Log2 Fold Change and Adjusted p Value proteins in infected MEF cells with clade II MPXV strains compared with noninfected MEFs. Upregulated proteins are colored red, downregulated blue, viral proteins are orange, and false values are gray. Most relevant hits are labeled. (B) Pie chart displaying the number of true and false values identified. (C) Venn diagram of differentially expressed proteins across MPXV strain infection in MEF cells. (D) Graph indicating quantity of upregulated/downregulated proteins (E) Heat Map displaying Log2 Fold Change values of viral proteins in infected MEF cells with Clade II MPXV strains compared with noninfected MEFs. Experiments were conducted with four replicates of infected protein extracts.
Comprehensive proteomic analysis using high-resolution mass spectrometry (Figure 1) revealed that the 2022 strain expresses lower viral protein levels compared to USA 2003 and WRAIR 7-61. Interestingly, certain proteins (e.g. O1, K1, and J2) were elevated in the 2022 strain, indicating a shift toward host immune evasion. Biological process pathway enrichment analysis of host proteins also showed increased modulation of the actin cytoskeleton and supermolecular fiber organization, which are critical for viral trafficking.
Phospho-proteomic analysis showed the highest amount of phosphorylayted proteins for the 2022 strain, with numerous altered phosphorylation sites. Most notably, the transcription factor FOXO1—associated with glucose metabolism and cellular stress responses—was hypophosphorylated following infection with 2022 MPX. This indicated that OXPHOS pathway is highly involved in viral replication as it has been previously shown.
It was noted that these molecular adaptations may explain the enhanced transmission of Clade IIb without requiring increased virulence. The authors caution that if similar adaptations arise in the more pathogenic Clade I viruses, the public health impact could be substantial.
This study highlights the importance of continuous molecular surveillance and the need for experimental systems that evaluate both viral fitness and host manipulation. Such insights are crucial for predicting the evolution of MPXV and prevention of MPOX outbreaks in the future.
