Comparative Analysis of 2022 Outbreak MPXV and Previous Clade II MPXV

Abstract

The 2022-2024 outbreak of MPOX is an important worldwide public health issue that has triggered significant concerns in the scientific community. MPOX is caused by monkeypox virus (MPXV) belonging to the Poxviridae family. The study of MPXV presents a multifaceted challenge due to the diverse viral formThis study was supported by ISIDORe consortium and Agencia Estatal de Investigación.s produced by this pathogen. Notably the intracellular mature viruses (MVs) primarily contribute to localized lesions and host-to-host transmission, while the extracellular enveloped viruses (EVs) are associated with systemic infection. Clinically, MPOX manifests as a vesiculopustular rash that initially emerges on the face and trunk, subsequently spreading throughout the body, with heightened severity observed in immunocompromised individuals. Results obtained in this manuscript indicate that the 2022 outbreak MPXV has a significantly slower viral cycle compared with previous Clade II strains, with WRAIR 7-61 being more intermediate and USA 2003 producing highest viral titers. Additionally, proteomic and phospho-proteomic analysis displays differences in protein expression between these three strains. These findings highlight key differences between the current Lineage B.1 MPXV and previous strains. Further studies will be undertaken to demonstrate if these differences are important for the apparent increased human-to-human transmission mechanisms observed in the Clade IIb MPXV outbreak.

Keywords: Clade II; EV; MPOX disease; MPXV; MV.

Figure 1

Low multiplicity infection demonstrates differences in viral growth and protein expression of 2022 MPXV compared with USA 2003 and WRAIR 7‐61. (A) MEFs were infected with the three MPXV strains (MOI 0.1), at 24 and 48 hpi, infectious viral particles from supernatants (extracellular virus) and cell extracts (intracellular virus) were titrated by plaque assay. Mean ± SD values from four independent experiments are represented. (B) Viral plaque phenotype of different clade II strains (2022 MPXV, USA 2003, and WRAIR 7‐61). Plaques were obtained through titration with BSC‐40 cells of intracellular virus and supernatant at dilutions indicated. (C) MEFs were infected with the three MPXV strain (MOI 0.1) and at the indicated times postinfection, equal amounts of proteins from cell extracts were analyzed by Western Blot. Specific antibodies for VACV early protein E3, intermediate protein F13, and late proteins D8 and A4 were used. β‐Actin was used as a loading control. Molecular weights (MW) in kilodaltons (kDa) are indicated, based on protein standards. (D) MEFs and A549 human cell lines were infected with 0.1 MOI of 2022 MPXV and VACV WR. Total virus produced at 24 hpi was quantified through plaque assay. Means ± the SD from three independent experiments are represented. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.0001.

Figure 2

Microscopy analysis of low multiplicity MPXV infected MEF cells at 24 hpi. (A) Fluorescence microscopy analysis of MEFs infected with the three MPXV strains (MOI 0.1, 24 hpi). Infected cells were visualized using Poli‐VACV Rabbit Antibody (in green), DAPI (4′,6‐diamidino‐2‐phenylindole) (in blue) was used to stain the nuclear DNA. The experiment has been carried out in triplicate and a representative image is shown. Objective used was ×20. (B) Brightfield microscopy was used to visualize the cytopathic effect caused by each virus. The objective used was ×10. The experiment has been carried out in triplicate and a representative image is shown.

Figure 3

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.

Figure 4

Pathway analysis indicates elevated enrichment of biological processes in 2022 MPXV infected cells when compared with USA 2003 and WRAIR. (A) Graph comparing fold enrichment of common biological process pathways produced in each MPXV strain infection. Fold enrichment indicates the number of proteins found to be associated with the indicated pathways. False discovery rate (FDR) has been colored from blue to red, with blue indicating less trustworthy and red indicating more certainty in values calculated. N displays the number of proteins found to be associated with the indicated pathway. (B) Network graphs displaying the most enriched biological process pathways in each strain infection in comparison to mock cells.

Figure 5

Phospho‐proteomic analysis indicated increased phosphorylation in 2022 MPXV infected cells. (A) Number of phosphoproteins and phosphosites identified in each strain infection. (B) Volcano plots of significantly altered phosphorylated proteins from Clade II MPXV infection in comparison to mock cells. (C) Western blot analysis of MPXV‐infected MEF lysates using an anti‐Tyrosine antibody. β‐Actin was used as a loading control. (D) Western blot analysis of MPXV infected MEF lysates using an anti‐Serine antibody. β‐Actin was used as a loading control.

Figure 6

Schematic representation of differences found between Clade II MPXV strains (WRAIR 7‐61, USA 2003, and 2022 MPXV) in MEF cell infection. Created with Biorender.