Opposing fates: a bipolar cellular model for FMDV replication shaped by 12C6+heavy-ion mutagenesis

Figures

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  Figure 1. Schematic representation of the cell screening process.

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  Figure 2. Phenotypic characterization and stability of FMDV replication in monoclonal BHK-21 cell lines following 12C6+ irradiation screening. (a) Initial screening of 63 monoclonal cell lines based on the ratio of FMDV 146S antigen content in each clone relative to parental BHK-21 cells post-infection. Clones are categorized by irradiation dose: 5 Gy (beige), 10 Gy (orange), and 15 Gy (dark red). Parental BHK-21 controls are shown as blue-outlined bars. (b) Stability of the viral replication phenotype across successive passages in 52 viable clones, presented as the 146S antigen ratio (χ) relative to control cells. Clonal stability was determined by monitoring the fluctuation of χ across three key passages (T3, T6, T9). A stable phenotype was defined by fluctuations within a ±0.2 range, with χ values progressively stabilizing close to 1. BHK-5 and BHK-7 (highlighted with green circles) were selected as stable anti-viral and pro-viral clones, respectively. (c) Relative FMDV RNA expression levels in the selected anti-viral (BHK-5) and pro-viral (BHK-7) clones compared to parental BHK-21 cells. (d) Viral titers (expressed as log10 TCID50/0.1 mL) produced by BHK-5, BHK-7, and parental BHK-21…

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  Figure 3. Transcriptomic divergence among BHK-5, BHK-7, and parental BHK-21 cells before and after FMDV infection. (a) Three-dimensional principal component analysis (PCA) of global gene expression profiles in BHK-5, BHK-7, and parental BHK-21 cells under uninfected and FMDV-infected (MOI = 0.1) conditions. (b) Hierarchical clustering heatmap of DEGs across all cell lines before and after infection with FMDV (MOI = 0.1).

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  Figure 4. Ultrastructure of BHK lines at 6 h postinfection with FMDV. Representative transmission electron microscopy (TEM) images of 12C6+-mutagenized BHK-7 and BHK-5 cells, and parental BHK-21 cells at 6 hours post-infection (hpi) with FMDV. (a) BHK-7 cells (pro-viral phenotype) exhibit extensive viral replication complexes and abundant mature virions, while maintaining overall cellular integrity. This morphology correlates with molecular mechanisms supporting viral immune evasion and metabolic exploitation by the virus. (b) BHK-5 cells (anti-viral phenotype) display severe cytopathic effects, including membrane blebbing indicative of apoptosis. This rapid cytopathic response aligns with enhanced interferon signaling and early cell death. (c) Parental BHK-21 cells show an intermediate response, marked by cytoplasmic vacuolation and entrapment of virions within double-membraned autophagosome-like structures—suggesting activation of autophagy-related defense mechanisms.

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  Figure 5. GO enrichment analysis of DEGs across BHK cell lines under uninfected and infected conditions. DEGs were functionally enriched across three GO categories: Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). The x-axis indicates significantly enriched GO terms (Top 30); the y-axis represents the enrichment significance as -log10(Pvalue). Bar color corresponds to the GO category (BP, MF, or CC). Each panel shows a specific comparison between parental BHK-21 and derivative cell lines (BHK-5 or BHK-7) under uninfected or FMDV-infected (MOI = 0.1, 6 hpi) conditions: (a) BHK-21 vs. BHK-7, downregulated (uninfected); (b) BHK-21 vs. BHK-7, upregulated (uninfected); (c) BHK-21 vs. BHK-5, downregulated (uninfected); (d) BHK-21 vs. BHK-5, upregulated (uninfected); (e) BHK-21 vs. BHK-7, downregulated (infected); (f) BHK-21 vs. BHK-7, upregulated (infected); (g) BHK-21 vs. BHK-5, downregulated (infected); (h) BHK-21 vs. BHK-5, upregulated (infected).

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  Figure 6. KEGG enrichment of DEGs across BHK cell lines under uninfected and FMDV-infected conditions. KEGG pathway enrichment was performed on DEGs identified via pairwise comparisons. The y-axis indicates significantly enriched pathways (Top 20); the x-axis represents the statistical significance as -log10(pvalue). Point size corresponds to the number of DEGs per pathway ("Gene number"), and color reflects the “Rich factor”. Each panel represents a specific comparison under uninfected or infected (MOI = 0.1, 6 hpi) conditions: (a) BHK-21 vs. BHK-7, downregulated (uninfected); (b) BHK-21 vs. BHK-7, upregulated (uninfected); (c) BHK-21 vs. BHK-5, downregulated (uninfected); (d) BHK-21 vs. BHK-5, upregulated (uninfected); (e) BHK-21 vs. BHK-7, downregulated (infected); (f) BHK-21 vs. BHK-7, upregulated (infected); (g) BHK-21 vs. BHK-5, downregulated (infected); (h) BHK-21 vs. BHK-5, upregulated (infected).

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  Figure 7. Statistics of differentially express transcription factor (TF) families. Bar charts show the number of differentially expressed TFs across various families in pairwise comparisons between parental BHK-21, anti-viral BHK-5, and pro-viral BHK-7 cells (with and without FMDV infection). (a) Key immune and developmental TFs; (b) TFs related to cell cycle and DNA damage; (c) The abundant zf-C2H2 family; (d) Other zinc-finger families; (e) TFs involved in development, metabolism, and signaling.

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  Figure 8. Protein–protein interaction (PPI) network of DEGs. The network illustrates functional modules enriched among DEGs. Nodes represent proteins (genes), and edges represent known interactions. Major functional clusters are highlighted and annotated: interferon signaling (orange), DNA damage response/p53 signaling (blue), metabolism (yellow), and myogenesis (green).

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  Figure 9. Integrated analysis of functional modules and gene expression. The core PPI network is shown in the center. Surrounding heatmaps correspond to key functional modules, displaying gene expression changes across four comparisons: BHK-7 vs. BHK-21 (uninfected), BHK-7 vs. BHK-21 (infected), BHK-5 vs. BHK-21 (uninfected), and BHK-5 vs. BHK-21 (infected). Red indicates upregulation; blue indicates downregulation. (a) Metabolism of xenobiotics by cytochrome P450; (b) Myogenic factors; (c) DNA damage response and p53 signaling; (d) Type I interferon production and defense response to virus.

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  Figure 10. Induction of polarized regulatory networks by heavy-ion mutagenesis in BHK cells upon FMDV infection. The diagram illustrates divergent molecular mechanisms underlying the anti-viral clone BHK-5 (left, green pathway) and pro-viral clone BHK-7 (right, gray pathway). Top: The initial irradiation event (5 Gy vs. 15 Gy) leads to distinct outcomes: DNA damage response (DDR)-induced immune pre-activation vs. selection for immune tolerance. This establishes a pre-configured TF network layer: BHK-5 is enriched in immune-activating IRFs, while BHK-7 is enriched in immunosuppressive Runt and metabolic MYC_N families. Bottom: The functional module layer shows BHK-5 activating the IFN–IRF axis (Map3k20, Irf3, Irf7) and ISGs (Mx1, Rsad2) while suppressing metabolism (P450, Acsf). BHK-7 suppresses the IFN–IRF axis (Irf3, RTP4), activates stress/metabolic pathways, and remodels cells to promote efficient FMDV replication.

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  Figure 11. Validation of DEGs by RT-qPCR. The relative expression fold change of five selected genes in BHK-7 and BHK-5 cells (before and after FMDV infection) was measured by RT-qPCR. Results are consistent with RNA-seq data, confirming the reliability of transcriptomic findings.