The impact of 12C6 heavy ion irradiation on the replication of foot-and-mouth disease virus

Wild-type BHK-21 cells were exposed to 12C6 heavy ions at doses of 5 Gy, 10 Gy, and 15 Gy. The impact of these three doses on cell viability was assessed, revealing a significant decrease in cell survival rates (Fig. 1A). Subsequently, surviving cells with stable morphology postirradiation were subjected to limiting dilution to generate monoclonal cell lines, which were then continuously passaged and screened. Throughout this process, the majority of cells exhibited reduced growth and underwent apoptosis due to 12C6 heavy ion exposure, while only a small fraction thrived. Ultimately, 14 stable monoclonal cell lines were established. Specifically, five cell lines originated from the 15 Gy dose group, designated BHK-7, 10-1, 15-4-f3, 15-4-g3, and 10-D4 mutants; five cell lines emerged from the 10 Gy dose group, identified as 10.59 C4, 15-f 8, 5–9–200 C6, 5A2, and 20imF10 mutants; and four cell lines arose from the 5 Gy dose group, named the 5:9 10 B2, 2-imB10, 5-h12, and BHK-5 mutants. After the infection of 14 stable mutant cell lines with FMDV, the levels of complete FMDV particles (146S) and FMDV gene expression were subsequently analyzed (Fig. 1C). Both the mutant cells and the control BHK-21 cells were infected with FMDV (MOI = 1.0) for 16 h, maintaining a consistent cell density. The results revealed that the 146S content in 4 mutant cell lines from the 5 Gy group and 5 mutant cell lines from the 10 Gy dose group was significantly lower than that in the control BHK-21 cells. Notably, the 146S content in BHK-5 cells was markedly reduced to 0.24067 ng/ml, representing an 81.07% decrease compared with that in BHK-21 cells (p < 0.0001). These findings suggest that, compared with control BHK-21 cells, mutant BHK-5 cells may have a potential inhibitory effect on FMDV. Conversely, when the mutant BHK-7, 15-f8, and 15-4-g5 cells from the 15 Gy dose group were infected with FMDV, their 146S content exceeded that of the control BHK-21 cells. Specifically, the 146S concentration in mutant BHK-7 cells increased to approximately 2.171 ng/ml, a 70.67% increase over that in control BHK-21 cells. This finding indicates that, under the same conditions, the mutant BHK-7 cells enhance FMDV replication to a significantly greater extent than the control BHK-21 cells do. The FMDV gene expression levels after infection were measured via RT-qPCR (Fig. 1D). The FMDV gene copy number was highest in the 15 Gy dose group, followed by the 10 Gy and 5 Gy dose groups, which was in line with the 146S detection results.

Fig. 1

A BHK-21 cells were induced with 12C6 heavy ions at doses of 5 Gy, 10 Gy, and 15 Gy. Cell viability was assessed via trypan blue staining and detected via a Count STAR (Countstar Rigel S2, RY074B2001), and the mortality rate was calculated. The data represent the means and standard errors from six independent experiments. B Changes in cell density over time (0 to 72 h) were monitored for control (BHK-21) cells and mutant BHK-7 and BHK-5 cells via a Count STAR. All the cells to be detected had the same initial density. Growth curves were plotted on the basis of the mean ± standard error from three independent experiments. C Fourteen mutant cell lines and control (BHK-21) cells were adjusted to the same initial density and infected with FMDV at an MOI of 1.0 for 16 h. The content of FMDV antigen 146 s was quantified. The results are presented as the means and standard errors from three independent experiments. D The expression level of the FMDV 3D gene in 14 mutant cell lines and control BHK-21 cells infected with FMDV (MOI = 1.0) for 16 h was measured by qRT-PCR with GAPDH as the reference. Relative expression levels were calculated via the 2−ΔΔCt method. The data represent the means and standard errors from three independent experiments. E An inverted microscope was used to capture the morphological characteristics of fully fused control BHK-21, BHK-7, and BHK-5 cells under bright-field conditions. The magnification was achieved by combining the appropriate eyepieces and a 20 × objective lens. For partially fused cells, magnification was obtained by combining the same eyepieces and a 40 × objective lens under the same bright-field conditions.. The data are presented as the means ± SDs from three independent experiments and were analyzed via Student's two-tailed unpaired t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Furthermore, the growth curves of the mutant BHK-5 and BHK-7 cells were compared with those of the control cells (Fig. 1B). The growth rates of the strains were essentially identical, thus ruling out any potential confounding effects of differential cell growth rates on viral replication in infected cells. Microscopic examination of the morphology of the fused and unfused cells (Fig. 1E) revealed that postfusion BHK-5 cells exhibited a spindle shape with a flattened center, whereas the morphology of BHK-7 and control BHK-21 cells remained similar. Unfused BHK-5 cells displayed a more prominent central region, whereas the morphology of BHK-21 and BHK-7 cells was similar but with minor variations.

Assessment of BHK-5 and BHK-7 in FMDV replication.

To assess the stability of the mutant cell lines BHK-5 and BHK-7, they were continuously passaged for nine generations. In every three passages, the cells were infected with FMDV (MOI = 1.0) for 16 h, and the levels of 146S and FMDV replication were monitored. The results indicated that the cell lines maintained consistent viral replication capabilities throughout the passage process. In summary, the screening process successfully identified BHK-7 cells as a cell line that promotes foot-and-mouth disease virus replication and BHK-5 cells as a cell line that inhibits replication (Fig. 2A, B).

Fig. 2

A Control BHK-21 cells and mutant BHK-7 and BHK-5 cells were passaged 9 times and infected with FMDV (MOI = 1.0) for 16 h every 3 passages to monitor changes in the content of the FMDV antigen 146S. B Control BHK-21 cells and mutant BHK-7 and BHK-5 cells were passaged 9 times and infected with FMDV (MOI = 1.0) for 16 h every 3 passages to assess changes in TCID50 values. The data represent the means ± standard deviations from three independent measurements. The data are presented as the means ± SDs from three independent experiments and were analyzed via Student's two-tailed unpaired t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Proteomic analysis of mutant BHK-7, BHK-5, and control BHK-21 cells before and after FMDV infection

A label-free 4D quantitative proteomics approach was employed to examine two groups: the FMDV-infected group and the non-FMDV-infected group. In the FMDV-infected group, BHK-21 cells infected with foot-and-mouth disease virus (FMDV) served as the control, whereas BHK-5 and BHK-7 cells infected with FMDV composed the experimental group of cells. In the control group, BHK-21 cells were the control, and BHK-5 and BHK-7 cells were the experimental group of cells.

For significant difference analysis, the fold change (FC) and P value of protein expression differences between BHK-5 and control BHK-21, as well as between BHK-7 and control BHK-21, in the FMDV-infected and non-FMDV-infected groups were calculated. The screening criteria were FC > 2 (upregulation greater than 2 times or downregulation less than 0.50 times) and a P-value < 0.05 to determine the number of upregulated and downregulated proteins among the groups.

Compared with control BHK-21, a total of 374 differentially expressed genes (DEGs) were identified in BHK-5, with 174 DEGs significantly downregulated and 200 DEGs significantly upregulated. In BHK-7, a total of 581 DEGs were identified, of which 312 DEGs were significantly downregulated, and 269 DEGs were significantly upregulated (Fig. 3A). Compared with control BHK-21, a total of 573 DEGs were identified in BHK-5 cells after FMDV infection, with 293 DEGs significantly downregulated and 280 DEGs significantly upregulated. In BHK-7, a total of 461 gene fragments were identified, with 264 gene fragments significantly downregulated and 197 gene fragments significantly upregulated (Fig. 3B). We subsequently plotted the overall changes in protein expression of control BHK-21 and mutant BHK-5 and BHK-7 cells before and after FMDV infection (Fig. 3C, D). The results demonstrated that there were significant differences in protein expression between control BHK-21 cells and mutant BHK-5 and BHK-7 cells.

Fig. 3

A Statistical graph of significantly different proteins between BHK-5, BHK-7, and the control (BHK-21); B Statistical graph of significantly different proteins after FMDV infection in BHK-5, BHK-7, and the control (BHK-21); C and D Heatmaps of significantly different protein expression before and after FMDV infection in BHK-5, BHK-7, and the control (BHK-21)

To elucidate the comprehensive metabolic pathway enrichment characteristics of all the differentially expressed proteins. Following the annotation steps, the studied proteins were blasted against the online Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://geneontology.org/) to retrieve their KEGG orthology identifications and subsequently mapped to pathways in KEGG. Enrichment analyses were performed via Fisher's exact test, considering all the quantified proteins as background datasets. Benjamini–Hochberg correction for multiple testing was further applied to adjust the derived p values. Only functional categories and pathways with p values under a threshold of 0.05 were considered significant. BHK-5 and BHK-7, induced by heavy ions, exhibited significant KEGG pathway alterations and intergroup proteomic differences before and after infection with foot-and-mouth disease virus (FMDV). In BHK-5, compared with those in the control BHK-21 without FMDV infection, the downregulated pathways involved primarily steroid biosynthesis, sesquiterpene and triterpene biosynthesis, and complement and coagulation cascades; the upregulated pathways involved mainly drug metabolism-related pathways (e.g., drug metabolism-other enzymes, cytochrome P450 for metabolizing xenobiotics) and arachidonic acid metabolism. Post-FMDV infection, cell adhesion molecules, the p53 signaling pathway, and autophagy-related pathways were significantly downregulated. The downregulation of cell adhesion molecules could impact intercellular connections and communication, changes in autophagy regulation were observed, and the downregulation of the p53 signaling pathway might influence cell apoptosis and DNA damage repair. Concurrently, metabolism-related pathways (drug metabolism-other enzymes, drug metabolism-cytochrome P450, and arachidonic acid) remained prominent, while new upregulated pathways, such as alcoholic liver disease and complement and coagulation cascades, emerged (Fig. 4A, C).

Fig. 4

A KEGG pathway map associated with proteins differentially expressed between BHK-5 and BHK-21 cells. B KEGG pathway map associated with proteins differentially expressed between BHK-7 and BHK-21 cells. C KEGG pathway map associated with proteins differentially expressed between BHK-5 and BHK-21 cells post-FMDV infection. D KEGG pathway map associated with proteins differentially expressed between BHK-7 and BHK-21 cells post-FMDV infection. The x-axis represents the -log10(p-value) derived from Fisher's exact test, indicating the statistical significance of pathway enrichment. The y-axis lists the names of the pathways. The upregulated and downregulated pathways are denoted by red (right) and blue (left) bars, respectively

When BHK-7 cells were not infected with FMDV, compared with those in control BHK-21 cells, the downregulated pathways included primarily cell cycle-related pathways (e.g., the cell cycle in yeast and the cell cycle), steroid biosynthesis, and ECM-receptor interactions, among others. The upregulated pathways included mainly the lysosome, glycosaminoglycan degradation, and retrograde endocannabinoid signaling pathways, among others. Upon infection with FMDV, ECM-receptor interactions, cell adhesion molecules, and the PI3K-Akt signaling pathway were significantly downregulated. Among the upregulated pathways, new pathways, such as folate biosynthesis, motor proteins, and lysosomes, emerged (Fig. 4B, D).

For the proteomic DEGs, we subsequently constructed a volcano plot of the DEGs via two criteria, the fold change and p-value, as shown in Fig. 5. The analysis revealed that GSTK1, TMEM59, LDIR, EGFR, F13A1, and LOC101822825 were significantly different. Notably, Cbr3 significantly differed in its upregulation and downregulation patterns between the groups (differential volcano maps are marked with green circles). Through KEGG enrichment analysis, Cbr3 was found to be enriched mainly in the arachidonic acid metabolism pathway, and this pathway was significant in the proteomic enrichment analysis. We subsequently analyzed the expression levels of the main enriched proteins in this KEGG pathway in BHK-5 and BHK-7 cells and control BHK-21 cells and found that Cbr3 in this pathway was significantly different (Appendices 1, 2). Therefore, Cbr3 may play a certain role in FMDV replication. To verify this hypothesis, we conducted further verification experiments.

Fig. 5

The multi-group differential volcano plots generated on the basis of the log fold change (logFC) and p-value of differentially expressed proteins (DEPs) in BHK-5, BHK-7, and control BHK-21 cells. The top 10 proteins from each group are labeled. The red dots indicate upregulated DEPs, the green dots denote downregulated DEGs, and the gray dots represent genes whose expression did not significantly change

The carbonyl reductase Cbr3 catalyzes the reduction of many carbonyl compounds with biological and pharmacological activities to the corresponding alcohols. This enzyme is classified as an NADPH-dependent monomeric oxidoreductase [22]. Cbr3 consists of three exons with a length of 11.2 kb and is closely related to another carbonyl reductase gene, Cbr1. Prostaglandin E2 (PGE2) plays a crucial role in immune and inflammatory responses. Elevated PGE2 levels are usually associated with intensified inflammatory responses and immunosuppression. Enrichment analysis revealed that Cbr3 participates in the degradation of PGE2 through the arachidonic acid metabolism pathway [23]. In BHK-5 virus-suppressing cells, an increase in Cbr3 expression may reduce PGE2 levels, thereby alleviating inflammatory responses and immunosuppression and enhancing the antiviral ability of cells. Conversely, in BHK-7 virus-promoted replication cells, a decrease in Cbr3 expression or inhibition of its activity may lead to an increase in PGE2 levels, thereby intensifying inflammatory responses and immunosuppression and providing a favorable environment for virus replication.

Establishment of a Cbr3 knockout BHK-21 cell line

To investigate the impact of Cbr3 knockout on FMDV replication, a BHK-21 cell line with Cbr3 knockout, designated BHK-21-KO-Cbr3, was constructed via the CRISPR/Cas9 system. The detailed procedures were as follows. An sgRNA expression plasmid was constructed and transfected into BHK-21 cells via Lipofectamine 2000. Puromycin selection was subsequently applied to screen for successfully transfected cells. PCR primers were designed to amplify regions 100 bp upstream and downstream of the sgRNA target site. DNA was extracted from the cells, and amplification and sequencing were performed (see supplementary materials for verification results). Compared with WT-BHK-21 cells, deletions were observed near the Cbr3 target site. Western blot analysis further confirmed that Cbr3 protein expression was nearly completely abolished in the knockout cell line (Fig. 6A). To assess the potential effects of Cbr3 knockout on cell growth, growth curves of the knockout cell line and WT-BHK-21 cells were compared. The results indicated that the growth patterns of both cell lines were essentially identical (Fig. 6C), suggesting that Cbr3 knockout does not impair cell proliferation. In summary, the Cbr3 knockout cell line was successfully established.

Fig. 6

A Cbr3 expression was detected via Western blotting via an anti-Cbr3 antibody, with β-actin serving as the loading control to ensure uniform sample loading. B WT-BHK-21 and BHK-21-KO-Cbr3 cells were infected with FMDV (MOI = 1.0). At 16 h postinfection, the protein levels of FMDV VP1 were assessed via Western blotting, with β-actin used as the internal reference. C WT-BHK-21 and BHK-21-KO-Cbr3 cells were seeded in T25 flasks at a density of 2.0 × 103 cells/mL. Trypsinized cells were counted every 4 h to construct growth curves. D: WT-BHK-21 and BHK-21-KO-Cbr3 cells were seeded at a density of 3.0 × 105 cells/mL and infected with FMDV (MOI = 1.0) for 16 h. The relative expression of FMDV 3D mRNA was quantified via qRT-PCR. All experiments were performed in triplicate, and the data are presented as the means ± SDs (n = 3). E: WT-BHK-21 and BHK-21-KO-Cbr3 cells were infected with FMDV (MOI = 1.0) at a density of 3.0 × 105 cells/mL for 16 h. The content of FMDV antigen 146 s was measured. The data are presented as the means ± SDs from three independent experiments and were analyzed via Student's two-tailed unpaired t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Knockout of Cbr3 increased FMDV replication.

To evaluate the replication efficiency of foot-and-mouth disease virus (FMDV) in wild-type BHK-21 cells (WT-BHK-21) and Cbr3 gene knockout BHK-21 cells (BHK-21-ko-Cbr3), the cells were infected with a multiplicity of infection (MOI) of 0.1 for 16 h. Total RNA was subsequently extracted from the cells and reverse transcribed to quantify the mRNA expression level of FMDV. Compared with that in WT-BHK-21 cells, the replication of FMDV in BHK-21-KO-Cbr3 cells was significantly greater (Fig. 6D), and the content of FMDV antigen 146 also significantly increased, as expected (Fig. 6E). In addition, the virus titers of BHK-21-ko-Cbr3 and WT-BHK-21 cells at 4, 8, and 16 h were measured and compared (Fig. 7A, B). Western blotting detection of FMDV VP1 also revealed that the expression of BHK-21-KO-Cbr3 infected with FMDV was greater than that of WT-BHK-21 at different time points. Finally, the replication of FMDV in these two cell types after 8 h of BHK-21-KO-Cbr3 and WT-BHK-21 infection with FMDV was visualized via confocal laser microscopy (Fig. 7C). These data indicate that knockout of the Cbr3 gene significantly enhances the replication of FMDV in BHK-21 cells.

Fig. 7

A WT-BHK-21 and BHK-21-KO-Cbr3 cells were infected with FMDV (MOI = 1.0). At 4, 8, and 16 h postinfection, the protein levels of FMDV VP1 were assessed via Western blotting, with β-actin used as the internal reference. B: TCID₅₀ values were determined by infecting WT-BHK-21 and BHK-21-KO-Cbr3 cells with FMDV. The experiments were conducted three times, and the data are expressed as the means ± SDs (n = 3). C: WT-BHK-21 and BHK-21-KO-Cbr3 cells were infected with FMDV (MOI = 1.0) for 8 h. Immunofluorescence staining followed by confocal microscopy was used to observe the signal of the FMDV protein VP1. The green signal represents VP1, the nucleus was stained with Hoechst 33,258, and the red signal represents β-actin. The data are presented as the means ± SDs from three independent experiments and were analyzed via Student's two-tailed unpaired t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Cbr3 influences FMDV replication through the regulation of PGE2 degradation

PGE2 is an eicosanoid lipid mediator with diverse functions, such as promoting cell proliferation and stem cell expansion and exerting immunosuppressive effects. Omics analysis revealed that the Cbr3 protein plays a role in the degradation of PGE2 within the arachidonic acid metabolism pathway (Fig. 8A). We measured the levels of PGE2 in both BHK-21-KO-Cbr3 cells and WT-BHK-21 cells. These findings indicate that Cbr3 knockout leads to increased intracellular PGE2 accumulation and reduced degradation of PGE2 to PGF2α (Fig. 8B). To investigate whether PGE2 influences FMDV replication, we added 0.005 nmol, 0.01 nmol, or 0.02 nmol of PGE2 to WT-BHK-21 cells and assessed their condition 16 h post-FMDV infection. The results demonstrated that as the concentration of added PGE2 increased, so did the replication rate of FMDV in WT-BHK-21 cells (Fig. 8D, E). In BHK-5 cells, which were previously selected for their ability to inhibit viral replication, the expression level of the Cbr3 protein was notably greater than that in control BHK-21 cells and the virus-promoting cell line BHK-7. These findings suggest that overexpression of the Cbr3 protein might facilitate the degradation of PGE2. To confirm this hypothesis, we supplemented BHK-5 cells with 0.02 nmol of PGE2, and the results aligned with our expectations: the addition of PGE2 led to a significant increase in FMDV replication in BHK-5 cells (Fig. 8C, F). Collectively, these findings suggest that the Cbr3 protein modulates FMDV replication by regulating cellular PGE2 levels.

Fig. 8

A represents the synthesis pathway of prostaglandin E2 (PGE2) in cells (AA arachidonic acid; PGH2 prostaglandin H2; PGE2 prostaglandin E2; PGF2α prostaglandin F2α). B PGE2 content in cells was determined via a PGE2 ELISA kit. The experiment included three replicates, and the results are expressed as the mean ± standard deviation (SD) (n = 3). The protein expression level of Cbr3 in BHK-21-ko-Cbr3 and control BHK-21 cells was evaluated. D. Wild-type BHK-21 cells were cultured in 6-well plates and treated with 0.005, 0.01, or 0.02 nmol/ml PGE2. The cells were infected with the foot-and-mouth disease virus (FMDV) for 16 h at a multiplicity of infection (MOI) of 1.0. The relative expression level of FMDV 3D mRNA was detected via real-time quantitative PCR (qRT-PCR). The experiment included three replicates, and the results are expressed as the means ± SDs (n = 3). When exogenous PGE2 was added to BHK-5 cells, the protein expression of Cbr3 was significantly greater than that in control BHK-21 cells. The cells were infected with FMDV (MOI = 1.0) for 16 h. The expression of the FMDV VP1 protein was detected by Western blotting, with β-actin used as the loading control. F. BHK-5 cells were cultured in 6-well plates, 0.02 nmol/ml PGE2 was added, and the cells were infected with FMDV (MOI = 1.0) for 16 h. The expression of the FMDV VP1 protein was detected by Western blotting, with β-actin serving as the internal reference. The data are presented as the means ± SDs from three independent experiments and were analyzed via Student's two-tailed unpaired t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001