Fig 1.
LSDV infection characteristics in kidney and mammary tissues and cells.
(A) Schematic diagram of the golden hamster infection model. Created in BioRender. wen, y. (2025) https://BioRender.com/5sa93q9 (B) Body weight changes in LSDV-infected and mock-infected hamsters over 14 days post-infection (dpi). (C) Representative image demonstrating characteristic pox-like skin lesions (red arrows) in LSDV-infected hamsters. (D) Viral loads in different tissues at 14 dpi quantified by qPCR (n = 5). (E) Growth kinetics of LSDV in MDBK and MAC-T cells infected at MOI = 1. (F) Phase-contrast microscopy images of morphological changes in LSDV-infected MDBK and MAC-T cells at 24 hpi compared to mock-infected controls. Scale bars = 20 μm. (G) TEM images illustrating ultrastructural changes in LSDV-infected cells at 24 hpi. Scale bars = 500 nm. Statistical significance was determined using one-way ANOVA with multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). Error bars represent mean ± SD.
Fig 2.
LSDV infection induces apoptosis in MDBK cells.
(A) KEGG pathway enrichment analysis of differentially expressed genes in LSDV-infected MDBK cells. Pathways associated with apoptosis, including the p53 signaling pathway and TNF signaling pathway, are significantly enriched. Circle size corresponds to the number of genes, and color intensity indicates statistical significance (-log10(q-value)). (B) TUNEL assay demonstrated DNA fragmentation in MDBK cells infected with LSDV, staurosporine (positive control), or mock treatment. Green TUNEL-positive signals indicated LSDV-induced apoptotic DNA fragmentation. Scale bars = 20 µm. (C) TEM images of MDBK cells infected with LSDV or mock-treated. Scale bars = 500 nm. (D) Quantification of early apoptotic, late apoptotic, and necrotic cells from flow cytometry analysis of MDBK and MAC-T cells. (E) Flow cytometry analysis of Annexin V-FITC and PI staining in MDBK and MAC-T cells infected with LSDV (12, 24, 48, 72, and 96 hpi) or mock-treated. (F) Western blot analysis of Caspase-3 expression and activation (Cleaved caspase-3) in MDBK and MAC-T cells following LSDV infection (12, 24, 48, 72 and 96 hpi) or mock treatment (96 h). α-tubulin was used as loading control.
Fig 3.
LSDV ORF117 interacts with GAPDH and induces apoptosis in MDBK cells.
(A) Fluorescence microscopy images of Vero cells transfected with plasmids expressing GFP-tagged LSDV ORFs. Scale bars = 100 µm. (B) Caspase-3/7 activity assay in Vero cells transfected with plasmids expressing the indicated LSDV ORFs. (n = 3) (C) Fluorescence microscopy images of MDBK cells stably expressing ORF117, demonstrating expression of ORF117-associated GFP fluorescence. Scale bars = 100 µm. (D) Western blot analysis confirming LSDV ORF117 expression in MDBK cells stably expressing ORF117. α-tubulin was used as the loading control. (E) Caspase-3/7 activity assay in MDBK cells stably expressing ORF117. (F) SDS-PAGE and silver staining of GST pull-down assay using recombinant GST-tagged ORF117 protein. (G) Mass spectrometry analysis of proteins interacting with ORF117. (H) Co-IP assay in 293T cells co-transfected with FLAG-tagged GAPDH and Myc-tagged ORF117. (I) Co-IP assay in MDBK cells infected with LSDV or mock-treated. (J) Confocal microscopy images of 293T cells co-transfected with Flag-GAPDH and Myc-ORF117 or MDBK cells infected with LSDV. Line-scan plot profiles of fluorescence intensity demonstrate overlapping signals of GAPDH and ORF117. Scale bars = 10 µm. Statistical significance was determined using one-way ANOVA with multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). Error bars represent mean ± SD.
Fig 4.
LSDV induces apoptosis in MDBK cells through ORF117 activation of the GAPDH-Siah1/p53 pathway.
(A) Immunofluorescence analysis of GAPDH nuclear translocation in LSDV-infected MDBK cells compared to mock controls. DAPI (blue) stains nuclei, GAPDH (red) detected using specific antibodies, and merged images highlight nuclear localization. Enlarged views indicate nuclear GAPDH (arrowhead). Scale bars = 10 µm. (B) Western blot analysis of GAPDH localization in nuclear and cytoplasmic fractions of LSDV-infected and mock-infected MDBK cells. Histone H3 and α-tubulin were used as nuclear and cytoplasmic markers, respectively. Quantification of nuclear GAPDH relative to Histone H3 is shown (n = 3). (C) Quantitative PCR analysis of pro-apoptotic genes (Bim, Puma, and Noxa) in MDBK cells infected with LSDV or mock-treated at 12, 24, and 48 hpi. LSDV infection significantly upregulates the expression of these genes in a time-dependent manner (n = 3). (D) Efficiency of GAPDH knockdown in MDBK cells using siRNA targeting GAPDH, confirmed by Western blot. (E) Western blot analysis of Cleaved caspase-3 and Siah1 levels in LSDV-infected MDBK cells following GAPDH knockdown. (F) Caspase-3 activity assay in LSDV-infected MDBK cells with GAPDH knockdown compared to siRNA-NC and mock controls. (G) qRT-PCR analysis of Bim, Puma, and Noxa mRNA levels in LSDV-infected MDBK cells following GAPDH knockdown compared to controls. (H) Western blot analysis of cleaved caspase-3 and Siah1 levels in MDBK cells overexpressing ORF117 compared to control cells. (I) Western blot analysis of GAPDH localization in nuclear and cytoplasmic fractions of MDBK-ORF117 and MDBK cells. Histone H3 and α-tubulin were used as nuclear and cytoplasmic markers, respectively. (J) qRT-PCR analysis of Bim, Puma, and Noxa mRNA levels in MDBK cells overexpressing ORF117 compared to control cells (n = 3). (K) Western blot analysis of Cleaved Caspase-3 in MDBK-ORF117 cells with or without GAPDH knockdown using siRNA. (L) qRT-PCR analysis of Bim, Puma, and Noxa mRNA levels in MDBK-ORF117 cells with or without GAPDH knockdown (n = 3). Statistical significance was determined using one-way ANOVA with multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). Error bars represent mean ± SD.
Fig 5.
LSDV induces apoptosis in kidney cells and pyroptosis in mammary cells of golden hamsters and bovine.
(A) Immunofluorescence analysis of Cleaved-Caspase-3 (green) in kidney tissues from LSDV-infected and mock-treated hamsters. LSDV (red) detected using specific antibodies, and DAPI (blue) indicates nuclear staining. Merged and enlarged images highlight apoptotic regions. Scale bars = 50 µm. (B) Western blot analysis of Cleaved-Caspase-3 expression in kidney tissues from LSDV-infected and mock-treated hamsters. (C) Caspase-3 enzymatic activity in kidney tissues from LSDV-infected and mock-treated hamsters. (D) Immunofluorescence analysis of GSDMC-N (green) in mammary tissues from LSDV-infected and mock-treated hamsters. LSDV (red) detected using specific antibodies, and DAPI (blue) indicates nuclear staining. Merged and enlarged images highlight pyroptotic regions. Scale bars = 50 µm. (E) Western blot analysis of GSDMC-F and GSDMC-N expression in mammary tissues from LSDV-infected and mock-treated hamsters. (F) Concentrations of pro-inflammatory cytokines IL-18 and IL-1β in plasma and mammary gland tissues from LSDV-infected and mock-treated hamsters. (G) Western blot analysis of Cleaved-Caspase-3 expression in kidney tissues from LSDV-infected and uninfected controls samples (left). Caspase-3 enzymatic activity in bovine kidney tissues was quantified using a specific assay kit (right). (H) Western blot analysis of GSDMC-N expression and concentrations of IL-18 and IL-1β in mammary gland tissues from LSDV-infected and mock-treated bovine samples. Statistical significance was determined using one-way ANOVA with multiple comparisons (*p < 0.05, ***p < 0.001). Error bars represent mean ± SD.
Fig 6.
LSDV infection induces pyroptosis in MAC-T cells.
(A) Cell viability analysis demonstrating dose-dependent protective effects of Z-VAD-FMK (0-50 μM) against LSDV-induced cell death in MAC-T cells. (B) Phase-contrast microscopy images of mock-infected cells (left), LSDV-infected cells exhibiting cellular swelling and rounding (middle), and Z-VAD-FMK-pretreated cells maintaining normal morphology after LSDV infection (right). Scale bars = 20 μm. (C) LDH release assay in MAC-T and MDBK cells at 24, 48, and 72 hpi. (D) ELISA quantification of IL-1β and IL-18 secretion in culture supernatants from LSDV-infected MAC-T cells at 24, 48, and 72 hpi. (E) TEM images of mock-infected (upper panel) and LSDV-infected (lower panel) MAC-T cells. Higher magnification insets (right) demonstrate normal cellular ultrastructure in mock-infected cells, while LSDV-infected cells exhibit characteristic features of pyroptosis including plasma membrane rupture (green arrows) and cytoplasmic vacuolization (red arrows). Scale bars = 20 µm. Statistical significance was determined using one-way ANOVA with multiple comparisons (**p < 0.01, ***p < 0.001, ****p < 0.0001). Error bars represent mean ± SD.
Fig 7.
LSDV infection of MAC-T activates Cleaved-Caspase 8 and GSDMC-N expression.
(A) Western blot analysis of GSDMD, GSDME and GSDMC active fragments after LSDV infection of MAC-T cells. (B) Dose- and time-dependent expression of GSDMC-F, GSDMC-N and Cleaved-Caspase 8 proteins in MAC-T cells after LSDV infection at different multiplicities of infection (MOIs: 0.1, 0.5, 1, 2, and 5) and time points (24, 48, and 72 hpi). (C-D) Immunofluorescence microscopy of MAC-T cells infected with LSDV or mock-infected, immunostained for key pyroptosis markers. Scale bars = 10 µm.
Fig 8.
LSDV induces MAC-T cell pyroptosis via the caspase 8-GSDMC pathway.
(A) Western blot analysis showing expression of Cleaved-Caspase 8, GSDMC-F, GSDMC-N, and LSDV proteins in MAC-T cells mock-infected or infected with LSDV, with or without Z-VAD-FMK treatment. (B) LDH release measuring pyroptotic cell death in response to LSDV infection, with or without Z-VAD-FMK treatment. (C) Knockdown efficiency of GSDMC using siRNA (siRNA-GSDMC1, siRNA-GSDMC2, siRNA-GSDMC3, and siRNA-NC [negative control]) evaluated using Western blot. (D) Cell viability of MAC-T cells after GSDMC knockdown and LSDV infection. (E) LDH release measuring pyroptosis in MAC-T cells following GSDMC knockdown and LSDV infection. (F) Viral load (log10 copies/mL) in MAC-T cells after GSDMC knockdown and LSDV infection. (G) Dose-dependent inhibition of Cleaved-Caspase 8 by Z-IETD-FMK (10–60 µM) in LSDV-infected MAC-T cells, determined by Western blot. (H-I) Western blot analysis showing expression of Cleaved-Caspase 8, GSDMC-F, GSDMC-N, and LSDV proteins in MAC-T cells mock-infected or infected with LSDV, with or without Z-IETD-FMK treatment. (J) LDH release measuring pyroptotic cell death in MAC-T cells treated with Z-IETD-FMK, with or without LSDV infection. Statistical significance was determined using one-way ANOVA with multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). Error bars represent mean ± SD.