Fig 1.
Construction and characterization of the recombinant LSDV reporter virus.
(A) The schematic illustration of the core elements of rPX458-sgRNA1 + 2 and the donor plasmid. (B) The schematic illustration of the construction and purification of rLSDV. This figure was created with BioRender.com. (C-H) MDBK cells were infected with either WT LSDV or rLSDV at a multiplicity of infection (MOI) of 0.01; viral titers (C), relative viral DNA abundance (D), and the expression of the viral early protein ORF35 and late protein ORF118 (E) were measured at the indicated time points; the morphology of viruses were compared at 72 hpi by TEM; the red arrow points to a typical virus particle (F); mCherry fluorescence was monitored from 24 to 144 hpi (G), and RLU was measured from 24 to 168 hpi (H). (I and J) Pearson correlation analysis was performed to assess the relationship between luciferase activity and viral titer (I), as well as between luciferase activity and relative DNA abundance (J). R² indicates the goodness of fit, and P values denote the significance of the correlation. (K) MDBK cells were infected with rLSDV at an MOI of 0.1, and the sensitivity of the two detection methods was compared during 0-5 hpi; viral ORF72 mRNA expression levels relative to bovine β-actin mRNA were quantified by qPCR, while luciferase activity was measured. The grayscale intensity of key protein bands in (E) was normalized to the intensity of the internal control. Scale bars are shown in the lower right corner in (F) and (G). Data are presented as mean ± SD, n = 3.
Table 1.
Primary screening results. The stored concentration of Amantadine was 2 mM. The inhibition rate represents expressed as mean ± SD, n = 3. For a single experiment, the inhibition rate is calculated as (1 - (luciferase activity of the compound group/ luciferase activity of the DMSO group)) × 100%.
Fig 2.
Screening of anti-LSDV compounds from an anti-orthopoxvirus drug library identifies six replication inhibitors.
(A) MDBK cells were treated with different concentrations of DMSO for 72 h, and cell viability was determined by CCK-8 assay. (B) rLSDV-infected (0.1 MOI) MDBK cells were treated with different concentrations of DMSO for 72 h, followed by measurement of luciferase activity. (C and D) rLSDV-infected (0.1 MOI) MDBK cells were treated with candidate compounds at concentrations indicated in Table 1 for 72 h, and antiviral activity was evaluated by luciferase activity (C) and mCherry fluorescence intensity (D). (E to G) MDBK cells were infected with WT LSDV (0.1 MOI), and were simultaneously treated with ENR (100 μM), IDU (20 μM), FIAU (5 μM), RBV (20 μM), AraC (0.5 μM), AraA (20 μM) or DMSO for 72 h. Antiviral efficacy was confirmed again through plaque assay for viral titers (E), qPCR for relative viral DNA abundance normalized to host β-actin (F), and western blot (G) analysis of viral protein expression (early protein ORF35 and late protein ORF118). Scale bars are shown in the lower right corner in (D). Data are presented as mean ± SD, n = 3. Two-sided Student’s t-test was used for statistical analysis in (E) and (F); **P < 0.01. The grayscale intensity of key protein bands in (G) was normalized to the intensity of the internal control. Data are presented as mean ± SD, n = 3.
Table 2.
Selectivity index of candidate compounds.
Fig 3.
CC50 and IC50 values of candidate compounds in MDBK and Vero cells.
(A) Cells were treated with 3-fold serially diluted compounds for 72 h, cell viability was determined by CCK-8 assay, dose-inhibition curves were generated using GraphPad Prism 7 to calculate CC50. rLSDV-infected (0.1 MOI) cells were treated with three-fold serially diluted compounds for 72 h, and viral replication was assessed by luciferase activity; dose-inhibition curves were generated using GraphPad Prism 7 to calculate IC50. (B) mCherry fluorescence signals were concurrently recorded for validation. Scale bars are shown in the lower right corner in (B). Data are presented as mean ± SD, n = 3.
Fig 4.
Time-of-addition assay for anti-LSDV activity of hit compounds in MDBK cells.
(A-D) Schematic illustration of the time-of-addition experiment. These figures were created with BioRender.com. MDBK cells were infected with rLSDV (0.1 MOI) and treated with ENR (100 μM), IDU (20 μM), FIAU (5 μM), RBV (20 μM), AraC (0.5 μM), or AraA (20 μM) at four distinct phases (Virucidal, Pre, During and Post); (E-H) Antiviral effects were quantified by relative luciferase activity; (I-L) mCherry fluorescence were simultaneously captured. Scale bars are shown in the lower right corner in (I, J, K and L). Data are presented as mean ± SD, n = 3. **P < 0.01.
Fig 5.
AraC suppresses LSDV DNA synthesis.
(A) Schematic diagram of the EdU-labeling assay used to detect newly synthesized viral and host DNA during LSDV infection. This figure was created with BioRender.com. (B and C) MDBK cells were infected with LSDV at 1 MOI. At 24 hpi, cells were incubated with AraC (0.5 μM) for 1 h, followed by an additional 1 h incubation with AraC in the continued presence of EdU to label newly synthesized DNA. Host DNA synthesis was visualized by fluorescence microscopy (B), in which white arrows indicate representative foci of newly synthesized viral DNA. Nuclear EdU relative fluorescence unit (RFU) values were quantified using ImageJ (C), in which viral proteins were detected using rabbit anti-LSDV polyclonal antibodies. (D-F) MDBK cells were infected with LSDV at 1 MOI. At 24 hpi, cells were treated with AraC (0.5 μM) for 1 h, followed by incubation with EdU for the indicated times in the continued presence of AraC (0.5 μM). Viral and host DNA synthesis was assessed by fluorescence microscopy (D). Cytoplasmic (E) and nuclear (F) EdU RFU values were quantified using ImageJ. Viral proteins were detected using rabbit anti-LSDV polyclonal antibodies. (G and H) MDBK cells were treated with AraC (0.5 μM) or an equivalent volume of DMSO for 1 h, followed by incubation with EdU for the indicated times in the continued presence of AraC (0.5 μM) to label newly synthesized DNA. Host DNA synthesis was examined by fluorescence microscopy (G), and nuclear EdU RFU values were quantified using ImageJ (H). (I) Schematic diagram of the qPCR assay used to analyze viral DNA replication during LSDV infection and under AraC treatment. This figure was created with BioRender.com. (J and K) MDBK and Vero cells were infected with LSDV at an MOI of 1. At 24 hpi, cells were treated with the indicated concentrations of AraC for 2 h. Viral replication in MDBK (J) and Vero (K) cells was quantified by qPCR and analyzed using the 2^–ΔΔCt method after normalization to host β-actin. Scale bars are shown in the lower right corner in (B, D and G). Data are presented as mean ± SD, n = 3. **P < 0.01.
Fig 6.
AraC elicits marginal cytotoxicity regardless of LSDV infection.
(A) PC1-PC2 scatter plot from PCA of variance-stabilized gene-expression data. Each point denotes one biological replicate, color-coded by condition (Mock, AraC, LSD, and LSD-AraC). (B) Volcano plot of differentially expressed genes (DEGs) for Mock vs AraC(significance thresholds defined in Methods); significantly up- and down-regulated genes are highlighted. (C) GO term enrichment of DEGs from Mock vs AraC; top 10 terms (FDR < 0.05) ranked by the number of enriched DEGs. (D) MDBK cells were treated with AraC (0.5, 1, or 2 μM) for 48 h. Cell lysates were collected for western blotting using antibodies against cleaved PARP1 (C-terminus; also detects full-length PARP1), phospho-Histone H2A.X (Ser139), cleaved caspase-3 (also detects pro-caspase-3), and β-Tubulin. (E) Volcano plot of DEGs for Mock vs LSD. (F) KEGG pathway enrichment of DEGs from Mock vs LSD; top 12 pathways (FDR < 0.05) ranked by the number of enriched DEGs. (G) Volcano plot of DEGs for LSD vs LSD-AraC. (H) MDBK cells were mock-infected or infected with LSDV at an MOI of 1 for 48 h in the presence of 0, 0.5, 1, or 2 μM AraC. Cell lysates were harvested for western blotting with the indicated antibodies. (I and J) Cells subjected to the same treatments as in Figs 6D, 6H and S5A were stained with Annexin V-APC/PI and analyzed by flow cytometry (I), and the percentages of apoptotic cells in each group were statistically analyzed using GraphPad software (J). (K) MDBK cells were mock-infected or infected with LSDV at an MOI of 1 for 48 h with or without 0.5 μM AraC. The pan-caspase inhibitor Z-VAD-FMK (50 μM) and the RIPK3 inhibitor GSK872 (5 μM) were added to the indicated groups. Cell lysates were collected for western blotting with the indicated antibodies. The grayscale intensity of key protein bands in (D, H, and K) was normalized to the intensity of the internal control. Data are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01; ns, not significant.
Fig 7.
AraC blocks the transcription of viral late genes.
(A-F) MDBK cells were infected with LSDV at an MOI of 10 and treated with DMSO or 0.5 μM AraC. Samples were collected at the indicated times and analyzed as follows: (A) virus titers determined by plaque assay; (B) viral DNA quantified by qPCR and normalized to host β-actin; (C) western blotting of viral proteins ORF35 (early) and ORF118 (late) with actin as a loading control; (D-H) RT-qPCR analysis of ORF11 (D), ORF35 (E), ORF40 (F), ORF61 (G), and ORF118 (H), normalized to actin mRNA. (I) Schematic illustrating the construction of early- and late- gene promoter driven reporter viruses. (J) MDBK cells were infected with the early gene promoter-driven reporter virus (top) or the late gene promoter-driven reporter virus (bottom) at an MOI of 10 and treated with DMSO or 0.5 μM AraC. EGFP fluorescence images were acquired at the indicated time points. The grayscale intensity of key protein bands in (C) was normalized to the intensity of the internal control. Scale bars are shown in the lower right corner in (J). Data are presented as mean ± SD, n = 3. **P < 0.01; ns, not significant.
Fig 8.
Structural basis of AraC-mediated inhibition of LSDV replication through targeting of viral DNA polymerase.
(A) Multiple sequence alignment of DNA polymerases from LSDV, bovine, and MPXV was performed, and key residues involved in AraC binding to MPXV DNA polymerase are highlighted with red solid circles. (B and C) Predicted three-dimensional structures of LSDV DNA polymerase (B) and bovine DNA polymerase α subunit (C) were predicted using AlphaFold3. (D and E) Structural alignment of MPXV DNA polymerase (purple, PDB: 8K8S) with predicted LSDV DNA polymerase (green, D) or predicted bovine DNA polymerase α subunit (brown, E) was performed; (F and G) Three-dimensional structural alignment of MPXV DNA polymerase with predicted LSDV DNA polymerase (F) or predicted bovine DNA polymerase α subunit (G) in the AraC binding pocket region was performed; amino acid residues involved in Ara-CTP binding are highlighted in red, and Ara-CTP is shown as yellow.
Fig 9.
Schematic diagram illustrating AraC inhibiting LSDV replication.
During LSDV infection, AraC targets the viral DNA polymerase to inhibit viral DNA synthesis, thereby preventing the activation of late-gene transcription and ultimately blocking progression of the replication cycle from the early to the late phase. This figure was created with BioRender.com. Created in BioRender. Gong, Z. (2026) https://BioRender.com/7ugrs73.