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LSDV001 positively regulates IL-1β-triggered signaling and has minimal effects on <t>TNFα-triggered</t> signaling in HEK293 cells. ( A ) Screening of LSDV proteins that regulate activation of NF-κB induced by IL-1β. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and LSDV eukaryotic expression plasmids (200 ng) for 24 hours. The cells were then treated with IL-1β or left untreated for 10 hours before reporter assays. ( B ) Effects of LSDV001 on IL-1β and TNFα-induced NF-κB activation. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and LSDV001 plasmid (1, 10, and 20 ng) for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for 10 hours before reporter assays. ( C ) Effects of LSDV001 on IL-1β and TNFα-induced transcription of inflammatory cytokines. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for the indicated times before qPCR experiments. ( D ) Effects of LSDV001 on IFN-β and IFN-γ-induced transcription of downstream genes. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IFN-β, IFN-γ, or left untreated for 6 hours before qPCR experiments. ( E-F ). Effects of LSDV001 on IL-1β and TNFα-induced phosphorylation of signaling molecules. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for the indicated times before immunoblot analysis. Data shown in B–D are mean ± SD ( n = 3) from one representative experiment. ns, nonsignificant; * P < 0.05; ** P < 0.01 (unpaired t-test).
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LSDV001 positively regulates IL-1β-triggered signaling and has minimal effects on <t>TNFα-triggered</t> signaling in HEK293 cells. ( A ) Screening of LSDV proteins that regulate activation of NF-κB induced by IL-1β. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and LSDV eukaryotic expression plasmids (200 ng) for 24 hours. The cells were then treated with IL-1β or left untreated for 10 hours before reporter assays. ( B ) Effects of LSDV001 on IL-1β and TNFα-induced NF-κB activation. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and LSDV001 plasmid (1, 10, and 20 ng) for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for 10 hours before reporter assays. ( C ) Effects of LSDV001 on IL-1β and TNFα-induced transcription of inflammatory cytokines. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for the indicated times before qPCR experiments. ( D ) Effects of LSDV001 on IFN-β and IFN-γ-induced transcription of downstream genes. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IFN-β, IFN-γ, or left untreated for 6 hours before qPCR experiments. ( E-F ). Effects of LSDV001 on IL-1β and TNFα-induced phosphorylation of signaling molecules. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for the indicated times before immunoblot analysis. Data shown in B–D are mean ± SD ( n = 3) from one representative experiment. ns, nonsignificant; * P < 0.05; ** P < 0.01 (unpaired t-test).
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LSDV001 positively regulates IL-1β-triggered signaling and has minimal effects on <t>TNFα-triggered</t> signaling in HEK293 cells. ( A ) Screening of LSDV proteins that regulate activation of NF-κB induced by IL-1β. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and LSDV eukaryotic expression plasmids (200 ng) for 24 hours. The cells were then treated with IL-1β or left untreated for 10 hours before reporter assays. ( B ) Effects of LSDV001 on IL-1β and TNFα-induced NF-κB activation. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and LSDV001 plasmid (1, 10, and 20 ng) for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for 10 hours before reporter assays. ( C ) Effects of LSDV001 on IL-1β and TNFα-induced transcription of inflammatory cytokines. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for the indicated times before qPCR experiments. ( D ) Effects of LSDV001 on IFN-β and IFN-γ-induced transcription of downstream genes. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IFN-β, IFN-γ, or left untreated for 6 hours before qPCR experiments. ( E-F ). Effects of LSDV001 on IL-1β and TNFα-induced phosphorylation of signaling molecules. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for the indicated times before immunoblot analysis. Data shown in B–D are mean ± SD ( n = 3) from one representative experiment. ns, nonsignificant; * P < 0.05; ** P < 0.01 (unpaired t-test).
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LSDV001 positively regulates IL-1β-triggered signaling and has minimal effects on TNFα-triggered signaling in HEK293 cells. ( A ) Screening of LSDV proteins that regulate activation of NF-κB induced by IL-1β. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and LSDV eukaryotic expression plasmids (200 ng) for 24 hours. The cells were then treated with IL-1β or left untreated for 10 hours before reporter assays. ( B ) Effects of LSDV001 on IL-1β and TNFα-induced NF-κB activation. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and LSDV001 plasmid (1, 10, and 20 ng) for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for 10 hours before reporter assays. ( C ) Effects of LSDV001 on IL-1β and TNFα-induced transcription of inflammatory cytokines. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for the indicated times before qPCR experiments. ( D ) Effects of LSDV001 on IFN-β and IFN-γ-induced transcription of downstream genes. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IFN-β, IFN-γ, or left untreated for 6 hours before qPCR experiments. ( E-F ). Effects of LSDV001 on IL-1β and TNFα-induced phosphorylation of signaling molecules. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for the indicated times before immunoblot analysis. Data shown in B–D are mean ± SD ( n = 3) from one representative experiment. ns, nonsignificant; * P < 0.05; ** P < 0.01 (unpaired t-test).

Journal: mBio

Article Title: Lumpy skin disease virus LSDV001 protein positively regulates inflammatory response by promoting assembly of the TAK1-TAB2/3 complex

doi: 10.1128/mbio.01677-25

Figure Lengend Snippet: LSDV001 positively regulates IL-1β-triggered signaling and has minimal effects on TNFα-triggered signaling in HEK293 cells. ( A ) Screening of LSDV proteins that regulate activation of NF-κB induced by IL-1β. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and LSDV eukaryotic expression plasmids (200 ng) for 24 hours. The cells were then treated with IL-1β or left untreated for 10 hours before reporter assays. ( B ) Effects of LSDV001 on IL-1β and TNFα-induced NF-κB activation. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and LSDV001 plasmid (1, 10, and 20 ng) for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for 10 hours before reporter assays. ( C ) Effects of LSDV001 on IL-1β and TNFα-induced transcription of inflammatory cytokines. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for the indicated times before qPCR experiments. ( D ) Effects of LSDV001 on IFN-β and IFN-γ-induced transcription of downstream genes. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IFN-β, IFN-γ, or left untreated for 6 hours before qPCR experiments. ( E-F ). Effects of LSDV001 on IL-1β and TNFα-induced phosphorylation of signaling molecules. HEK293 cells (5 × 10 5 ) were transfected with either an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were then treated with IL-1β, TNFα, or left untreated for the indicated times before immunoblot analysis. Data shown in B–D are mean ± SD ( n = 3) from one representative experiment. ns, nonsignificant; * P < 0.05; ** P < 0.01 (unpaired t-test).

Article Snippet: Fetal bovine serum (SA211.02, Cellmax); penicillin and streptomycin (SV30010, HyClone); Dulbecco’s modified Eagle’s medium (C11965500BT, Gibco); Puromycin (AMR-J593, VWR); Protein A + G agarose (P2055, Beyotime); PMSF (P7626, Sigma); recombinant human TNFα (10601, Sino Biological); recombinant human IL-1β (200-01B, PeproTech); recombinant bovine TNFα (2279-BT/C, R&D); Dual-Specific Luciferase Assay Kit (E1980, Promega); SYBR Green supermix (Q312, Vazyme); HiScript II Select RT SuperMix for qPCR (R323, Vazyme); ELISA MAX Deluxe Set Mouse TNFα and IL-6 (430904 and 431304, BioLegend); the inhibitors aprotin, leupeptin, β-glycerophosphate disodium salt, and sodium orthovanadate (HY-P0017, HY-18234A, HY-126304 and HY-D0852, MCE) were purchased from the indicated manufacturers.

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Phospho-proteomics, Western Blot

LSDV001 positively regulates IL-1β- and TNFα-triggered signaling in MDBK cells. ( A and B ). Effects of LSDV001 on IL-1β and TNFα-induced transcription of inflammatory cytokines. MDBK cells (2 × 10 5 ) were transduced with either an empty vector or LSDV001-Flag plasmid to establish stable cell lines. The cells (2 × 10 5 ) were treated with IL-1β, TNFα, or left untreated for the indicated times before qPCR experiments. ( C ) Effects of LSDV001 on IFN-β and IFN-γ-induced transcription of downstream genes. MDBK cells (2 × 10 5 ) stably transduced with an empty vector or LSDV001-Flag plasmid were treated with IFN-β, IFN-γ, or left untreated for 6 hours before qPCR experiments. ( D and E ). Effects of LSDV001 on IL-1β and TNFα-induced phosphorylation of IKKα/β, IκBα, and p65. MDBK cells (2 × 10 5 ) stably transduced with an empty vector or LSDV001-Flag plasmid were treated with IL-1β, TNFα, or left untreated for the indicated times before immunoblot analysis. Data shown in A–C are mean ± SD ( n = 3) from one representative experiment. ns nonsignificant; * P < 0.05, ** P < 0.01 (unpaired t-test).

Journal: mBio

Article Title: Lumpy skin disease virus LSDV001 protein positively regulates inflammatory response by promoting assembly of the TAK1-TAB2/3 complex

doi: 10.1128/mbio.01677-25

Figure Lengend Snippet: LSDV001 positively regulates IL-1β- and TNFα-triggered signaling in MDBK cells. ( A and B ). Effects of LSDV001 on IL-1β and TNFα-induced transcription of inflammatory cytokines. MDBK cells (2 × 10 5 ) were transduced with either an empty vector or LSDV001-Flag plasmid to establish stable cell lines. The cells (2 × 10 5 ) were treated with IL-1β, TNFα, or left untreated for the indicated times before qPCR experiments. ( C ) Effects of LSDV001 on IFN-β and IFN-γ-induced transcription of downstream genes. MDBK cells (2 × 10 5 ) stably transduced with an empty vector or LSDV001-Flag plasmid were treated with IFN-β, IFN-γ, or left untreated for 6 hours before qPCR experiments. ( D and E ). Effects of LSDV001 on IL-1β and TNFα-induced phosphorylation of IKKα/β, IκBα, and p65. MDBK cells (2 × 10 5 ) stably transduced with an empty vector or LSDV001-Flag plasmid were treated with IL-1β, TNFα, or left untreated for the indicated times before immunoblot analysis. Data shown in A–C are mean ± SD ( n = 3) from one representative experiment. ns nonsignificant; * P < 0.05, ** P < 0.01 (unpaired t-test).

Article Snippet: Fetal bovine serum (SA211.02, Cellmax); penicillin and streptomycin (SV30010, HyClone); Dulbecco’s modified Eagle’s medium (C11965500BT, Gibco); Puromycin (AMR-J593, VWR); Protein A + G agarose (P2055, Beyotime); PMSF (P7626, Sigma); recombinant human TNFα (10601, Sino Biological); recombinant human IL-1β (200-01B, PeproTech); recombinant bovine TNFα (2279-BT/C, R&D); Dual-Specific Luciferase Assay Kit (E1980, Promega); SYBR Green supermix (Q312, Vazyme); HiScript II Select RT SuperMix for qPCR (R323, Vazyme); ELISA MAX Deluxe Set Mouse TNFα and IL-6 (430904 and 431304, BioLegend); the inhibitors aprotin, leupeptin, β-glycerophosphate disodium salt, and sodium orthovanadate (HY-P0017, HY-18234A, HY-126304 and HY-D0852, MCE) were purchased from the indicated manufacturers.

Techniques: Transduction, Plasmid Preparation, Stable Transfection, Phospho-proteomics, Western Blot

LSDV001 interacts with TAK1 and TAB2/3. ( A ) Schematic diagram of IL-1β and TNFα signaling. ( B ) Effects of LSDV001 on NF-κB activation mediated by various components. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and indicated plasmids for 24 hours before reporter assays. Data shown are mean ± SD ( n = 3) from one representative experiment. ns, nonsignificant; * P < 0.05, ** P < 0.01 (unpaired t-test). ( C ) The interaction of LSDV001 with TAK1 and TABs in the overexpression system. HEK293 cells (1 × 10 7 ) were transfected with the indicated plasmids for 24 hours and then lysed for coimmunoprecipitation with control IgG or HA antibody, followed by immunoblot analysis with the indicated antibodies. ( D ) The interaction of LSDV001 with endogenous TAK1 and TAB2/3. HEK293 cells (2 × 10 7 ) were transfected with an empty vector or LSDV001-Flag plasmid for 24 hours and then lysed for coimmunoprecipitation with Flag antibody, followed by immunoblot analysis with the indicated antibodies. ( E ) Co-localization of LSDV001 with TAK1 and TAB2/3. HeLa cells (2 × 10 4 ) were transfected with the indicated plasmids for 20 hours and then fixed for immunostaining before being subjected to confocal microscopy. ( F ) The interaction of endogenous LSDV001 with TAK1 and TAB2 during LSDV infection. MDBK cells (5 × 10 7 ) were left uninfected or infected with LSDV (MOI = 2) before cells were harvested for immunoprecipitation with control IgG or antibodies against TAK1 or TAB2. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies.

Journal: mBio

Article Title: Lumpy skin disease virus LSDV001 protein positively regulates inflammatory response by promoting assembly of the TAK1-TAB2/3 complex

doi: 10.1128/mbio.01677-25

Figure Lengend Snippet: LSDV001 interacts with TAK1 and TAB2/3. ( A ) Schematic diagram of IL-1β and TNFα signaling. ( B ) Effects of LSDV001 on NF-κB activation mediated by various components. HEK293 cells (1 × 10 5 ) were transfected with pRL-TK (20 ng), NF-κB reporter (20 ng), and indicated plasmids for 24 hours before reporter assays. Data shown are mean ± SD ( n = 3) from one representative experiment. ns, nonsignificant; * P < 0.05, ** P < 0.01 (unpaired t-test). ( C ) The interaction of LSDV001 with TAK1 and TABs in the overexpression system. HEK293 cells (1 × 10 7 ) were transfected with the indicated plasmids for 24 hours and then lysed for coimmunoprecipitation with control IgG or HA antibody, followed by immunoblot analysis with the indicated antibodies. ( D ) The interaction of LSDV001 with endogenous TAK1 and TAB2/3. HEK293 cells (2 × 10 7 ) were transfected with an empty vector or LSDV001-Flag plasmid for 24 hours and then lysed for coimmunoprecipitation with Flag antibody, followed by immunoblot analysis with the indicated antibodies. ( E ) Co-localization of LSDV001 with TAK1 and TAB2/3. HeLa cells (2 × 10 4 ) were transfected with the indicated plasmids for 20 hours and then fixed for immunostaining before being subjected to confocal microscopy. ( F ) The interaction of endogenous LSDV001 with TAK1 and TAB2 during LSDV infection. MDBK cells (5 × 10 7 ) were left uninfected or infected with LSDV (MOI = 2) before cells were harvested for immunoprecipitation with control IgG or antibodies against TAK1 or TAB2. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies.

Article Snippet: Fetal bovine serum (SA211.02, Cellmax); penicillin and streptomycin (SV30010, HyClone); Dulbecco’s modified Eagle’s medium (C11965500BT, Gibco); Puromycin (AMR-J593, VWR); Protein A + G agarose (P2055, Beyotime); PMSF (P7626, Sigma); recombinant human TNFα (10601, Sino Biological); recombinant human IL-1β (200-01B, PeproTech); recombinant bovine TNFα (2279-BT/C, R&D); Dual-Specific Luciferase Assay Kit (E1980, Promega); SYBR Green supermix (Q312, Vazyme); HiScript II Select RT SuperMix for qPCR (R323, Vazyme); ELISA MAX Deluxe Set Mouse TNFα and IL-6 (430904 and 431304, BioLegend); the inhibitors aprotin, leupeptin, β-glycerophosphate disodium salt, and sodium orthovanadate (HY-P0017, HY-18234A, HY-126304 and HY-D0852, MCE) were purchased from the indicated manufacturers.

Techniques: Activation Assay, Transfection, Over Expression, Control, Western Blot, Plasmid Preparation, Immunostaining, Confocal Microscopy, Infection, Immunoprecipitation

LSDV001 promotes assembly of the TAK1-TAB2/3 complex. ( A ) Effects of LSDV001 on the association of TAK1 with TABs. HEK293 cells (1 × 10 7 ) were transfected with the indicated plasmids for 24 hours, followed by coimmunoprecipitation and immunoblot analysis. ( B and C ) Effects of LSDV001 on the endogenous association of TRAF6 or TRAF2 with TAK1 and TAB2/3, as well as TAB2/3 with TAK1 following IL-1β or TNFα stimulation. HEK293 cells (2 × 10 7 ) were transfected with an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were left untreated or treated with IL-1β or TNFα for 15 minutes before cells were harvested for immunoprecipitation with control IgG or antibodies against TAK1, TAB2, TRAF6, or TRAF2. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies.

Journal: mBio

Article Title: Lumpy skin disease virus LSDV001 protein positively regulates inflammatory response by promoting assembly of the TAK1-TAB2/3 complex

doi: 10.1128/mbio.01677-25

Figure Lengend Snippet: LSDV001 promotes assembly of the TAK1-TAB2/3 complex. ( A ) Effects of LSDV001 on the association of TAK1 with TABs. HEK293 cells (1 × 10 7 ) were transfected with the indicated plasmids for 24 hours, followed by coimmunoprecipitation and immunoblot analysis. ( B and C ) Effects of LSDV001 on the endogenous association of TRAF6 or TRAF2 with TAK1 and TAB2/3, as well as TAB2/3 with TAK1 following IL-1β or TNFα stimulation. HEK293 cells (2 × 10 7 ) were transfected with an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were left untreated or treated with IL-1β or TNFα for 15 minutes before cells were harvested for immunoprecipitation with control IgG or antibodies against TAK1, TAB2, TRAF6, or TRAF2. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies.

Article Snippet: Fetal bovine serum (SA211.02, Cellmax); penicillin and streptomycin (SV30010, HyClone); Dulbecco’s modified Eagle’s medium (C11965500BT, Gibco); Puromycin (AMR-J593, VWR); Protein A + G agarose (P2055, Beyotime); PMSF (P7626, Sigma); recombinant human TNFα (10601, Sino Biological); recombinant human IL-1β (200-01B, PeproTech); recombinant bovine TNFα (2279-BT/C, R&D); Dual-Specific Luciferase Assay Kit (E1980, Promega); SYBR Green supermix (Q312, Vazyme); HiScript II Select RT SuperMix for qPCR (R323, Vazyme); ELISA MAX Deluxe Set Mouse TNFα and IL-6 (430904 and 431304, BioLegend); the inhibitors aprotin, leupeptin, β-glycerophosphate disodium salt, and sodium orthovanadate (HY-P0017, HY-18234A, HY-126304 and HY-D0852, MCE) were purchased from the indicated manufacturers.

Techniques: Transfection, Western Blot, Plasmid Preparation, Immunoprecipitation, Control

LSDV001 deficiency attenuates LSDV-triggered inflammatory response and pathogenesis in vivo . ( A ) Effects of LSDV001 deficiency on LSDV-induced production of TNFα and IL-6 in serum. Seven-week-old C57BL/6 mice ( n = 5 for wild-type LSDV001 infection group, n = 6 for LSDVΔ001 infection group) were intravenously injected with wild-type LSDV or LSDVΔ001 (1 × 10 6 pfu) for 24 hours, followed by orbital blood collection for ELISA. ( B ) Effects of LSDV001 deficiency on LSDV-induced formation of skin nodules. Three-week-old LVG hamsters ( n = 3) were intradermally injected with wild-type LSDV or LSDVΔ001 (5 × 10 5 pfu) for 5 days, and then the skin nodules were fixed and photographed. ( C ) Effects of LSDV001 deficiency on LSDV-induced histopathological changes of skin nodules. LVG hamsters ( n = 3) were intradermally injected with wild-type LSDV, LSDVΔ001 (5 × 10 5 pfu), or PBS for 5 days, and then the skin nodular lesions were fixed and stained with HE to observe pathological characteristics. Three representative fields from PBS, wild-type LSDV- or LSDVΔ001-injected groups were shown. The red box showed the tissue damage of skin nodular lesions. Any diagonal lines present in the images are stitching artifacts generated by the whole-slide scanner software during the assembly of the panoramic image from multiple fields of view. ( D ) Effects of LSDV001 deficiency on the viral loads of the skin nodules. LVG hamsters ( n = 3) were intradermally injected with wild-type LSDV or LSDVΔ001 (5 × 10 5 pfu) for 5 days, and then the skin nodular lesions were fixed and stained with DAPI for microscopy analysis. Three representative fields from wild-type LSDV- or LSDVΔ001-injected group were shown. ( E ) Effects of LSDV001 deficiency on the viral loads of the skin nodules. LVG hamsters ( n = 3) were intradermally injected with wild-type LSDV or LSDVΔ001 (5 × 10 5 pfu) for 5 days, and then the skin nodular lesions were harvested. Total DNA was extracted using a commercial viral DNA extraction kit. Viral loads were quantified by qPCR targeting the viral LSDV031 and LSDV063 genes. Data shown in A ( n = 5 for wild-type LSDV001 infection group, n = 6 for LSDVΔ001 infection group) and E ( n = 3) are mean ± SD from one representative experiment. ns, nonsignificant; * P < 0.05 (unpaired t-test).

Journal: mBio

Article Title: Lumpy skin disease virus LSDV001 protein positively regulates inflammatory response by promoting assembly of the TAK1-TAB2/3 complex

doi: 10.1128/mbio.01677-25

Figure Lengend Snippet: LSDV001 deficiency attenuates LSDV-triggered inflammatory response and pathogenesis in vivo . ( A ) Effects of LSDV001 deficiency on LSDV-induced production of TNFα and IL-6 in serum. Seven-week-old C57BL/6 mice ( n = 5 for wild-type LSDV001 infection group, n = 6 for LSDVΔ001 infection group) were intravenously injected with wild-type LSDV or LSDVΔ001 (1 × 10 6 pfu) for 24 hours, followed by orbital blood collection for ELISA. ( B ) Effects of LSDV001 deficiency on LSDV-induced formation of skin nodules. Three-week-old LVG hamsters ( n = 3) were intradermally injected with wild-type LSDV or LSDVΔ001 (5 × 10 5 pfu) for 5 days, and then the skin nodules were fixed and photographed. ( C ) Effects of LSDV001 deficiency on LSDV-induced histopathological changes of skin nodules. LVG hamsters ( n = 3) were intradermally injected with wild-type LSDV, LSDVΔ001 (5 × 10 5 pfu), or PBS for 5 days, and then the skin nodular lesions were fixed and stained with HE to observe pathological characteristics. Three representative fields from PBS, wild-type LSDV- or LSDVΔ001-injected groups were shown. The red box showed the tissue damage of skin nodular lesions. Any diagonal lines present in the images are stitching artifacts generated by the whole-slide scanner software during the assembly of the panoramic image from multiple fields of view. ( D ) Effects of LSDV001 deficiency on the viral loads of the skin nodules. LVG hamsters ( n = 3) were intradermally injected with wild-type LSDV or LSDVΔ001 (5 × 10 5 pfu) for 5 days, and then the skin nodular lesions were fixed and stained with DAPI for microscopy analysis. Three representative fields from wild-type LSDV- or LSDVΔ001-injected group were shown. ( E ) Effects of LSDV001 deficiency on the viral loads of the skin nodules. LVG hamsters ( n = 3) were intradermally injected with wild-type LSDV or LSDVΔ001 (5 × 10 5 pfu) for 5 days, and then the skin nodular lesions were harvested. Total DNA was extracted using a commercial viral DNA extraction kit. Viral loads were quantified by qPCR targeting the viral LSDV031 and LSDV063 genes. Data shown in A ( n = 5 for wild-type LSDV001 infection group, n = 6 for LSDVΔ001 infection group) and E ( n = 3) are mean ± SD from one representative experiment. ns, nonsignificant; * P < 0.05 (unpaired t-test).

Article Snippet: Fetal bovine serum (SA211.02, Cellmax); penicillin and streptomycin (SV30010, HyClone); Dulbecco’s modified Eagle’s medium (C11965500BT, Gibco); Puromycin (AMR-J593, VWR); Protein A + G agarose (P2055, Beyotime); PMSF (P7626, Sigma); recombinant human TNFα (10601, Sino Biological); recombinant human IL-1β (200-01B, PeproTech); recombinant bovine TNFα (2279-BT/C, R&D); Dual-Specific Luciferase Assay Kit (E1980, Promega); SYBR Green supermix (Q312, Vazyme); HiScript II Select RT SuperMix for qPCR (R323, Vazyme); ELISA MAX Deluxe Set Mouse TNFα and IL-6 (430904 and 431304, BioLegend); the inhibitors aprotin, leupeptin, β-glycerophosphate disodium salt, and sodium orthovanadate (HY-P0017, HY-18234A, HY-126304 and HY-D0852, MCE) were purchased from the indicated manufacturers.

Techniques: In Vivo, Infection, Injection, Enzyme-linked Immunosorbent Assay, Staining, Generated, Software, Microscopy, DNA Extraction