Journal: Nature Microbiology

Article Title: Evolution of enhanced innate immune suppression by SARS-CoV-2 Omicron subvariants

doi: 10.1038/s41564-023-01588-4

Figure Lengend Snippet: a – g , Calu-3 infection with 2,000 E copies per cell of Delta (yellow, Ο), BA.1 (blue, Ο), BA.2 (blue, Δ), BA.4 (purple, O) and BA.5 (purple, Δ), n = 3: mean viral E copies at 2 h.p.i. across three independent experiments ( a ); viral replication over time measured by RT–qPCR for intracellular E copies per microgram RNA ( b ); infection levels measured by nucleocapsid expression (% N+ by flow cytometry) ( c ); expression of IFNB , CXCL10 , IFIT1 , IFIT2 , RSAD2 , MX1 , MX2 and DDX58 in infected cells over time ( d ); IFNβ ( e ) and CXCL10 ( f ) secretion from infected Calu-3 cells measured by ELISA at 48 h.p.i.; rescue of viral replication by JAK1-inhibitor ruxolitinib in Calu-3 cells at 48 h.p.i., where relative infection levels are shown across three independent experiments determined by E copies per microgram RNA normalized to the median infection level of the untreated control ( g ). h – k , Primary bronchial HAEs were infected with the indicated variants at 1,500 E copies per cell: viral replication measured by intracellular E copies at 72 h.p.i. ( h ) and viral release into apical washes over time ( i ), with three biological replicates shown; expression of IFNB , CXCL10 , IFIT1 , IFIT2 , DDX58 and RSAD2 in HAEs at 72 h.p.i., with six biological replicates shown ( j ); intracellular viral E copies in HAEs in the presence or absence of 5 μM ruxolitinib at 72 h.p.i., with three biological replicates shown ( k ). For a , one-way analysis of variance (ANOVA) with Bonferroni post-test was used. n.s., not significant at P > 0.05 for all comparisons. For b – h and j , one-way ANOVA and Dunnett’s post-test were used. For i , two-way ANOVA with a Bonferroni post-test was used. For k , one-tailed unpaired Student’s t -test was used. Replicate measurements from one of three independent experiments. Fold change over mock is shown. Mean ± s.e.m. or individual datapoints are shown. h.p.i., hours post infection.

Article Snippet: IFNβ, IFNλ1/IFNλ3 and CXCL10 were measured using Human IFN-β Quantikine ELISA Kit, Human IL-29/IL-28B (IFNλ1/IFNλ3) DuoSet ELISA or Human CXCL10/IP-10 DuoSet ELISA reagents (Bio-Techne R&D Systems) according to the manufacturer’s instructions.

Techniques: Infection, Quantitative RT-PCR, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: Clinical Epigenetics

Article Title: The immunomodulatory anticancer agent, RRx-001, induces an interferon response through epigenetic induction of viral mimicry

doi: 10.1186/s13148-017-0312-z

Figure Lengend Snippet: RRx-001 induced IFN response through upregulation of type I and III IFN expression and JAK/STAT pathway. Cells were transiently (24 h) treated with 0.5 μM RRx-001 or 0.5 μM 5-AZA and subsequently maintained in drug-free medium for an additional 7 days. RRx-001 induced a significant increase in type I IFN (IFN-β) ( a ) and type III IFN (IL-29/IL-28B) ( b ) secretion into culture medium by HCT 116 cells as measured by ELISA. Transcript levels of IL29 / IL28A were also increased as determined by qPCR ( c ). ISGs ( IFI27 , IFi44 , IFI44L , and IFI6 ) were upregulated by 5-AZA and RRx-001 but blocked by the JAK/STAT inhibitor ruxolitinib (rux) at 2 μM concentration ( d ). Expression of ISGs ( IRF7 , ISG15 , DDX58 , and OASL ) was also upregulated in HCT 116 cells cultured in conditioned medium containing secreted IFNs induced by 5-AZA and RRx-001 as determined by qPCR ( e )

Article Snippet: Levels of type I IFN (IFN-β) and type III IFN (IL-29/IL-28B) were determined using a VeriKine Human IFN-beta ELISA Kit (PBL Assay Science, Piscataway Township, NJ, USA) and a Human IL-29/IL-28B (IFN-lambda 1/3) DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to manufactures’ instructions, respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: Nature Communications

Article Title: WDR77 inhibits prion-like aggregation of MAVS to limit antiviral innate immune response

doi: 10.1038/s41467-023-40567-5

Figure Lengend Snippet: a Immunoblot analysis of MAVS expression in P5 fraction containing crude mitochondria. b Purification procedure for Flag-MAVS. c Silver staining of purified Flag-MAVS. The Flag-MAVS band is indicated by an arrow and the asterisk indicated interested bands. d Peptides identified by mass spectrometry. e Plasmids as indicated were co-transfected into HEK293T cells. 36 h after transfection, cells were harvested for immunoprecipitation and immunoblotting. Asterisk indicated nonspecific bands. f–h HEK293T cells were transfected with luciferase reporters and increasing amounts of plasmids as indicated for 24 h, and then stimulated with or without SeV for 12 h. Cells were collected and IFNB1 promotor activation was detected by luciferase assay ( f ). IFNB1 induction was measured by quantitative PCR (qPCR) ( g ). Culture medium was collected and IFN-β was detected by ELISA ( h ) (For f , IFNB1 : **** p < 0.0001, **** p < 0.0001, ns p = 0.9540, ns p = 0.4002 in sequence; For g , IFNB1 : **** p < 0.0001, **** p < 0.0001, ns p = 0.6511, ns p = 0.1233 in sequence; For h , IFNB1 : *** p = 0.0002, **** p < 0.0001, ns p = 0.6413, ns p = 0.0503 in sequence). i HEK293T cells were transfected with luciferase reporters and plasmids as indicated for 24 h, and then stimulated with or without SeV for 12 h. IFNB1 promotor activation was analyzed by luciferase assay (For i, IFNB1 : **** p < 0.0001, ns p = 0.0861, **** p < 0.0001, ns p = 0.1511 in sequence). j–k HEK293T cells were transfected with luciferase reporters and Flag-WDR77 plasmids for 24 h, and then stimulated with or without SeV, VSV or poly(I:C) for 12 h. IFNB1 promotor activation was analyzed by luciferase assay ( j ). IFN-β was detected by ELISA in ( k ) (For j, IFNB1 : ns p = 0.9994; **** p < 0.0001, **** p < 0.0001, *** p = 0.0001 in sequence; For k , IFN-β: ns p > 0.9999, * p = 0.0377, ** p = 0.0059, * p = 0.0312 in sequence). Data are representative of one experiment ( c and d ), two independent experiments with similar results ( e ), or three independent experiments ( f – k ). P -value was determined by two-tailed Student’s t -test. n.s. indicates no statistical significance. Source data are provided as a Source Data file.

Article Snippet: Concentrations of the IFN-β in cell culture medium and mouse serum were measured by Human Interferon β ELISA kit (Cusabio, CSB-E09889h) or Mouse Interferon β ELISA kit (Cusabio, CSB-E04945m) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Purification, Silver Staining, Mass Spectrometry, Transfection, Immunoprecipitation, Luciferase, Activation Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Sequencing, Two Tailed Test

Journal: Nature Communications

Article Title: WDR77 inhibits prion-like aggregation of MAVS to limit antiviral innate immune response

doi: 10.1038/s41467-023-40567-5

Figure Lengend Snippet: a , b WT or WDR77 -/- HEK293T cells were stimulated with SeV ( a ) or VSV ( b ) for 12 h before IFNB1 induction was measured by qPCR (For a and b , IFNB1 : all **** p < 0.0001). c WT or WDR77 -/- HEK293T cells were stimulated with SeV or VSV for 12 h. Cells were harvested and subjected to immunoblotting. d – f WT or WDR77 -/- #1 HEK293T cells were stimulated with SeV, VSV or poly(I:C) for 12 h before IFNB1 ( d ), ISG56 ( e ) and CXCL10 ( f ) induction was measured by qPCR (For d , IFNB1 : ** p = 0.0046, **** p < 0.0001, **** p < 0.0001 in sequence; For e , ISG56 : *** p = 0.0001, **** p < 0.0001, **** p < 0.0001 in sequence; For f , CXCL10 : * p = 0.0261, **** p < 0.0001, **** p < 0.0001 in sequence). g WT, MAVS -/- or WDR77 -/- #1 HEK293T cells were infected with VSV-GFP (MOI = 0.05) for 12 h before microscopic imaging (left panel) and plaque assay (right panel) were performed. The dilution fold of VSV is displayed in the upper right corner of the plaque analysis. Scale bars indicate 100 μm. h GFP-VSV titers were quantitated in plaque assay (For d , VSV: * p = 0.0123, **** p < 0.0001 in sequence). i – k WT or WDR77 -/- HEK293 cells were stimulated with SeV, VSV, poly(I:C) or HT-DNA for 12 h before IFNB1 induction was measured by qPCR ( i ). Culture medium was collected and IFN-β was detected by ELISA ( j ). Following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimerization ( k ) (For i , IFNB1 : **** p < 0.0001, **** p < 0.0001, *** p = 0.0007, ns p = 0.7439 in sequence; For j , IFN-β: ** p = 0.0014, **** p < 0.0001, ns p = 0.1199, ns p = 0.9973 in sequence). l WT or WDR77 -/- #1 HEK293T cells were transfected with plasmids as indicated for 24 h, and then stimulated with or without SeV for 12 h. IFNB1 induction was measured by qPCR (For l , IFNB1 : **** p < 0.0001, *** p = 0.0001, ns p = 0.9957 in sequence). Data are representative of three independent experiments with similar results ( c , g and k ) or three independent experiments ( a , b , d – f , h – j and l ). P- value was determined by two-tailed Student’s t -test. n.s. indicates no statistical significance. Source data are provided as a Source Data file.

Article Snippet: Concentrations of the IFN-β in cell culture medium and mouse serum were measured by Human Interferon β ELISA kit (Cusabio, CSB-E09889h) or Mouse Interferon β ELISA kit (Cusabio, CSB-E04945m) according to the manufacturer’s instructions.

Techniques: Western Blot, Sequencing, Infection, Imaging, Plaque Assay, Enzyme-linked Immunosorbent Assay, Fractionation, Clear Native PAGE, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: WDR77 inhibits prion-like aggregation of MAVS to limit antiviral innate immune response

doi: 10.1038/s41467-023-40567-5

Figure Lengend Snippet: a – f PEMs from WT, Wdr77 CKO and Mavs -/- mice respectively were collected. PEMs were then stimulated for 6 h with or without VSV, SeV, poly(I:C), HSV or HT-DNA as indicated. Culture mediums were then collected and IFN-β was measured by ELISA ( a ). Ifnb1 ( b ), Ifna4 ( c ), Isg56 ( d ), Cxcl10 ( e ), Il-6 ( f ) induction were measured by qPCR (For a , IFN-β: ** p = 0.0021 **** p < 0.0001, **** p < 0.0001, ns p = 0.0790, ns p = 0.9993 in sequence; for b , Ifnb1 : all **** p < 0.0001, ns p = 0.1813, ns p = 0.8208 in sequence; for c , Ifna4 : all **** p < 0.0001, ns p = 0.9997, ns p = 0.9998 in sequence; for d , Isg56 : all **** p < 0.0001, ns p = 0.1378; For e, Cxcl10 : all **** p < 0.0001, ns p = 0.5755, ns p = 0.7986 in sequence; for f , Il-6 : ns p = 0.9872, **** p < 0.0001, ns p = 0.9932, ns p = 0.7531, ns p = 0.9849 in sequence). g BMDMs from WT, Wdr77 CKO and Mavs -/- mice respectively were collected. BMDMs were then stimulated for 6 h with or without VSV, SeV, poly(I:C), HSV or HT-DNA as indicated. Ifnb1 induction was measured by qPCR. (All **** p < 0.0001, ns p = 0.1224, ns p = 0.9878 in sequence). h PEMs from WT, Wdr77 CKO and Mavs -/- mice were collected respectively. PEMs were infected with or without SeV for 6 h. Cells were collected for subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer. P5 fractions were subjected to SDD-AGE to examine MAVS aggregation and WCL were subjected to SDS-PAGE to examine IRF3 phosphorylation. Data are representative of two independent experiments with similar results ( h ), or three independent experiments ( a – g ) (mean ± SEM of three biological replicates). P -value was determined by two-tailed Student’s t -test. n.s. indicates no statistical significance. Source data are provided as a Source Data file.

Article Snippet: Concentrations of the IFN-β in cell culture medium and mouse serum were measured by Human Interferon β ELISA kit (Cusabio, CSB-E09889h) or Mouse Interferon β ELISA kit (Cusabio, CSB-E04945m) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Sequencing, Infection, Fractionation, Clear Native PAGE, SDS Page, Phospho-proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: WDR77 inhibits prion-like aggregation of MAVS to limit antiviral innate immune response

doi: 10.1038/s41467-023-40567-5

Figure Lengend Snippet: a – d WT, Wdr77 CKO and Mavs -/- mice ( n = 3 each) were infected intravenously with VSV at 2 × 10 7 PFU per mouse. The spleens, livers, and lungs were collected 4 h or 8 h after infection. Ifnb1 ( a ), Ifna4 ( b ) inductions and VSV RNA levels ( c ) were measured respectively by qPCR. The sera were collected and used for the measurement of IFN-β by ELISA ( d ) (For a , Ifnb1 : ** p = 0.0013, **** p < 0.0001, **** p < 0.0001, ns p = 0.0815, *** p = 0.0006, ns p = 0.1397 in sequence; for b , Ifna4 : *** p = 0.0004, **** p < 0.0001, **** p < 0.0001, ns p = 0.6194, *** p = 0.0008, *** p = 0.0008 in sequence; For c , VSV: ns p = 0.7616, ** p = 0.0010, ns p = 0.3889, ns p = 0.1316, ns p = 0.8740, ** p = 0.0023 in sequence; for d , IFN-β: * p = 0.0216, *** p = 0.0003 in sequence). e Hematoxylin-eosin staining of lung sections from WT and Wdr77 CKO mice infected intravenously with VSV at 2 × 10 7 PFU per mouse for 12 h. Scale bars indicate 50 μm. f WT, Wdr77 CKO and Mavs -/- mice ( n = 10 each) were injected intravenously with VSV at 5 × 10 7 PFU per mouse, and the survival rates were monitored for 15 days (* p = 0.049). g WT, Wdr77 CKO and Mavs -/- mice ( n = 10 each) were injected intranasally with IAV at 8 × 10 2 PFU per mouse, and the survival rates were monitored for 15 days (* p = 0.045). h WT, Wdr77 CKO and Mavs -/- mice ( n = 3 each) were infected intravenously with HSV at 1.5 × 10 8 PFU per mouse. The sera were collected 12 h or 24 h after infection and used for the measurement of IFN-β by ELISA (IFN-β: ns p = 0.5412, ns p = 0.9892 in sequence). i WT, Wdr77 CKO and Mavs -/- mice ( n = 10 each) were injected intravenously with HSV at 1.5 × 10 8 PFU per mouse, and the survival rates were monitored for 15 days (* p = 0.445). Data are representative of three independent experiments with similar results ( e ), or three independent experiments ( a – d , and h ) (mean ± SEM of three biological replicates). P -value was determined by two-tailed Student’s t -test, two-sided log-rank (Mantel-Cox) test ( f , g and i ). n.s. indicates no statistical significance. Source data are provided as a Source Data file.

Article Snippet: Concentrations of the IFN-β in cell culture medium and mouse serum were measured by Human Interferon β ELISA kit (Cusabio, CSB-E09889h) or Mouse Interferon β ELISA kit (Cusabio, CSB-E04945m) according to the manufacturer’s instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Sequencing, Staining, Injection, Two Tailed Test

Journal: BMC Veterinary Research

Article Title: Adjuvant screening of the Senecavirus A inactivated vaccine in mice and evaluation of its immunogenicity in pigs

doi: 10.1186/s12917-024-03949-5

Figure Lengend Snippet: Evaluation of the immunogenicity of the SVA inactivated vaccine in mice. ( A ) Neutralizing antibody responses of mice to SVA vaccine vaccination. ( B ) Total IgG antibody responses of mice to SVA vaccination. ( C ) IgG subtype detection. IgG1 antibody (left), IgG2a antibody (middle), and IgG2b antibody (right) responses of mice to SVA vaccination. ( D ) Concentration of IL-4 detected in mice. ( E ) Concentration of IFN-γ detected in mice

Article Snippet: Cytokine test kits for IL-4 and IFN-γ(mouse derived) were purchased from Invitrogen (Shanghai, China), and cytokine test kits for IL-4 and IFN-γ (pig derived) were purchased from Cloud-clone (Wuhan, China).

Techniques: Concentration Assay

Journal: BMC Veterinary Research

Article Title: Adjuvant screening of the Senecavirus A inactivated vaccine in mice and evaluation of its immunogenicity in pigs

doi: 10.1186/s12917-024-03949-5

Figure Lengend Snippet: Evaluation of the immunogenicity of the SVA inactivated vaccine in pigs. ( A ) Neutralizing antibody responses of pigs to vaccination with the SVA vaccine. ( B ) Total IgG antibody responses of pigs to vaccination with the SVA vaccine. ( C ) Concentration of IL-4 detected in pigs. ( D ) Concentration of IFN-γ detected in pigs

Article Snippet: Cytokine test kits for IL-4 and IFN-γ(mouse derived) were purchased from Invitrogen (Shanghai, China), and cytokine test kits for IL-4 and IFN-γ (pig derived) were purchased from Cloud-clone (Wuhan, China).

Techniques: Concentration Assay