Journal: The Journal of Experimental Medicine

Article Title: Role for NF-κB in herpes encephalitis pathology in mice genocopying an inborn error of IRF3-IFN immunity

doi: 10.1084/jem.20250064

Figure Lengend Snippet: Elevated proinflammatory cytokine levels in the brain of Irf3 R278Q/R278Q mice upon HSV-1 infection. (A–F) Expression of Ifnb , Mx1 , Tnfa , Il1b , Il6 , and Ccl2 in brain stems from WT or Irf3 R278Q/R278Q mice on days 1–4 after infection, measured by RT-qPCR. UI, uninfected control. WT, n = 5–7; Irf3 R278Q/R278Q , n = 5–12. (G–J) Mesoscale analysis of cytokine levels (TNFα, CCL2, and IFNγ) (G–I) and HSV-1 titer in brain stem homogenates on day 5 after infection quantified by plaque assay (J) (WT UI, n = 4, WT, n = 9; Irf3 R278Q/R278Q UI, n = 4; Irf3 R278Q/R278Q , n = 8). (K) Expression of Tnfa in brain stems from WT ( n = 7), Irf3 −/− ( n = 5), Irf3 R278Q/R278Q ( n = 9), and Irf3 WT/R278Q ( n = 6) mice on day 5 after HSV-1 infection, measured by RT-qPCR. (L–N) Ifnb, Tnfa , and Il6 response 16 h after infection with 1.0 MOI HSV-1 in MBCs treated with 0.5 μM PLX5622 (PLX) for microglia depletion. (O and P) Tnfa and Il6 response 16 h after infection with 1.0 MOI HSV-1 in MBCs treated with NF-κB inhibitors BMS-345541 (2 μM), PDTC (25 μM), or saline. UI controls were included. (Q and S) Ifnb and Tnfa mRNA levels in murine microglia from WT, Irf3 WT/R278Q , and Irf3 R278Q/R278Q mice 24 h after HSV-1 infection at increasing virus MOI as indicated. (R and T) IFNB1 and TNFA mRNA levels in iPSC-derived microglia from the IRF3 WT/R285Q HSE patient and controls 24 h after HSV-1 infection at increasing virus MOI as indicated. Expression data were normalized to β-actin and shown as fold change compared with the UI control. All in vitro experiments were performed in triplicates and independently repeated at least three times. Cytokine expression kinetics were compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype) (A–F), error bars; SEM. Statistical analyses of cytokine and virus levels in brain stem homogenates (G–J and L–T) were analyzed by two-tailed two-way ANOVA for difference of means, followed by an unpaired t test of means, error bars; SD. CNS cell experiments (K) were analyzed with a two-tailed one-way ANOVA for difference of means followed by an unpaired t test of means. P values <0.05 were considered statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001. UT, untreated; MOI, multiplicity of infection.

Article Snippet: Quantitative PCR was performed using the following TaqMan Gene Expression Assays (Applied Biosystems): ACTB (Hs01060665_g1), 18S (Hs03003631_g1), IFNB (Hs01077958_s1), IL6 (Hs00174131_m1), TNFA (Hs00174128_m1), IL1B (Hs01555410), MX1 (Hs00895598_m1), CXCL10 (Hs00171042), and ISG15 (Hs01921425). mRNA levels of interest were normalized to the housekeeping gene ACTB or 18S (as indicated) using the ΔΔCt method.

Techniques: Infection, Expressing, Quantitative RT-PCR, Control, Plaque Assay, Saline, Virus, Derivative Assay, In Vitro, Two Tailed Test

Journal: Nature Communications

Article Title: The potential of H5N1 viruses to adapt to bovine cells varies throughout evolution

doi: 10.1038/s41467-025-67234-1

Figure Lengend Snippet: a Identification of virus and Mx1 expressing cells by immunohistochemistry. Udder slices infected with r-Tx/37-B3.13. NP (brown) is visible in the epithelial layer lining the duct, while Mx1 is localised in the cytoplasm of ductal epithelial cells as shown by immunohistochemistry. Images are representative of experiments carried out in 8 different sections from two different animals. Scale bars, 100 μm. b Image showing detection of virus NP and Mx1 by immunofluorescence. NP and Mx1 show miminal colocalization in the duct epithelium. Scale bars, 20 μm. Immunofluorescence was performed on sections from the tissue in ( a ) as a final validation. c Western blot of lysates from A549 and BSF cells stably expressing human (MxA), bovine, chicken and rat Mx1. d Single-cycle infection of ZsGreen-expressing reporter viruses on modified A549 cells at 7 hpi. Titres normalised to Chicken Mx1 control cells. e, f A549 ( e ) or BSF ( f ) cells overexpressing Mx1 were infected at an MOI of 0.001 and infectious titres at 48 ( e ) or 24 ( f ) hpi were determined by plaque assay on MDCK cells. g As described in ( e, f ) except recombinant 4:4 viruses (Table ) were used and peak titres of each virus (24 h or 48 h) are shown. h Western blot of lysates from MDCK cells overexpressing human BTN3A3. i Virus titres on modified MDCK cells overexpressing human BTN3A3 or transduced with an empty vector control. j Alignment of NP residues at positions associated with Mx1 or BTN3A3 resistance. Residues are aligned to r-Bovine-B3.13 (bold). Changes associated with resistance are boxed (bold). The data in ( d –f ) and ( g, i ) are mean +/- s.d. three biological repeats (n = 3), except for ( f ) where n = 4. Data in ( e –g and i ) were log-transformed and statistical significance was determined by two-way ANOVA with Tukey’s ( d –f ) or Šídák ( g, i ) multiple comparisons tests (two-tailed; α = 0.05). Only comparisons relative to untreated or chicken Mx1 controls are shown. P-values in bold indicate statistical significance. Gating data for ( d ) can be found in Fig. .

Article Snippet: The primary antibodies used in this study are: PB2 - GeneTex (GTX125926), NP - MRC PPU Reagents and Services, Dundee (DA183, 5 th Bleed), NS1 – (MRC PPU Reagents and Services, Dundee (DA182, 2 nd Bleed), pSTAT1 (Tyr701) - Cell Signalling (9167S), IFIT1 - Origene (TA500948), RSAD2 - Proteintech (28089-1-AP), GAPDH - Cell Signalling (2118S), a-tubulin - Proteintech (66031-1-Ig), b-actin - Proteintech (66009-1-Ig), Mx1 - Proteintech (13750-1-AP) and puromycin (Millipore; MABE343).

Techniques: Virus, Expressing, Immunohistochemistry, Infection, Immunofluorescence, Biomarker Discovery, Western Blot, Stable Transfection, Modification, Control, Plaque Assay, Recombinant, Transduction, Plasmid Preparation, Transformation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: The potential of H5N1 viruses to adapt to bovine cells varies throughout evolution

doi: 10.1038/s41467-025-67234-1

Figure Lengend Snippet: Immortalised BSF were pre-treated for 24 h with increasing concentrations of type I IFN prior to low MOI infection (0.001 PFU/cell). a Western blot of interferon-stimulated gene expression (pSTAT1, Mx1, RSAD2) at 24 h post IFN-treatment. b Virus titres in supernatants were determined by plaque assay on MDCK cells at indicated times post-infection. c, d As in ( a ) and ( b ) but including r-Goose-B3.13. e, f Replication of r-Bovine-B3.13, r-Avian-B1.1 and reciprocal segment swaps in the absence or presence of universal IFN. BSF were pre- or mock-treated with 2 U/mL IFN for 24 h prior to infection at MOI of 0.001. Western blot for Mx1 at 24 hours post-treatment ( e ), viral titres at 24 hpi were determined by plaque assay on MDCK cells ( f ) . g , Immortalised udder skin fibroblasts expressing an ISRE-firefly luciferase reporter infected with 2.3.4.4b 2:6 viruses at an MOI of 5 PFU/cell. Luminescence was measured at 24 hpi and normalised to mock controls. h , Western blot of viral proteins and ISGs in udder fibroblasts infected at an MOI of 5 for 24 hours. Data are mean +/- s.d. from three biological replicates (n = 3); each point is an average of technical duplicates. In g , n = 6, error bars show s.d. For b, d and f , data were log-transformed; statistical significances in b, d and f between IFN treated and untreated groups was assessed by two-way ANOVA with Dunnett’s ( b and d ) or Šídák ( f ) multiple comparisons tests (two-tailed; α = 0.05). For g , comparisons were determined by one-way ANOVA with Tukey’s test (two-tailed; α = 0.05). For b, d and f , P values in bold indicate statistical significance. For g , only significant values are shown.

Article Snippet: The primary antibodies used in this study are: PB2 - GeneTex (GTX125926), NP - MRC PPU Reagents and Services, Dundee (DA183, 5 th Bleed), NS1 – (MRC PPU Reagents and Services, Dundee (DA182, 2 nd Bleed), pSTAT1 (Tyr701) - Cell Signalling (9167S), IFIT1 - Origene (TA500948), RSAD2 - Proteintech (28089-1-AP), GAPDH - Cell Signalling (2118S), a-tubulin - Proteintech (66031-1-Ig), b-actin - Proteintech (66009-1-Ig), Mx1 - Proteintech (13750-1-AP) and puromycin (Millipore; MABE343).

Techniques: Infection, Western Blot, Gene Expression, Virus, Plaque Assay, Expressing, Luciferase, Transformation Assay, Two Tailed Test