Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: PHEV infection activates the innate immune response. ( A ) Viral load in subcultures harvested at different time points post-infection was quantified by qRT-PCR targeting the viral N gene. All the experiments were performed in triplicate. ( B ) IFN-β levels in PHEV-infected samples were determined by ELISA. Cells infected with VSV (MOI = 1) or treated with Poly(I:C) (20 μM, 24 h) served as positive controls. ( C ) QRT-PCR analysis of IFNA and IFNB1 mRNA expression in N2a cells at various time points (0–48 h) post-PHEV infection. ( D ) QRT-PCR analysis of ISGs (Mx, OAS, GBP, STAT) expression in N2a cells at 24 and 48 h post-PHEV infection. ( E ) QRT-PCR analysis of RIG-I, MDA5, IRF3, and IRF7 expression in N2a cells at various time points (0–48 h) post-PHEV infection. ( F ) WB analysis of RIG-I, IRF7, MAVS, and viral N protein levels in N2a cells at indicated times (0–48 h) after PHEV infection. ( G ) Detection of PHEV, RIG-I, IRF3, IRF7, and IFNB1 in brain tissues from mice at 5 days post-PHEV infection. Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test).

Article Snippet: The IFN-β recombinant protein was purchased from MCE.

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: IRF3 initiates the interferon response to suppress viral replication. ( A ) Inhibition of PHEV replication by recombinant IFN-β. WB analysis of viral protein levels to evaluate the suppression of PHEV replication by recombinant IFN-β (0.5 or 1 μg/mL). ( B ) QRT-PCR analysis of IRF3 mRNA in N2a cells overexpressing Myc-tagged IRF3 (1, 2, or 4 μg) for 24 h, followed by 24 h PHEV infection. ( C ) QRT-PCR analysis of IRF7 mRNA in N2a cells overexpressing Myc-tagged IRF7 (1, 2, or 4 μg) for 24 h, followed by 24 h PHEV infection. ( D ) QRT-PCR analysis of PHEV mRNA expression as described in panels B and C . ( E ) WB analysis of PHEV N protein as described in panels B and C . ( F ) QRT-PCR analysis of IFNB1 mRNA expression as described in panels B and C . Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test). ns, not significant.

Article Snippet: The IFN-β recombinant protein was purchased from MCE.

Techniques: Inhibition, Recombinant, Quantitative RT-PCR, Infection, Expressing, Two Tailed Test

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: PHEV N protein targets IRF3 to suppress IFN production. ( A ) Schematic illustration of the experimental design for the dual-luciferase assay using HT1080 cells with a stably integrated IFN-β promoter reporter. ( B ) PHEV N protein inhibits the IFN-β promoter. HT1080 cells were transfected with PHEV proteins, cultured for 24 h, and then stimulated with Poly(I:C) (20 μM) for another 24 h to induce IFN-β promoter activity. IFN-β-Luc reporter activity is normalized to that of Renilla luciferase and shown. Detection of viral protein expression by WB. ( C ) WB analysis demonstrated that the protein levels of RIG-I and IRF3 in N2a cells were unaltered by transfection with a gradient of N protein (0.5–4 μg). ( D ) Nuclear-cytoplasmic fractionation. N2a cells were transfected with 2 μg GFP-N recombinant plasmid for 24 h, and then treated with Poly(I:C) (20 μM) for 24 h or infected with VSV (MOI = 1) for 12 h. Cells were collected for cytoplasmic and nuclear isolation. WB analysis of IRF3 nuclear translocation using cytoplasmic and nuclear fractions prepared from harvested cells. ( E ) N2a cells were infected with PHEV for 24 h (with Poly(I:C) treatment (20 μM, 24 h) as a positive control), followed by immunostaining with anti-N (red) and anti-IRF3 (green) antibodies. Nuclei were counterstained with Hoechst (blue). Scale bar, 10 μm. Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test).

Article Snippet: The IFN-β recombinant protein was purchased from MCE.

Techniques: Luciferase, Stable Transfection, Transfection, Cell Culture, Activity Assay, Expressing, Fractionation, Recombinant, Plasmid Preparation, Infection, Isolation, Translocation Assay, Positive Control, Immunostaining, Two Tailed Test