Molecular cloning and antiviral function analysis of pigeon interferon lambda(piIFN-λ)

Fig. 1. Multiple sequence alignment of piIFN-λ with avian and mammalian IFN-λ Proteins. The amino acid sequence of IFN-λ was aligned with IFNλ sequences from: shearwater (XP_075010086.1), chicken (ABU82742), duck (AII23257), goose (KX377964), human (AAI30315), and mouse (AAX58715). The alignment was performed using CLUSTAL OMEGA and edited with the ESPript program.

Fig. 2. Phylogenetic analysis and predicted structure of piIFN-λ. (A) Homology comparison of IFN-λ amino acid sequences from different species. (B) A neighbor-joining phylogenetic tree of IFNλ was constructed using MegAlign software, based on IFNλ sequences obtained from the following species: shearwater (XP_075010086.1), chicken (ABU82742), turkey (XP_003207426), goose (KX377964), duck (AII23257), ferret (XP_004810653), pig (NP_001159962), cat (XP_047707765), human (AAI30315), and mouse (AAX58715). (C) Predicted structure of piIFN-λ based on homology modeling.

Fig. 3. Expression, purification, and identification of recombinant piIFN-λ. (A) Coomassie-stained SDS-PAGE analysis of purified recombinant piIFN-λ. Lane Marker: protein molecular weight marker; Lane 1: purified piIFN-λ (∼21 kDa). (B) Western blot analysis of recombinant piIFN-λ expression and purification using anti‑His antibody. Lane 1: recombinant piIFN-λ expressed as inclusion bodies; Lane 2: refolded protein from inclusion bodies; Lane 3: recombinant piIFN-λ purified by Ni‑NTA affinity chromatography; Lane 4: dialyzed protein sample after imidazole removal.

Fig. 4. Antiviral activity of recombinant piIFN-λ against VSV in DF-1 cells. DF-1 cells were pretreated with fourfold serial dilutions of recombinant piIFN-λ (starting concentration: 1.7 mg/mL) for 24 h, followed by infection with VSV at 100 TCID50. Representative images show CPE at dilutions of (A) 4⁻⁴, (B) 4⁻⁵, (C) 4⁻⁶, and (D) 4⁻⁷. (E) Negative control: cells treated with PBS only (no virus). (F) Positive control: cells treated with PBS and infected with VSV. All images were acquired at a total magnification of 200 × (20 × objective).

Fig. 5. Antiviral activity of recombinant piIFN-λ against H9N2 AIV in DF-1 cells. DF-1 cells were pretreated with fourfold serial dilutions of recombinant piIFN-λ (starting concentration: 1.7 mg/mL) for 24 h, followed by infection with H9N2 AIV at 100 TCID50. Representative images show CPE at dilutions of (A) 4⁻⁴, (B) 4⁻⁵, (C) 4⁻⁶, and (D) 4⁻⁷. (E) Negative control: cells treated with PBS only (no virus). (F) Positive control: cells treated with PBS and infected with H9N2 AIV. All images were acquired at a total magnification of 200 × (20 × objective).

Fig. 6. Quantitative antiviral activity of recombinant piIFN‑λ against VSV‑GFP and H9N2 AIV in DF‑1 cells. DF‑1 cells were pretreated with 1000 U/mL recombinant piIFN‑λ or PBS (mock control) for 24 h, then infected with 100 TCID50 of VSV‑GFP or H9N2 AIV. At 24 h post‑infection, progeny virus titers in culture supernatants were determined by TCID₅₀ assay, and viral protein expression in cell lysates was analyzed by Western blot with GAPDH as the loading control. (A) VSV‑GFP titers; (B) H9N2 AIV titers; (C) Western blot analysis of viral GFP protein levels; (D) Western blot analysis of H9N2 AIV NP protein levels.

Fig. 7. Activation of ISRE and Mx promoters by piIFN-λ and chIFN-α in DF-1 cells. DF-1 cells were co‑transfected with pGL3‑chISRE‑Luc or pGL3‑chMx‑Luc reporter plasmids along with pRL‑TK as an internal control. At 24 h post‑transfection, cells were treated with PBS (mock control), 100 U/mL or 1000 U/mL of recombinant piIFN‑λ (A, B) or the positive control chIFN‑α (C, D). Luciferase activity was measured 12 h post‑treatment and normalized to Renilla luciferase activity. (A) ISRE promoter activation by piIFN‑λ; (B) Mx promoter activation by piIFN‑λ; (C) ISRE promoter activation by chIFN‑α; (D) Mx promoter activation by chIFN‑α.

Fig. 8. Induction of ISGs by piIFN‑λ and chIFN‑α in DF‑1 cells. DF‑1 cells were treated with PBS (mock control), 100 U/mL or 1000 U/mL of recombinant piIFN‑λ (A‑C) or the positive control chIFN‑α (D‑F) for 24 h. The mRNA levels of Mx, OAS, and PKR were quantified by qRT‑PCR and normalized to GAPDH. (A) Mx expression (piIFN‑λ); (B) OAS expression (piIFN‑λ); (C) PKR expression (piIFN‑λ); (D) Mx expression (chIFN‑α); (E) OAS expression (chIFN‑α); (F) PKR expression (chIFN‑α).