Antiviral potential and stability analysis of chicken interferon-α produced by Newcastle disease virus in chicken embryo fibroblast cells

Abstract

Chicken interferon-α (chIFN-α) is an important antiviral cytokine and represents one of the first lines of the chicken's innate immune system. The current study is the first-ever report of chicken IFN (chIFN) production in Pakistan. In this study, we have used live and UV-irradiated Newcastle disease virus (NDV) to induce the expression of chIFN-α in chicken embryo fibroblast (CEF) cells. ChIFN-α was partially purified in a two-step protocol; ultracentrifugation followed by treatment with anti-chIFN-β antibodies. The purified chIFN-α was ana-lysed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the in vitro antiviral potential of chIFN-α was determined against the H9N2 avian influenza virus (AIV) via a cytopathic inhibition assay. The relative mRNA level of the IFN-stimulated genes (ISGs) in the IFN-stimulated CEF cells was measured at various time intervals by a quantitative polymerase chain reaction (qPCR). The stability of natural chIFN-α to the temperature, pH, and ultraviolet (UV) light was also determined. The in vivo therapeutic potential of chIFN-α was determined in 7-day-old broiler chickens challenged with AIV. We found that a higher chIFN-α expression level was induced by the UV-irradiated NDV in the CEF cells as compared to the live NDV. The UV-irradiated NDV induced the maximum IFN production in the CEF cells at 24 h post-infection. Two bands of 21 kDa on SDS-PAGE confirmed the presence of the chIFN-α protein. The cytopathic inhibition assay indicated the strong antiviral activity of chIFN-α against AIV. Our results of the stability analysis showed that chIFN-α was stable at a wide range of temperatures and pH levels. However, a little exposure to UV-light resulted in a significant loss of antiviral activity. We also observed that the antiviral activity of chIFN-α is related to the expression levels of the antiviral ISGs. The results of the in vivo study showed that the chIFN-α therapy via the oral route resulted in a significant improvement in the tracheal pathology of chickens challenged with AIV. In conclusion, we suggest that chIFN-α could be an important therapeutic tool to control avian influenza infection in poultry.

Keywords: IFN-stimulated genes; Newcastle disease virus; antiviral; chicken embryo fibroblast; chicken type I IFNs; cytopathic inhibition assay; innate immunity.

Figure 1. (A) Normal cell morphology of the chicken embryo fibroblast (CEF) cells at 24 h observed under an inverted microscope (× 100). (B) Crude chIFN-α yield in the cell culture supernatant harvested at 6, 12, 24, 36, and 48 h post-stimulation of the CEF cells by the live and UV-irradiated NDV

Figure 2. SDS-PAGE analysis of the protein fractions containing IFN

L1 is the protein ladder, while L3 and L4 represent the 21 kDa protein bands of chIFN-α obtained after the purification of the CEF supernatant while L2 indicates the blank control

Figure 3. No cytopathic effects (CPEs) were observed in the CEF cells pre-treated with chIFN-α in comparison to the untreated CEF, i.e., positive control

Protection from CPEs was observed in the cells pre-treated with chIFN-α compared with the non-treated cells that were infected with AIV only

Figure 4. The relative mRNA levels of the IFN-stimulated genes assayed by qPCR at various time intervals post-stimulation by chIFN-α

After the CEF cells were incubated with chIFN-α, the CEF cells were harvested at the indicated time intervals for the RNA extraction and cDNA preparation. The transcriptional levels of PKR (B), Mx (C), 2'-5' OAS (D), and STAT1 (E) were assayed by qPCR

Figure 5. Effect of the temperature, pH and UV-light on the biological activity of chIFN-α produced by NDV in the CEF cells

Figure 6. H&E stained histological micrograph (× 100) of the trachea collected from various chicken groups of the in vivo study

Ep = epithelium; sm = submucosa; tc = tracheal cartilage Group A = AIV challenged + chIFN-α therapy (500 IU for 3 consecutive days); Group B = AIV challenged + chIFN-α (1 000 IU for 3 consecutive days); Group C = AIV challenged only (positive control); Group D = PBS treatment only (negative control) A mild disruption of the epithelium and submucosa was seen in the trachea of the group A birds while the trachea of the group B birds exhibited a normal tracheal histology. Severe degenerative changes, i.e., mucosal sloughing of the epithelium and submucosal oedema along with the infiltration of inflammatory cells were observed in the virus challenged group C birds. A normal trachea with mucosal epithelium, cilia and plates of cartilage was observed in the group D birds. The submucosa contains normal glands and connective tissue