Anjum Faisal Rasheed, Ur Rahman Sajjad, Aslam Muhammad Aamir, Qureshi Anas Sarwar
Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan.
Department of Anatomy, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan.
Vet Med (Praha). 2021 May 3;66(5):197-207. doi: 10.17221/106/2020-VETMED. eCollection 2021 May.
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 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 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 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.
鸡α干扰素（chIFN-α）是一种重要的抗病毒细胞因子，是鸡先天免疫系统的第一道防线之一。本研究是巴基斯坦首次关于鸡干扰素（chIFN）产生的报告。在本研究中，我们使用活的和紫外线照射的新城疫病毒（NDV）诱导鸡胚成纤维细胞（CEF）中chIFN-α的表达。chIFN-α通过两步法进行部分纯化；先进行超速离心，然后用抗chIFN-β抗体处理。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析纯化的chIFN-α，并通过细胞病变抑制试验测定chIFN-α对H9N2禽流感病毒（AIV）的抗病毒潜力。通过定量聚合酶链反应（qPCR）在不同时间间隔测量IFN刺激的CEF细胞中IFN刺激基因（ISG）的相对mRNA水平。还测定了天然chIFN-α对温度、pH和紫外线（UV）的稳定性。在感染AIV的7日龄肉鸡中测定chIFN-α的治疗潜力。我们发现，与活的NDV相比，紫外线照射的NDV在CEF细胞中诱导了更高水平的chIFN-α表达。紫外线照射的NDV在感染后24小时在CEF细胞中诱导了最大的IFN产生。SDS-PAGE上的两条21 kDa条带证实了chIFN-α蛋白的存在。细胞病变抑制试验表明chIFN-α对AIV具有强大的抗病毒活性。我们的稳定性分析结果表明，chIFN-α在广泛的温度和pH水平下是稳定的。然而，少量暴露于紫外线会导致抗病毒活性显著丧失。我们还观察到chIFN-α的抗病毒活性与抗病毒ISG的表达水平有关。研究结果表明，通过口服途径给予chIFN-α可显著改善感染AIV的鸡的气管病理状况。总之，我们认为chIFN-α可能是控制家禽禽流感感染的重要治疗工具。
