Establishment And Application Of Molecular Fluorescence Quantitative PCR Detection Method For Chicken Innate Immune

Innate immunity is the first line of host resistance to virus infection. Chicken innate immune cells identify virus infection through pattern recognition receptors, and activate related signaling pathway, which further induce many kinds of cytokines and inflammatory mediators, and eventually lead to the secretion of antiviral substance. Innate immunity functions in a few minutes to a few hours after exposure to harmful stimulus without the participation of specific immune cells and antibodies, and plays important role in host defense at the early stage of viral infection.The aim of this study is to develop real-time PCR methods for the detection of chicken innate immune-related molecules. For this purpose, we designed and screened primers for the amplification of twelve genes(MDA5?TLR3?TLR7?IFN-??IFN-??IFN-??IL-1??IL-2?IL-6?IFIT5?PKR and OAS). Specific PCR products of the genes were utilized to construct recombinant plasmids, which served as template standards to establish standard curves for the assays. The assays developed in this study were sensitive, specific and could stably detect related genes of concentrations ranging from 109 to several copies.Newcastle disease virus(NDV) is the cause of Newcastle disease(ND), which is one of the main hazards in poultry industry, and causes serious economic losses. For a better understanding of the pathogenesis of NDVs, SPF chicken embryos were infected with different doses of a virulent strain in genotype VII of Class II, and an avirulent vaccine strain-LaSota, respectively. At the early stage of NDV infection, the dynamics of viral proliferation and the transcriptional level of innate immune-related molecules were detected with fluorescence quantitative PCR methods established in this study. The results showed that both the virulent and avirulent NDV strains could activate the innate immune signaling pathways, and resulted in the transcriptional changes of genes encoding cellular pattern recognition receptors, interferons, interleukins and antiviral proteins. NDV infection of high doses in comparison with low doses resulted in quicker viral proliferation and earlier peaks of innate immune-related gene expression. The virulent NDV strain induced higher levels of interferons and inflammatory cytokines in chickens, which is consistent with the severe clinical symptoms and pathological damages caused by virulent viral infection. In conclusion, this study found that the response pattern of chicken innate immune signaling pathway varied chicken embryo cells infected with NDV response patterns of the due to virus infection titer and virulence vary, and the genotype VII NDV strain could induce strong cellular inflammatory responses, which may contribute to its high pathogenicity to chickens.