Utilizing RNA-Seq To Characterize The Chicken Interferon Lambda Mediated Immune Signaling In Organs And Cells Of Chicken

Baculovirus expression vector system is an efficient,time saving,cost effective and mass-scale expression system.It is wildly employed to express foreign proteins especially eukaryotic proteins.The most extensively utilized and deeply researched baculoviruses are Bombyx mori nucleopolyhedrovirus(BmNPV)and Autographa Califomica nucleopolyhedrovirus(AcNPV).Moreover,the Baculoviruscell/larvae expression systems based on BmNPV and AcNPV are wildly used in research and industry.Interferons(IFNs)are pleiotropic cytokines that establish the first line of defense against viral infections in vertebrates.Several types of IFN have been identified;however,limited information is available in poultry,especially using live animal experimental models.IFN-lambda(IFN-λ)has recently been shown to exert a significant antiviral impact against viral pathogens in mammals.In order to investigate the in vivo and in vitro potential of chicken IFN-λ(chIFN-λ)as a regulator of innate immunity,and potential antiviral therapeutics,we expressed,characterized and profiled the chIFN-λ-stimulated immune response in chicken immune organs(in vivo)and compared it with primary chicken embryo fibroblasts(in vitro).First of all,we tried to express the chicken interferon lambda gene in silkworm baculovirus expression vector system by employing a novel defective-rescue BmBac(reBmBac)approach.In order to assure the successful construction and expression of reBmBac,the luciferase reporter assay was used as a reporter gene.Secondly,after successful expression,we attempted to characterize the antiviral role of recombinant interferon lambda against Newcastle disease virus in vitro.Thirdly,using the advanced tools of deep sequencing(RNA-Seq),we attempted to elaborate the possible signaling pathways in the cascade of chIFN-λ-induced immune signaling in both primary chicken fibroblasts(CEF)and immune organs of a live chicken.Briefly,we were able to identify an array of IFN-regulated genes in both CEF and immune organs.The transcriptional profiling of immune organs of the chicken(bursa of Fabricius and thymus)using RNA-Seq and subsequent bioinformatics analysis(Gene Ontology,differentially expressed genes(DEGs),and KEGG analysis)demonstrated an upregulation of fundamental immune-related genes(viperin,IKKB,CCL5,IL1β,and AP1)as well as the potent antiviral signaling pathways.Interestingly,this experimental approach revealed contrasting evidence of the antiviral potential of chIFN-λ in both in vivo and in vitro models.Taken together,our data signifies the potential of chIFN-λ as a potent antiviral cytokine and highlights its future possible use as an antiviral therapeutic in poultry.