IFNb of black carp functions importantly in host innate immune response as an antiviral cytokine

Type I interferons (IFN-Is) play an important role in the antiviral immune response in teleost fishes. In this study, one type I interferon (bcIFNb) from black carp (Mylopharyngodon piceus) has been cloned and characterized. The full-length cDNA of bcIFNb gene consists of 806 nucleotides and the predicted bcIFNb protein contains 188 amino acids. Basing on the cysteine number and evolutionary position, bcIFNb was classified into group II type I IFN. q-PCR analysis demonstrated that bcIFNb mRNA level varied in vivo and ex vivo in response to different stimuli. bcIFNb was detected in both the whole cell lysate and the supernatant media of HEK293T cells or EPC cells transfected with bcIFNb through immunoblot assay. IFN stimulated genes (ISGs) were greatly upregulated when the host cells were treated with the bcIFNb-containing conditioned media. EPC cells showed greatly enhanced antiviral ability when the cells were transfected with bcIFNb or treated with the bcIFNb-containing conditioned media before GCRV or SVCV infection. Glycosidase digestion analysis determined that bcIFNb was modified with N-linked glycosylation, which occurred on the Asn (N) of 92 site of this cytokine. The un-glycosylated mutant bcIFNb-N92Q presented the similar antiviral ability as that of wild type bcIFNb, which demonstrated that N-linked glycosylation did not contribute directly to the antiviral property of this fish cytokine.