Identification,Expression Profiles And Antiviral Activities Of A Type?IFN From Gibel Carp Carassius Auratus Gibelio

Gibel carp?Carassius auratus gibelio?is one of the major fish species in freshwater aquaculture in China.Cyprinid herpesvirus 2?CyHV-2?has been identified as the causative agent of the disease named haematopoietic necrosis in gibel carp and has led to huge economic losses in aquacultural industry.The study of the antiviral defense mechanism of gibel carp has great practical significance for the control of the viral disease in future.As important secreted cytokines,interferons?IFNs?have intricate functions in both innate and adaptive immunity,and they can inhibit the propagation of virus and regulate the immune response.To date,type I interferons?IFN-I?have been studied in a variety of teleost fish,but the research on gibel carp IFN-I have not been carried out in depth.In this study,a novel type I IFN of gibel carp?CagIFNa?was identified and characterized.The expression patterns of CagIFNa gene both in vivo and in vitro were profiled,and the antiviral activities of CagIFNa against CyHV-2 in gibel carp brain cells?GiCB?were investigated.The study herein aims to further reveal the fish immune defense system and the innate antiviral mechanism,which will lay a theoretical foundation for the effective prevention and control of the herpesviral haematopoietic necrosis in gibel carp.The main results are as follows:1.Cloning and sequence structure analysis of CagIFNaBased on RACE PCR amplification technology,the full-length cDNA sequence of CagIFNa gene was cloned from total RNA that extracted from the gibel carp kidneys.The full-length cDNA of CagIFNa is 724 bp.The length of predicted ORF is552 bp and encodes a CagIFNa protein of 183 amino acids,in which the first 23amino acids were identified as a signal peptide.And,there are two highly conserved cysteine residues(C1:C²⁴,C2:C¹²⁰)in the deduced protein.Amino acid sequence similarity analysis showed that CagIFNa had the highest similarity to Cyprinidae IFN.Phylogenetic tree analysis showed that CagIFNa was clustered with other piscine IFNa subgroup genes of group I type I IFNs.2.Expression profiles of CagIFNa in vivoReal-time PCR results indicated that CagIFNa was broadly expressed in nine examined tissues and highly expressed in the liver,intestine,spleen and head kidney of healthy gibel carp.Following CyHV-2 infection,CagIFNa expression level significantly decreased in the head kidney at 12,24,and 48 h but dramatically increased up to 6-fold higher at 120 h post-injection compared to sham-infected controls.In the spleen,CagIFNa expression level significantly decreased at 24,48and 120 h post CyHV-2 injection.3.Expression profiles of CagIFNa post poly I:C stimulation and CyHV-2infection in vitroAfter stimulation of GiCB cells with poly I:C,real-time PCR results indicated that the expression level of CagIFNa was rapidly up-regulated at 6 h,peaked the highest level at 12 h with a 347-fold induction and subsequently down-regulated,but the expression level was still higher than that in control group.After CyHV-2infection,the expression level of CagIFNa was significantly up-regulated at 6 h and then down-regulated,but at 120 h,the expression was significantly up-regulated to the highest level?10.7-fold?compared with control group.4.Antiviral activities of CagIFNaConstructing the expression plasmid of pCMV-HA-CagIFNa and transfecting the GiCB cells,then western blot analysis confirmed that CagIFNa protein could be synthesized and secreted extracellularly.After infection with CyHV-2,the CPE in pCMV-HA-CagIFNa transfected GiCB cells was significantly decreased compared with control group.And the viral titer was remarkably inhibited at 24,48 and 120 h post CyHV-2 infection,showing a 6.8,18.6 and 69.2-fold inhibition compared with control group,respectively.Meanwhile,real-time PCR results showed that the expression levels of viral genes CTP,MCP and TK were significantly decreased in tested group at 24,48 and 120 h post CyHV-2 infection compared to control group.CagIFNa can effectively prevent CyHV-2 infection.5.Activation of type I IFN signaling pathway associated genes by CagIFNaReal-time PCR results showed that the expression of a series of genes associated with IFN-I system including IRF3,IRF7,IRF9,STAT1,Mx1 and PKR were significantly induced in CagIFNa-overexpressed GiCB cells infected with CyHV-2.CagIFNa can effectively induce the expression of IFN-I system related genes to promote the activation of host antiviral immune response.