The Structure And Characteristics Of The Domestic Pig Type I Interferon Gene Family Of Interferon-alpha On The Foot And Mouth Disease Genetically Engineered Vaccine Enhancement,

The availability of data on the pig genome sequence prompted us to characterize the porcine IFN-α(PoIFN-α) multigene family.Fourteen functional PoIFN-αgenes and two PoIFN-αpseudogenes were detected in the porcine genome. Multiple sequence alignment revealed a C-terminal deletion of eight residues in six subtypes.A phylogenetic tree of the porcine IFN-αgene family defined the evolutionary relationship of the various subtypes.In addition,analysis of the evolutionary rate and the effect of positive selection suggested that the C-terminal deletion is a strategy for preservation in the genome.Eight PoIFN-αsubtypes were isolated from the porcine liver genome and expressed in BHK-21 cells line.We detected the level of transcription by real-time quantitative RT-PCR analysis.The antiviral activities of the products were determined by WISH cells/Vesicular Stomatitis Virus(VSV) and PK 15 cells/Pseudorabies Virus(PRV) respectively.We found the antiviral activities of intact PoIFN-αgenes are approximate 2-50 times higher than those of the subtypes with C-terminal deletions in WISH cells and 15-55 times higher in PK 15 cells.There was no obvious difference between the subtypes with and without C-terminal deletion on acid susceptibility.For investigating the virus-inducing expression profile of PoIFN-αsubtypes,the expression of PoIFN-αwas detected using the two PCR strategies in three systems,namely,the Poly(I). Poly(C)-DEAE-dextran-induced PK 15 cells,the Pseudorabies Virus-infected PK 15 cells and the infected PK 15 cells with attenuated strain of Swine Fever Virus respectively.In Poly(I).Poly(C)-DEAE-dextran induced PK 15 cells,the expression of IFN-α2,-α3,-α4,-α8,-α9 after 6h/24h inducement in PK 15 cells were observed. In Pseudorabies Virus-infected PK 15 cells,the expression of PoIFN-α2,-α3,-α8,-α9, -α10,and -α13 was observed after 6h/24h infection and in attenuated strain of Swine Fever Virus-infected PK 15 cells,the upregulation of PoIFN-α2,-α3,-α4,-α8,-α9, and -α10 was detected.The results of Real-time quantitative PCR analysis suggested that the expression was time-dependent in Pseudorabies Virus/Poly(I). Poly(C)-DEAE-dextran induced PK 15 cells.But in attenuated strain of Swine Fever Virus-infected PK 15 system,the expression level of IFN-αsubtypes was not obviously time dependent. From the data of the pig genome sequence,we obtained a group of new interferon-like sequences,which have intact ORF to code 167AA-184AA peptides. The peptides have 23%-59%amino acid identities to the typeⅠinterferon as known. The prediction of conserved domain suggested those interferon-like peptides possess the typeⅠinterferon conserved domain,and then the phylogenetic analysis showed that those sequences have close relationship with mouse IFN-ζand porcine IFN-δ. Therefore,we classified those peptides to typeⅠinterferon family and nominated them as interferon-θ.IFN-θmultigene family consists of eight subtypes,including five functional subtypes and three pseudogenes.In functional genes,IFN-θ2/-θ3 and IFN-θ4/-θ5 have high sequence homology respectively.However,IFN-θ1 has about 80%sequence similarity to the other four subtypes.In this work,we designed the PCR primers and obtained the five IFN-θfunctional genes from porcine liver genomic DNA.Three subtypes(IFN-θ1,IFN-θ2,IFN-θ5) were chosen to be inserted into the pcDNA4 vactor,and then the reconstructed plasmids were used to transfect BHK-21 cell lines.After 48hr inoculation,we collected the supernatants to determine the antiviral activity of IFN-θ.The results showed that all IFN-θs possess antiviral activities in PK-15 and IBRS-2 porcine kidney cell lines,but the three IFN-θsubtypes had obvious difference in antiviral activity.For investigating the virus-inducing expression profile of IFN-θsubtypes,we induced IBRS-2 cell using PRV,attenuated strain of SFV and Poly IC.After 6hr and 18hr inducing,we detected the up-regulation of all IFN-θmRNA using quantitative RT-PCR analysis.The receptor analysis and ISGs gene inducing analysis suggested that IFN-θs especially bind to extra-cellular domain of IFNαR and activate the JAK/STAT signaling pathway to up-regulate ISGs expression.At last,we analyzed the cytokines expression after IFN-θinducement.The results showed that IFN-θstimulate the expression of IL-6,IL-12 and IFN-γ,which suggested that IFN-θcould have the regulative function to immune system.On the basis of our results,IFN-θnot only has the sequence homology to other typeⅠinterferons,but also matches the primary properties of typeⅠinterferons in antiviral, IFNαR receptor binding and virus inducing,etc.So we figured that IFN-θis a new subtype of typeⅠinterferon family. The adjuvant effect of porcine interferon alpha(PoIFN-α) was examined in swine vaccinated with a recombinant FMD protein vaccine named IgG-FMDV, which contains the swine IgG single heavy chain constant region and an immunogenic peptide of serotype O FMDV.The PoIFN-αgene was cloned into pcDNA3 vector and the recombinant plasmid was incorporated into cationic liposomes by a dehydration and rehydration procedure to use as an adjuvant,injected together with low-dose IgG-FMDV.This procedure resulted in strong induction of FMDV-specific neutralizing antibody and significant T cell-mediated immune responses,whereas only a modest humoral and cellular response was observed with low dose vaccine alone.As an adjuvant for the protein vaccine,PoIFN-αinduced strong inflammatory cytokine production in vivo and the results denoted that IFN-adjuvant and our vaccines could drive the immune response toward Th1 type responses.The data of ELISA suggests that the recombinant protein vaccine synergizes with the IFN-adjuvant to produce endogenous IFN in vivo.In response to viral challenge,all control animals developed viremia and lesions,whereas all animals received IFN-adjuvant+IgG-FMDV were protected and nonstructural protein antibody in this group could not be detected by 14 days post-challenge(dpc).Our studies indicate that porcine IFN-αis a powerful adjuvant for recombinant FMD protein vaccine and could aid in vaccination against FMDV in swine.