| Newcastle disease(ND),caused by virulent Newcastle disease virus(NDV),seriously threatens the poultry industry.Interferons(IFNs)are key factors of the host's natural immunity.After NDV infecting the host,the cell's pathogen recognition receptor(PRR)recognizes pathogen-related molecular patterns(PAMPs)and binds to the adaptor protein(MAVS)in the outer mitochondrial membrane activating TANK binding kinase 1(TBK1).Since chicken lacks IFN regulator IRF3,TBK1 intends to activate IFN regulator IRF7.The activated IRF7 translocates to the nucleus and combines with ISRE inducing the expression of type I interferonand activating the expression of a large number of ISGs through the JAK/STAT pathway,including chicken-derived interferon stimulating gene 12-2(Interferon-stimulated gene 12(2),ISG12(2)),which plays an anti-viral effect.Our previous results found that NDVcould significantly up-regulate the expression of ISG12(2)after infecting chickens and confirmed that ISG12(2)could inhibit the replication of NDVand IFNs also stimulated the ISG12(2)expression in chicken embryo fibroblasts cell(DF1).Interestingly,ISG12(2)could also promote the expression of a series of interferon and interferon signal pathway molecules.In this study,firstly by chimerizing ISG12(2)onto the NDV attenuated vector LaSota,and then using the NDV reverse genetic operating system skillfully applied in our laboratory,the ISG12(2)chimeric strain was successfully rescued and the chimerism was verified.The strain not only has NDV activity,but also successfully expresses ISG12(2)protein.Through detection of MDT and ICPI,it infects 6-week-old low-antibody chickens.The experimental results show that the ISG12(2)chimeric strain can significantly reduce the pathogenicity of the virus.Finally,the rescued ISG12(2)chimeric strain was used to immunize 6-week-old low-antibody chickens.The HI antibody titer can reach the immune protection line above 24,which can play an immune protection effect.After three weeks of immunization,the virulent strain F48E9 is used to challenge the virus.Chickens can survive 100%.The in vitro detoxification results also show that the ISG12(2)chimeric strain can significantly shorten the detoxification time.The transcriptome results show that the ISG12(2)chimeric strain can not only activate humoral immunity,but also activate cellular immunity.Better immune protection effect.In summary,ISG12(2)LaSota chimeric strain can stimulate the body to produce better immune protection effects,and ISG12(2)has the ability to act as a molecular adjuvant.The main research content and results of this paper are as follows:1.Research on the mechanism of ISG12(2)positive regulation of IFNsIn order to preliminarily explore how ISG12(2)regulates the expression of IFN and its pathway related factors,the plasmid pCMV-ISG12(2)-EGFP that can express both ISG12(2)protein and EGFP protein was first constructed.plasmid could inhibit the replication of NDV and up-regulate the expression of IFN and its pathway-related factors.In order to test which pathway involvs ISG12(2)inducing IFN expression,The transcriptome sequencing was employed.The plasmid was first transfected into DF-1 cells,36 h later,cells expressing ISG12(2)protein were sorted by flow cytometry,RNA was extracted,and transcriptome sequencing was performed.The results showed that,comparing control group,,compared with the control group that did not express ISG12(2),there were a total of 1327 differentially expressed genes in gourp of over-expression of ISG12(2),of which 709 were up-regulated and618 were down-regulated,and KEGG pathway enrichment analysis found that the main enrichment pathways were about immune pathway,such as m TOR signaling pathway,TGF-?signaling pathway,Toll-like receptor signaling pathway,cytokine interaction signaling pathway,influenza A pathway and so on.The differentially expressed genes,IFNGR1,IL1R2,IFNAR2,IFNAR1,GHR,CXCR1,CXCL12,CSF1,SMURF2,ACVR2 A,SKP1 and RPS6KB1,were selected for q RT-PCR verification.The q RT-PCRresults were consistent with the transcriptome sequencing results,indicating that ISG12(2)was indeed involving in immunity,suggesting that it has the function of activating natural immunity and enhancing adaptive immunity.2.Rescue of ISG12(2)chimeric NDV strain and Determination determination of viral biological characteristicsThe ISG12(2)was cloned into full-genome vector of NDV LaSota strain.Through molecular cloning methods,such as PCR,restriction enzyme digestion and ligation,ISG12(2)was inserted into the full-genome vector of the NDV LaSota strain(r LaSota)and the F protein cleavage site mutant strain(r LaSota/Fmut).Then,utilizing the laboratory NDV reverse genetic system,the r LaSota,r LaSota/Fmut,r LaSota-ISG12(2),r LaSota-ISG12(2)/Fmut strains were successfully rescued.Through Western Blot and indirect immunofluorescence,it was verified that the rescued chimeric strain could not only express ISG12(2)protein efficiently,but also had NDV activity.Cytokine detection,virus proliferation ability and pathogenicity determination found that the chimeric strain expressing ISG12(2)could stimulat more interferon,IL-16,IL-18 IL-22 and other cytokines expression in vitro and in vivo compared with non-expressing NDV.These cytokines inhibited the proliferation of NDV,and then reduced the pathogenicity of the virus.Observation of pathological tissue slices revealed that the chimeric ISG12(2)strain did not cause related inflammation and pathological changes in the spleen and bursa of Fabricius,avoiding adverse reactions caused by cytokine storm.3.Evaluation of immune protection of ISG12(2)chimeric NDV StrainThe previous rescued strains were immunized 6-week-old low-antibody chickens via intraocular-nasal route.The three weeks after vaccination,chicken was challenged by NDV virulent strain F48E9.The ND antibody titer was weekly monitored by HI,and found that chickens immunized with these strains could produce high ND antibodies and 100% protect chicken from death caused by virulent strain challenge.Compare other three strains,r LaSotaISG12(2)could reduce viral shedding and viral loading in different tissures and prevent the pathological damage of the NDV virulent strain in various tissues including small intestine and bursa of Fabricius.Further research found that the r LaSota-ISG12(2)vaccinated group,compared with the r LaSota group,significantly increased the expression of immune-related factors,such as IFN?,IFN?and cytokines,such as IL-16,IL-18,IL-22,Transcriptome sequencing analysis of the spleen of the immunized chicken found that the r LaSota-ISG12(2)could better activate the humoral and cellular immunity of the chicken In summary,this study successfully constructed and rescued the NDV chimeric strain expressing ISG12(2).The chimeric strain,with the help of ISG12(2),to promote the expression of interferon,interleukin and other cytokines,reduces the pathogenicity of the virus,and enhances the innate and adaptive immune pathways of immunized chickens,to better prevent l chickens from NDV virulent strain infection.These results lay a foundation for using chicken ISG12(2)as a molecular adjuvant to enhance the immunogenicity of poultry vaccines and improve protective effects of vaccines. |
PDF Full Text Request