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Isolation And Identification Of Bovine Viral Diarrhea Virus From Deer And Molecular Mechanism Of Cp BVDV Activated NF-Kappa B

Posted on:2019-06-27Degree:DoctorType:Dissertation
Country:ChinaCandidate:K ShiFull Text:PDF
GTID:1360330599963025Subject:Prevention of Veterinary Medicine
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Bovine viral diarrhea is a multi-clinical disease caused by bovine viral diarrhea virus.Cattles of all ages are susceptible to BVDV,and its clinical symptoms mainly include acute bleeding syndrome,persistent infection,immunosuppression,immune tolerance,respiratory and gastrointestinal diseases,and reproductive insufficiency.The World Organisation for Animal Health?OIE?lists it as a Class B infectious disease and lists it as a second-class infectious disease in the list of imported animal infectious diseases of China.The BVDV was subsequently isolated,followed by BVD was first discovered in New York,USA.Since then,the disease has been reported around the world.Chinese scholar Youmin Li isolated and identified BVDV from the aborted fetus spleen in 1983,which is the first report of the disease in China.BVD presents regionally prevalent in the world,and the animal infection rate in the developed areas of animal husbandry is high,which has become one of the major infectious diseases that harm the cattle industry,causing huge economic losses to the world's aquaculture industry.1.Isolation,identification and genetic variation analysis of deer source bovine viral diarrhea virusThe suspected diarrhea samples collected from the deer of Jilin province,then treated with bovine kidney cells?MDBK?,followed the virus was isolated.They were identified as deer-source bovine viral diarrhea virus after the morphological,immunological and molecular biological identifications including:BVDV JL01,BVDV F3,BVDV Y3,BVDV de4 and BVDV SY4.The isolates were further detected by immunoperoxidase unilateral cell assay and the TCID50.The results showed that the TCID50 of BVDV JL01,BVDV F3,BVDV Y3,BVDV de4 and BVDV SY4 were 105.3TCID50/mL,105.8TCID50/mL,104.7TCID50/mL,105.5TCID50/mL and 104.4TCID50/mL.BVDV JL01,BVDV F3,BVDV Y3,BVDV de4 are cp BVDV,and BVDV SY4 is ncp BVDV.The primers were designed to amplify the Npro and E2 genes according to the reference sequence of different strains of BVDV registered by Genbank,and using DNAStar analysis the nucleotide homology comparison and genetic evolution of the BVDV Npro and E2 genes between the isolates and the BVDV genotypes registered in Genbank.The results showed that BVDV JL01,BVDV F3,BVDV Y3,BVDV de4 were BVDV1 type,and BVDV SY4 was BVDV 2 type.The forecast of glycosylation sites of the isolated strain E2 protein showed that different from there were 9 potential N-glycosylation sites in BVDV JL01 strain and BVDV de4 strain,BVDV Y3 strain and BVDV SY4 strain both had ten potential N-glycosylation sites.The BVDV F3 strain has 11 potential glycosylation sites.2.Molecular mechanism of activation of NF-?B by bovine viral diarrhea virusThe activation of NF-?B in MDBK cells by different biotype BVDV infections of cp and ncp was analyzed by dual luciferase reporter assay system.The results showed that cp BVDV was significantly activated compared with uninfected group,and pathway was significantly activated after infected by cp type BVDV JL01 strain of 48h,ncp BVDV SY4 strain failed to activate NF-?B,cpBVDV even had a dose-effect relationship with NF-?B activation.The results of laser confocal analysis showed that cp-type BVDV infection significantly activated NF-?B expression with increasing infection time compared with uninfected control group,and NF-?B p65 showed obvious nuclear translocation at 4 h after infection.No p65 nuclear translocation was observed in ncp-type BVDV infection.It is indicated that cp type BVDV infection activates NF-?B expression and causes nuclear translocation.For laid the foundation for the study of BVDV pathogenesis,Real-time PCR and Western blot analysis of signal transduction pathway of TL3,TLR4 and RIG-I BVDV activation of NF-?B signaling pathway.3.Activation of NF-?B signaling pathway for bovine viral diarrhea virus protein screeningIn order to identify the role of structural and non-structural proteins which was encoded BVDV in the activation of NF-?B,eukaryotic expression vectors of BVDV genes were constructed,and co-transfected with NF-?B reporter plasmid and Renilla internal reference plasmid.The reporter system was analyzed and the results showed that BVDV NS5B protein can significantly activate NF-?B.4.Molecular mechanism of NF-?B activation by bovine viral diarrhea virus NS5BTo explore the activation mechanism of NF-?B by BVDV NS5B.The eukaryotic expression plasmid of NS5B protein was transfected into cells,indicating that NS5B has a dose-effect relationship with NF-?B activation.NS5B protein can induce I?B degradation,cytoplasmic p65 and phosphorylation of p65 protein in nucleus.To further clarify that the NS5B protein activates the functional domain of NF-?B,based on the NS5B protein domain,NS5B is truncated into 6 segments for segmental expression,NS5B-NT,NS5B-Core,NS5B-finger,NS5B-palm,NS5B-thumb And NS5B-CT.The dual luciferase reporter assay system measures NF-?B and Renilla luciferase activity in transfected cells.The results showed that BVDV NS5B protein,NS5B-Core,NS5B-finger and NS5B-palm can activate NF-?B.The difference between the NF-?B and the empty vector control group was extremely significant,indicating that the region that activates NF-?B is located at the RdRp polymerase catalytically active center of the NS5B protein.Real-time PCR was used to detect the mRNA expression level of IFN-I after transfection of NS5B truncation mutants.The results showed that the truncation mutant of NS5B induced the increase of IFN-I mRNA level by activating NF-?B signaling pathway.
Keywords/Search Tags:deer, bovine viral diarrheal virus, isolation and identification, NF-kappa B, nonstructural protein 5B, signaling pathway
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