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Genetic Variation Analysis Of Bovine Viral Diarrhea Virus In Shandong Province And Establishment Of Reverse Genetic Platform For Strain SDTA-1

Posted on:2022-10-08Degree:MasterType:Thesis
Country:ChinaCandidate:Z SunFull Text:PDF
GTID:2480306575469504Subject:Veterinarians
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Bovine viral diarrhea(BVD)is an infectious disease caused by bovine viral diarrhea virus(BVDV),which is characterized by mucosal inflammation,erosion,necrosis and diarrhea,leading to serious economic loss of cattle industry worldwide.Vaccine immunization is important for preventing cattle from BVD.However,due to the numerous subtypes and the persistent variation of BVDV,the cross-protection of current vaccines for BVD are less effective,limiting the application of current vaccines.Therefore,it is necessary to develop novel vaccines based on the epidemic isolates.Reverse genetics are critical methods for RNA virus study at cDNA level.With availability of full-length cDNA clones of RNA viruses,such as BVDV,it is possible to develop novel recombinant vaccines or marker vaccines.Herein,10 BVDV-1 strains isolated from cattle in Shandong province were analyzed according to the sequence of 5'-UTR.While multiple sutypes of BVDV-1 exist in cattle herds in Shandong province,BVDV-1d is the most popular subtype.By comparing the replication capacity of BVDV-1d isolates,a candidate vaccine strain,named SDTA-1,was identified.Based on the analysis of whole genome sequence and genetic evolution of SDTA-1,five cDNA fragments covering the full-length genome of SDTA-1 were obtained by RT-PCR,in which overlapping regions between the adjacent fragments were designed.In addition,a CMV promoter sequence was added in the left of 5'-UTR,and a hepatitis delta virus ribozyme sequence(HDVr)together with the bovine growth hormone Poly A sequences were introduced to the end of the 3'-UTR of the virus genome.Finally,the cDNA of SDTA-1full-length genome was cloned into the pBR322 vector by homologous recombination to obtain the infectious cDNA clone pBR322-SDTA-1.Consequently,rescued viruses were successfully generated by transfection the infectious clone into bovine kidney cells MDBK.In summary,we have identified a candidate vaccine strain of BVDV-1d SDTA-1.Based on the whole genome analysis,a full-length infectious cDNA clone of SDTA-1 were generated,which eventually results in the successful production of the stable rescued virus of BVDV-1d.Hence,the established platform of reverse genetics of BVDV-1d in this study provides an effective technology for further study of BVDV and development of novel vaccines for BVD.
Keywords/Search Tags:Bovine, Bovine viral diarrhea virus, Reverse genetics, Infectious cDNA clone, Rescue of virus
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