Regulation Of Untranslated Regions And Nonstructural Protein 5B On The Replication Of Classical Swine Fever Virus Vaccine C Strain

Classical swine fever virus（CSFV）is the pathogen of classical swine fever（CSF）.CSF is a highly contagious,highly lethal disease of pigs.The outbreak and epidemic of CSF impede the development of pig industry seriously.CSFV belongs to the genus Pestivirus in the family Flaviviridae,along with bovine viral diarrhea virus（BVDV）and border disease virus（BDV）.The genome of CSFV is a single positive-strand RNA approximately 12.3 kb in length,containing a large open reading frame（ORF）flanked by two untranslated regions（5′UTR and 3′UTR）.The ORF of CSFV encodes a large polyprotein which is processed into at least 12 mature proteins by cellular and viral proteases,including four structural proteins and eight non-structural proteins.The non-structural protein 5B,as an RNA-dependent RNA polymerase（RdRp）,plays key roles in viral genome replication.Vaccination with attenuated CSF vaccine（C strain）is the most effective strategy to prevent and control CSF.It is significant for the innovation of CSF vaccine to understand the biological characteristics,genetic stability and replication regulation of attenuated CSFV vaccine strain.In this study,we replaced the 5′UTR and 3′UTR of CSFV vaccine C strain with 5′UTR and 3′UTR of BVDV separately or simultaneously using the reverse genetics based on infectious CSFV vaccine strain clone p ST_I/C to generate infectious chimeric clones,p ST_I/C/b5?UTR,p ST_I/C/b3?UTR and p ST_I/C/b UTRs.The recombinant viruses vC/b5?UTR,vC/b3?UTR and vC/b UTRs were successfully rescued in PK-15 cells transfected with these chimeric c DNA clones.The recombinant CSFV C strains containing UTRs substitutions of Shimen strain,vC/SM5?UTR,vC/SM3?UTR and vC/SMUTRs,were successfully rescued using the infectious clones p ST_I/C/SM5?UTR,p ST_I/C/SM3?UTR and p ST_I/C/SMUTRs preserved in our laboratory.The recombinant CSFV vaccine strains vC/b5?UTR,vC/b3?UTR,vC/b UTRs,vC/SM5?UTR,vC/SM3?UTR and vC/SMUTRs were continuously passaged 30 times in PK-15 cells.The representative passaged viruses,1^st passage（p1）,11^th passage（p11）,21^stpassage（p21）and 31^st passage（p31）,were characterized.After the continuously passaging,the characteristics of the 6 recombinant viruses were significantly changed,including the increased infectious virus production,the enhanced viral genome replication efficiency and the larger plaque size.The genomes of six 31^st recombinant viruses were sequenced.The mutations located in 31^st viruses were further investigated in the genomes of p11 and p21 viruses by sequencing.Data showed that 5,8,8,6,8 and 7 nucleotide mutations,and 4,5,5,3,5and 4 amino acid mutations occurred during the serial passage of vC/SM5?UTR,vC/SM3?UTR,vC/SMUTRs,vC/b5?UTR,vC/b3?UTR and vC/b UTRs,respectively.The recombinant viruses harboring substitution of 3?UTR exhibited later significant change of viral characteristics and higher genome mutation frequency.It has been speculated that 3?UTR and the NS5B 162^nd amino acid of CSFV played key roles in determining the characteristics of passaged viruses by analyzing the viral characteristics and genome sequences.We constructed three recombinant virus mutants,vC_P162T,vC/SM3?UTR_P162T and vC/b3?UTR_P162T based on infectious clones p ST_I/C,p ST_I/C/SM3?UTR and p ST_I/C/b3?UTR using the reverse genetics.Analyses of characteristic demonstrated that NS5B P162T mutation increased the viral genome replication efficiency and infectious virus production in PK-15 cells.Animal expereiment showed that NS5B P162T mutation increased the mutant virus replication in vivo and reduced the fever response in rabbits.To further understand the regulatory mechanism of P162T mutation on NS5B RdRp activity and viral genomic replication,we prepared wild-type NS5B_WT and mutant NS5B_P162T by expressing in E.coli and affinity chromatography purification.The effects of NS5B P162T mutation on the process of RNA synthesis were analyzed with RdRp assays in vitro using T30,poly C and（+）CSFV/3?UTR templates.We found that P162T mutation increased NS5B RdRp activity.The RdRp elongation activity and stability of elongation complex（EC）were not affected by P162T mutation in the single nucleotide elongation assay using T30 template.We finally found that the NS5B P162T mutation enhanced the initiation efficiency of RNA synthesis by characterizing the NS5B RdRp kinetics during the initiation stage with T30 template.Our works identified the role of untranslated regions of pestivirus in determining genetic stability of the genome,and deepened the understanding of the structure and function of NS5B.Our results will contribute to develop new CSFV prevention and control strategies and the novel attenuated CSFV vaccines.