A Novel Reverse Genetics System For Classical Swine Fever Virus And The Viral Replication Regulation

Classical swine fever is an economically important, highly contagious disease of pigs caused by the classical swine fever virus (CSFV). CSFV belongs to the genus Pestivirus within the family Flaviviridae, together with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). The genome of CSFV is a single1 positive-strand RNA of about 12.3 kb which contains 5'-untranslated region (5'UTR), 3'UTR and a single large open reading frame (ORF). The ORF codes a polyprotein of approximately 3,898 amino acids, which is processed co-and post-translationally by host cell and viral proteases to produce structural and nonstructural proteins.Reverse genetics system is an important platform to study the structure and function of RNA viruses. Reverse genetics system allows to investigate molecular genetic basis of RNA virus replication, assembly and release of viral particle and pathogenesis by genome modification. In this study, a novel plasmid-based single step reverse genetics system for CSFV was developed. The recombinant plasmid harboring CSFV strain Shimen (pSPT1/SM), or Chinese (C) strain (pSPT1/C) full-length cDNA flanked by a swine RNA pol I promoter and murine pol I terminator was constructed, respectively. After transfection with the recombinant plasmid, an infectious CSFV with accurate ends was gererated in the transfected-PK15 cells. Compared with parental CSFV or vSM from in vitro transcripts RNA system, the CSFV rescued from the novel recombinant plasmid maintained same growth characteristics and plaque formation. The novel plasmid-based single step reverse genetics system for CSFV contributes to investigate molecular genetic basis of replication, assembly and release and pathogenesis of CSFV and the development of modified CSFV live vaccines, and provides potential basis for other pestiviruses.To understand the effect of UTR from highly virulent Shimen strain on viral replication of vaccine C-strain, several recombinant chimeric plasmids pC/SM UTRs, pC/SM 5'UTR and pC/SM 3'UTR were constructed based on the infectious cDNA clone pSPT1/C. The chimeric CSFVs were rescued from PK15 cells transfected with recombinant plasmids and characterization of the chimeric CSFVs was further evaluated. The results demonstrated that compared with CSFV C strain, the recombinant chimeric CSFVs containing the UTRs of the Shimen strain exhibited higher replication efficiency. Among them, vC/SM UTRs containing both 5'UTR and 3'UTR replicated higher than that of vC/SM 3'UTR, or vC/SM 5'UTR. After serial passages in PK-15 cells, the chimeric recombinant CSFVs maintained similar characteristics with parental recombinant viruses.As a CSFV-encoded autoprotease, NS2 creates in cis its own C terminus associating with a cellular chaperone termed Jiv or its fragment Jiv90 and thereby releases an essential viral replication complex component NS3. However, the additional function of cleaved NS2 for CSFV replication regulation is unknonwn. To understand the function for CSFV replication regulation, a series of mutations in NS2 N-terminus were constructed and the effect of the mutaions on infectious virus production and viral genome RNA replication was investigated. Our results showed that two aspartic acids mutations NS2/D60A, NS2/D60K and NS2/D78K abolished infectious virus production and the mutation NS2/R100A significantly decreased virus production. The mutantsNS2/T37A, NS2/K52A, NS2/D78A and NS2/W85A displayed similar virus titers compared with wt CSFV. The results based on the monocistronic replicon showed that no Rluc activity was detected in cells electroporated with the replicon containing NS2/D60A, NS2/D60K and NS2/D78K, and Rluc activities of NS2/R100A were significantly reduced at 72 h post-electroporation. In addition, the genomic RNA copies of the rescued vCSFV-Rluc and vCSFV-Rluc/R100A were detected using qRT-PCR after infection. The data showed that mutant NS2/R100A showed significantly reduced viral RNA copies, to the similar Rluc activities after infection viruses. These results suggested that the effect of NS2 N-terminal mutations on impairment of infectious virus production mainly resulted from the regulation of viral genome RNA replication level.Western blot analysis showed NS2-3 proteins containing wt and mutated NS2s had similar cleavage efficiency in vitro, and the interaction between NS2-NS2 and the stability of NS2 protein were not affected by the mutations. The correlation between cleavage efficiency of NS2-3 and viral RNA replication was further investigated based on the bicistronic replicon. Compared with NS3-NS5B/Rluc, reduced Rluc activity was observed from replicon NS2-NS5B/Rluc. Mutant replicon analysis showed that the RNA replication regulated by NS2 N-terminal mutations based on monocistronic replicon was similar to bicistronic replicons. These results demonstrated that the single amino acid within NS2 N-terminus modulated the viral RNA replication independently of the cleavage efficiency of precursor NS2-3.The serial passages of PK15 cells transfected with mutated CSFV genomic RNAs produced infectious virus particles. The genome sequencing exhibited that revertants of the NS2/D60A, NS2/D60K or NS2/D78K were obtained from passaged viruses. After a serial passages, the reduced infectious virus titer of mutant NS2/R100A was reversed by NS2/I90L compensatory second-site mutation, suggesting a possible interaction between transmembrane regions of NS2.Our works will contribute to explore the structure and function of CSFV genome, pathogenicity, and to develop CSFV genetically engineered vaccines.