Genetic Rescuing And Characterization Of Recombinant Classical Swine Fever Virus Vaccine Strains Containing The E2 Gene Representing The Prevalent Field Isolates

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine and wild boar, causing significant economic losses to the swine industry. The causative agent of the disease is classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family. CSFV is an enveloped RNA virus with its genome size of approximately 12.3 kb. The structural glycoprotein E2 is the most immunogenic among the CSFV proteins, inducing neutralizing antibodies and protection against lethal challenge. It also plays multiple roles in viral life cycle, such as virus attachment, entry into target cells, cell tropsim as well as virulence determinant. Currently, the switch of virus populations from historical group 1 to group 2 has been reported and a trend to chronic form of the disease, even in a certain proportion of vaccinated pigs has been reported in the endemic areas. Therefore, the efficacy of the classical group 1 lapinized C-strain vaccine is facing challenge. The present study was aimed (1) to investigate the CSFV infection status in swine population in Zhejiang, (2) to establish an RFLP method for differential identification of the vaccine strain and field isolates, (3) to explore the genetic and antigenetic diversity of the E2 of field isolates, and (4) to develop recombinant C-strain-based vaccine candidate strains containing E2 genes from prevalent field isolates using the reverse genetics technology.1.Differential identification of field CSFV isolates and the vaccine strain by RFLPRT-nested PCR (RT-nPCR) using the consensus-degenerate hybrid oligonucleotide primers targeted on the full-length E2 was used for detection of CFSV. The assay was able to detect as low as 1400 copies of CSFV genomic RNA. Vaccinated and infected CSFV strains could be differentiated by Mspl-based restriction fragment length polymorphism (RFLP) analysis. The assay was applied to identify CSFV isolates from 309 clinical specimens from 2003-2008 in Zhejiang. In 91 CSFV isolates,22 were identified as the C-strain,60 as field strains, and 9 as having both the C-strain and the field ones. RFLP of the RT-nPCR amplicons by Bglâ… , Ddeâ… , Draâ… and Pstâ… was used for subtyping of the field CSFV isolates. Thirty-eight field isolates were divided into 11 subtypes by this RFLP scheme, indicating the genetic diversity of the prevalent isolates in Zhejiang.2. Growth kinetics of prevalent field CSFV isolates and classical virulent isolate Shimen in PK-15 and ST cellsTwo field strains QZ-07 and HZ1-08 were isolated and their replication kinetics in PK-15 and swine testicle ST cell lines were compared with the classical virulent Shimen strain. The Shimen strain replicated more efficiently in PK-15 cells than in ST cells (107 TCID50/ml vs 105.25 TCID50/ml at 48 h post-infection). In contrast, two field strains displayed decreased replication in PK-15 cells (102.75 TCID50/ml for isolate HZ1-08 vs 105 TCID50/ml in ST cells). This was consistent with the protein expression kinetics. Only a few fluorescent foci were detected by IFA in HZ1-08-infected cells, while all cells were positive for Shimen-infected cells at 48h post-infection. Although the cell-associated virus titers were similar among three strains, the ratio of secreted virus versus cell-associated virus was different. The virulent strain Shimen secreted more progeny virus to the culture supernatants than the recent isolates. Considering the increased release of progeny virus particles from the cells as an attribute of high virulence, the prevalent isolates did not seem to have high virulence.3.Characterization of genetic variations of prevalent CSFV isolates in their full genomes and E2 genesThe genome of the strain QZ-07, vaccine C-strain and virulent Shimen were determined. Phylogenetic analysis revealed two major clusters of 25 isolates including those retrieved from the GenBank representing other parts of China and other countries. C-strain and Shimen strain were clustered together, while strain QZ-07 fell into another cluster far away from these historical strains. The variability of the full-length polyprotein of these 25 isolates was analyzed by the differences between non-synonymous (dN) and synonymous (dS) rates and the entropy values. The first one third of the polyprotein covering all structural proteins was more variable than the last two thirds containing the nonstructural proteins essential for RNA replication. When the individual proteins were compared, three structural proteins and NS5A were more variable as compared with the relative conserved NS3, NS4B and NS5B proteins.Phylogenetic analysis of the E2 gene of 34 CSFV isolates from Zhejiang was further revealed that genotype 2.1b viruses became predominant in Zhejiang with 33 isolates clustered in 2.1b and only 1 isolate belonged to 2.2. Pairwise comparisons demonstrated that isolates in this study had an identity of 94.6%-99.8% at the nucleotide level and 94.9%-99.7% at the amino acid level. The identity ranged from 81.6% to 82.6% for nucleotide and 87.4%-89.3% for amino acids, as compared to the C-strain. Two variable regions in the antigenic domains as well as some positive selected positions located in the identified neutralizing epitopes or related to monoclonal antibodies (mAb) binding were defined. Moreover, these residues in mAb related positions were different between prevalent isolates and vaccine C-strain.4. Characterization of monoclonal antibodies against E2 of the CSFV vaccine strainMonoclonal antibodies to antigenic domains of glycoprotein E2 of C-strain were prepared and used to examine the effect of the variable regions and the specific positive selected positions on antigenic diversity between C-strain and recent field isolates. Three hybridoma cell lines secreting mAb (1E7,2B6 and 6B8) were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized with the purified recombinant E2 protein (rE2). Western blot and ELISA analysis showed that only the mAb 2B6 could react with rE2. Six Cystine residues important for maintenance of the structure of antigenic domains of E2 were mutated in the backbone of nature C-strain E2 and expressed in PK-15 cells. Mutagenic analysis revealed that 1E7 and 6B8 reacted with the conformational epitopes located in antigenic domain B/C, while 2B6 reacted with all Cys mutant E2s independent of the secondary structure. Thus, mAb 2B6 was confirmed to react with a linear epitope. The reactivity of these three mAb with Shimen, QZ-07 and XS-08 indicated that the linear epitope recognized by 2B6 was specific for group 1 strains, while 1E7 and 2B6 reacted weakly with recent group 2 isolates. The reactivity patterns for prevalent isolates were further confirmed by the expression of different field isolates E2 in PK-15 cells. MAbs 1E7 and 2B6 did not react with mutants with substitution of the E2-Cys737 with Arg in the isolates LS-05 and QZ2-06 possibly due to the abolishment of the structure of antigenic domain B/C. This Cys mutant virus may escape from neutralizing antibodies against domain B/C in vivo.5.Genetic rescuing and characterization of recombinant classical swine fever virus vaccine strains containing the E2 gene representing the prevalent field isolatesSince the genetic and antigenic diversity was apparent between recent isolates from Zhejiang and the vaccine C-strain, the vaccine efficacy could be compromised. Thus, we used reverse genetics to engineer recombinant CSF viruses based on the vaccine C-strain and the genetic features of the isolates circulating in Zhejiang. The cDNA fragments covering the genome of the C-strain were assembled and inserted downstream of a T7 promoter to obtain the full-length cDNA clone of pA-FL22. The in vitro synthesis of full-length viral RNA derived from pA-FL22 proven to be replication-competent and infectious after electroporated into ST and PK-15 cells. Real-time PCR and IFA revealed that the RNA replication and protein translation was more efficiently in ST cells than in PK-15 cells. The recombinant virus FL22 recovered from the electroporated cells retained the properties of the parental C-strain in rabbit, fever and enlargement of the spleen. A silent point mutation at position 8036 of the genome which introduced an additional Ncoâ… restriction site as genetic tag was also retained during virus replication in vitro and in vivo. Two infectious recombinant CSF viruses were generated by exchanging the 830-bp region including the antigenic domains in pA-FL22 with the equivalent region of CSFV strains Shimen and HZ 1-08, respectively. The resulting recombinant viruses FL22-SM-E2 and FL22-HZ-E2 could be differentiated from FL22 by Mspâ… based RFLP assay, and virus FL22-HZ-E2 also changed mAb pattern same as the donor strain HZ 1-08. Therefore, the recombinant virus FL22-HZ-E2 may serve as the candidate strain for further marker vaccine development.In summary, our studies revealed the divergence of the prevalent CSFV isolates in Zhejiang and their antigenic variations to the vaccine C-strain. The RT-nPCR-based RFLP could be used for subtyping and differentiation of the field isolates from the vaccine strain. Successful rescuing of the recombinant viruses based on the vaccine strain would be of great use for development of anti-CSFV marker vaccines and for in-depth studies on the replication and pathogenesis of CSFV.