Construction And Immunogenicity Evaluation Of Classical Swine Fever Virus Marker C-strain Based On Reverse Genetic Manipulation Technology

Classical swine fever?CSF?is a highly contagious disease of pigs,which is characterized by high fever retention and extensive bleeding,causing huge economic losses to the pig industry in China and even many countries around the world.At present,the main measure to prevent CSF in China is to immunize the Chinese hog cholera lapinized virus?HCLV?,also called C-strain.This vaccine is a highly safe and efficacious rabbit-adapted vaccine,which plays an important role in the prevention and control of CSF in the world.However,C-strain lacks the capacity for the serological differentiation between infected and vaccinated animals?DIVA?,which impairs the eradication of CSF.Our previous study has identified a novel conserved monoclonal antibody?MAb?HQ06-recognized linear B-cell epitope ¹¹⁶LFDGTNP¹²² on the E2 protein of CSFV and ¹¹⁷F,¹¹⁹G,and¹²²P constitute the core of the epitope.The purpose of this study is to develop C-strain as a marker vaccine by reverse genetic manipulation for CSFV.In this study,we constructed three CSFV full-length infectious clones in the background of C-strain,containing the single amino acid mutation of ¹¹⁷F,¹¹⁹G,or ¹²²P of MAb HQ06-recognized epitope on E2 glycoprotein into alanine?A?respectively,resulting in rHCLV-E2F117A,rHCLV-E2G119A and rHCLV-E2P122A and evaluated the phenotype of mutants in vitro.Then,we evaluated the biological characteristics of the mutants in rabbits by intravenous,including typical fever responses and replicating in spleens of rabbits,the ability of the antibody induced by mutant and the reactivity of the antibody with the LFDGTNP antigen peptide.Finally,the mutant virus rHCLV-E2P122A screened in rabbit experiments was inoculated into weaned piglets by intramuscular,and evaluated the ability of the virus to induce piglets to produce neutralizing antibodies and the reactivity of the antibodies with LFDGTNP antigen peptide.The results of indirect immunofluorescence assay?IFA?showed that three mutants lost the reactivity with the MAb HQ06 completely.Sequencing analysis revealed that these three mutant viruses retain the mutant phenotype and no additional mutations were seen during additional passages in SK6cells.The results of the growth kinetics of the mutants in vitro showed that rHCLV-E2P122A had the same titer in the final virus progeny yield relative to parental rHCLV at 60 hpi,while the growth ability of others was delayed.Intravenous administration of these three viruses in rabbits demonstrated that all the mutants retain the phenotype of C-strain,which could induce fever response and replicate in rabbits.Sequencing results confirmed that the mutant virus remained stable in rabbits without reversion of the MAb HQ06-recognized epitope in rabbits.Anti-E2 antibodies seroconverted in the sera of all vaccinated rabbits at 7 days post-inoculation?dpi?.Notably,the antibodies in the sera of the rabbits vaccinated with rHCLV-E2F117A,rHCLV-E2G119A,or C-strain were strongly reacted with the LFDGTNP antigen peptide,whereas the antibodies against the peptide were not detected in the sera from the rabbits immunized with rHCLV-E2P122A.Intramuscular administration of rHCLV-E2P122A or C-strain in weaned piglets showed that no clinical signs were noted.The sera obtained from vaccinated pigs showed that all anti-CSFV E2 antibodies were positive and the titer of the neutralizing antibodies of was reached over 1:240 at 28 dpi.Notably,the sera from the pigs inoculated with rHCLV-E2P122A were not reactive with the LFDGTNP antigen peptide,whereas the antibodies against LFDGTNP antigen peptide were detected in the sera of pigs inoculated with C-strain.Taken together,our data demonstrated that the mutant virus rHCLV-E2P122A derived from C-strain by CSFV reverse genetics platform,containing the single amino acid mutation of ¹²²P of MAb HQ06-recognized epitope on E2 glycoprotein into alanine?A?,was conferred the potential DIVA capability,which indicating it was a promising candidate to be used as a live attenuated marker vaccine against CSF.This study provides a feasible idea and method for the development of attenuated marker vaccines against CSF,and provides a strong technical support for the eradication of CSF in China.