Generation And Characterization Of Recombinant Classical Swine Fever Viruses Based On C-strain Expressing The Cap Protein Of Porcine Circovirus Type 2

Classical swine fever（CSF）is an economically important disease of pigs in many countries and is caused by classical swine fever virus（CSFV）.C-strain,also known as the hog cholera lapinized virus（HCLV）,was developed through hundreds of passage of a highly virulent CSFV strain in rabbits in China.C-strain as an efficacious live vaccine is able to protect pigs against lethal challenge with CSFV.Porcine circovirus type 2（PCV2）,the agent of porcine circovirus-associated disease（PCVAD）,results in tremendous economic losses in the swine industry worldwide.The characteristic symptoms of PCVAD are weight loss,dyspnea and enlarged lymph nodes.PCV2 infection and replication in lymphoid tissues can destroy the architecture of lymphoid follicle,which leads to lymphoid depletion.Under experimental condition,clinical PCVAD is often difficult to reproduce in pigs infected with PCV2 alone.Generally,CSFV is the most common agent coinfecting with PCV2.The genome of PCV2 contains two major open reading frames（ORFs）,one coding for the replicase protein and the other coding for the capsid protein（Cap）.The Cap protein is a major immunogen and induces protection against a subsequent challenge with PCV2.A number of Cap-based subunit vaccines have been developed and evaluated in mice or pigs,indicating that Cap can induce protective immunity against PCVAD.In our study,to explore the potential of C-strain as a useful viral vector,we used the following several strategies to construct four recombinant C-strain expressing the Cap gene of PCV2.The recombinant viruses rHCLV-Cap(harboring the Cap gene in fusion with N^pro)and rHCLV-2ACap(harboring the Cap gene between the N^pro and C genes)were constructed.The other two recombinant viruses rHCLV-uspCap（containing the signal peptide of the ubiquitin-specific peptidase gene）and rHCLV-pspCap（containing the signal peptide of the prolactin gene）expressed the secreted Cap gene without the nuclear localization signal（NLS）.Then,we evaluated the genetic stability of the inserted genes in vitro and the biological characteristics of the recombinant viruses in rabbits.The Cap protein was expressed in the nuclei of SK6 cells infected with rHCLV-Cap or rHCLV-2ACap,while the Cap protein was detected in the cytoplasm of the cells infected with rHCLV-uspCap or rHCLV-pspCap by indirect fluorescence assay.The inserted genes of the recombinant viruses of passages 15 to 20 in SK6cells were detected by RT-PCR.The inoculated rabbits developed fever at 36 h post inoculation,which lasted for 18 to 24 h.At 3 d post inoculation（dpi）,the viral RNA was detected in the spleens of the inoculated rabbits.At 10 dpi,compared with the DMEM group,high-level anti-E2 antibodies were detected in the inoculated rabbits.However,rHCLV-uspCap and rHCLV-pspCap but not rHCLV-Cap or rHCLV-2ACap elicited detectable anti-PCV2 antibodies in rabbits.In conclusion,we constructed four recombinant C-strain viruses expressing the Cap protein.The recombinant viruses are genetically stable in vivo.Furthermore,the animal experiment results showed that the anti-CSFV antibodies in rabbits were similar between the all recombinant viruses and the parental virus.Interestingly,the two recombinant viruses expressing the secreted Cap protein elicited detectable anti-PCV2 antibodies in rabbits.Our study provides a foundation for developing bivalent vaccines to control PCV2 and CSFV co-infections.