Construction And Immunogenicity Of Recombinant Pseudorabiesvirus Variant Co-expressing Porcine Circovirus Type 2 Capsid Protein And Interleukin2

Porcine circovirus type 2(PCV2)is considered the major pathogen of Postweaning Multisystemic Wasting Syndrome(PMWS)that causes large economic losses for the swine industry.PCV2-infected pigs can cause severe immunosuppression,resulting in easy concurrent and secondary infections,thereby increasing the morbidity and mortality of the herd.IL-2 is a kind of cytokine with a wide range of biological activities.IL-2 induces proliferation of natural killer cells and augments their cytolytic activity as well as that of lymphokine-activatedkiller cells,promotes antibody production and proliferation by B cells,and is essential for activation-induced cell death.Therefore IL-2 can be used as a good adjuvant.Porcine Pseudorabies is an acute infectious disease caused by Pseudorabies virus(PRV).More seriously,since late 2011,PRV variants have emerged in many regions of China.PRV has a linear DNA genome of approximately 150 kb.It contains several envelope glycoproteins that interact with host cells.Foreign genes can be stably inserted and expressed in the PRV genome,making attenuated PRV strain a promising live vector for multivalent vaccines against pseudorabies and other swine diseases.1 Phylogenetic Analysis of Prevailing Pathogenic porcine circovirus in Henan province during 2016-2017In order to determine the genotype and genetic variation of pathogenic porcine circovirus(PCV)in partial region of Henan province,65 suspected cases of porcine circovirus disease from different region of Henan province were collected during2016-2017.The gene fragments were amplified by PCR with the specific primers of PCV2 and PCV3.There were 43 PCV2 positive samples with positive rate of 66.15%and 3 PCV3 positive samples with positive rate of 4.61%.13 PCV2 positive samples and 3 PCV3 positive samples were selected for cloning,sequencing and sequence analysis of ORF2 gene.Sequence alignment showed that variation among 16 samples was small and there was only single point mutations,no insertion or deletion changes.The phylogenetic analysis based on nucleotide sequence of ORF2 showed that 13PCV2 were classified into two genotypes(7 PCV2 b and 6 PCV2d),indicating that PCV2 b and PCV2 d were the predominant epidemic strains in Henan.2 Construction of a recombinant pseudorabiesvirus variant co-expressing porcine circovirus type 2 capsid protein andinterleukin IL-2.The PCV2 YY strain ORF2 gene and IL-2 gene were inserted into the TK locus deleted from the r PRV-g E-/TK-/EGFP+ virus.The recombinant virus r PRV-g E-/TK-/ORF2+/IL-2+was obtained by homologous recombination.The presence of PCV2 Cap and IL-2 proteins in the recombinant virus r PRV-g E-/TK--ORF2+-IL2+ was confirmed by Western blot analysis.PCV2 Cap(27.8k Da)and IL-2(15k Da)were identified based on their approximate molecular weights.The recombinant virus r PRV-g E-/TK-/ORF2+/IL2+ has similar proliferation ability with the parent strain PRV QYY strain and r PRV-g E-/TK-/EGFP+.This recombinant virus was safe for mice.This research provides the basic materials for evaluating its immunogenicity.3 Immunogenicity of recombinant pseudorabiesvirus variant co-expressing porcine circovirus type 2 capsid protein andinterleukin 2Immunogenicity of the recombinant virus r PRV-g E-/TK-/ORF2+/IL2+ was evaluated by anti-PCV2 ELISA,PRV-neutralizing assay,PCV2 specific lymphocyte proliferation assay and flow cytometry.Our data clearly showed that mice vaccinated with r PRV-g E-/TK-/ORF2+/IL2+ produced high titer of PCV2 specific antibodies.PRV neutralizing anti-bodies titer was 1:64.The lymphocytes from mice immunized with r PRV-g E-/TK-/ORF2+/IL2+proliferated significantly more than lymphocytes from the mice immunized with DMEM.The percentages of CD3+,CD4+and CD8+T lymphocytes were higher after immunization r PRV-g E-/TK-/ORF2+/IL2+ compared to the r PRV-g E-/TK-/ORF2+group,PCV2 Subunit vaccine group and DMEM control group.Similar results were obtained regarding the changes in the percentages of IFN??IL-4 ? IL-12.IL-2 should be regarded as a unique cytokine that enhance sinnate immunity,and induce Th1-type cytokines and Th2-type cytokines for Cap protein.This study provided data support for the evaluation of the immunogenicity of the recombinant virus r PRV-g E-/TK-/ORF2+/IL2+ in pigs.The novel recombinant virus described here is an attractive candidate vaccine to prevent PCV2 and PRV infections in pigs.