| Since porcine circovirus type 3(PCV3)was detected in the tissues of sows with Porcine dermatitis and nephropathy syndrome(PDNS)in 2016,the detection rate of PCV3 has been increasing,which pose a potential threat to pig industry worldwide.At present,the research of PCV3 mainly focused on epidemiology and diagnostic methods et al,while the research on immune mechanism and vaccine development are limited.The capsid protein(Cap)encoded by ORF2 gene of PCV3 can induce specific immune response.It can be used in the study of pathogenic mechanism and vaccine development.Although PCV2 vaccine have been commercialized,they cannot cross protect pigs from PCV3 infection.Pseudorabies virus(PRV)can infect pigs of all ages,causing various clinical symptoms and pathological changes.Since the outbreak of pseudorabies(PR)variant strains in many provinces of China in 2011,the variant strain PRV-HNX has been isolated and identified in our laboratory.Co-infection PRV and PCV3 make disease more complex and difficult to control.Therefore,it is very important to develop a vaccine that can prevent PCV3 and PRV at the same time.Therefore,we have conducted the following study:1.Construction and identification of recombinant pseudorabies virus expressing PCV3-Cap protein(HNX-(35)TK/(35)g E-ORF2)Firstly,sg RNA recombinant plasmids TK-sg RNA-PX458 targeting PRV TK gene and m Cherry-p UC19 recombinant plasmids were constructed.Then PRV-HNX-(35)g E genome constructed before,TK-sg RNA-PX458 and m Cherry recombinant plasmids were co-transfected into HEK 293T cells.The m Cherry positive plaques were picked and purified by plaque purification.The HNX-(35)TK/(35)g E-m Cherry recombinant virus was verified by PCR.Then,the genome of HNX-(35)TK/(35)g E-m Cherry recombinant virus was co-transfected with m Cherry-sg RNA-PX458 and PCV3-Cap-p UC19 recombinant plasmids into HEK 293T cells.The m Cherry negative plaques were picked and purified by plaque purification.The recombinant virus with PCV3 ORF2 gene was obtained and named HNX-(35)TK/(35)g E-ORF2.PCR results showed that the recombinant virus was pure;Western blot and indirect immunofluorescence showed that PCV3-Cap protein was successfully expressed;The one-step growth curves showed that there was no significant difference in growth characteristics between the recombinant virus and its parent strain PRV-HNX.2.Safety evaluation of HNX-(35)TK/(35)g E-ORF2 recombinant virusSix weeks old BALB/C mice were inoculated with PRV-HNX,HNX-(35)TK/(35)g E,HNX-(35)TK/(35)g E-ORF2,DMEM,respectively.PRV DNA was detected in brain,spleen and kidney of mice in the PRV-HNX inoculation group by q PCR,but not in HNX-(35)TK/(35)g E,HNX-(35)TK/(35)g E-ORF2 inoculation groups;ELISA results showed that at4 days post inoculation,IL-6 in plasma of mice inoculated with HNX-(35)TK/(35)g E and HNX-(35)TK/(35)g E-ORF2 were 35.00?14.19pg/m L and 30.40?17.18pg/m L,respectively,and G-CSF was 375.43?113.28 pg/m L and 392.40?191.53pg/m L,respectively.There was no significant difference between the two groups(P>0.05),but it was significantly lower than the level of IL-6(353.89?75.88pg/ml)and G-CSF(7229.25?1042.38pg/ml)in the plasma of PRV-HNX inoculated mice;RT-q PCR results showed that IL-6 in brain of mice inoculated with PRV-HNX,HNX-(35)TK/(35)g E,HNX-(35)TK/(35)g E-ORF2 was 7.35?1.96,1.63?0.44 and 1.64?1.02 fold higher than that of mice inoculated with DMEM,respectively,and G-CSF was 82.30?25.00,2.03?1.64 and 1.62?0.52 fold higher than that of mice inoculated with DMEM.There was no significant difference in the level of IL-6and G-CSF between HNX-(35)TK/(35)g E and HNX-(35)TK/(35)g E-ORF2 groups(P>0.05),but it was significantly lower than that in PRV-HNX group(P<0.01);All the mice in PRV-HNX group died on the day 6 post inculation,while none mice in HNX-(35)TK/(35)g E and HNX-(35)TK/(35)g E-ORF2 groups died during the observation period,nor showed any pruritus,ruffled fur or other clinical symptoms,indicating that even after inserting the PCV3 ORF2 gene,the recombinant virus still maintains good safety profile,and there is no obvious difference in their ability in inducing inflammatory responses.3.Immunogenicity of HNX-(35)TK/(35)g E-ORF2 recombinant virusSix weeks old BALB/C mice were immunized with DMEM,HNX-(35)TK/(35)g E,HNX-(35)TK/(35)g E-ORF2,respectively.Three days later,1m L of blood was collected from the mice orbit.The cells in blood were collected and incubated with PE anti-mouse CD3,FITC anti-mouse CD4,APC anti-mouse CD8a and APC/cyamine7 anti-mouse CD69antibodies at room temperature for 30 min,then analyzed by flow cytometry.The results showed that the percentages of CD3~+T cells in peripheral blood of mice inoculated with DMEM,HNX-(35)TK/(35)g E and HNX-(35)TK/(35)g E-ORF2 were 19.42?1.75%,38.98?0.97%,35.78?2.65%,CD3~+CD4~+T cells were 42.35?2.98%,56.23?2.01%,56.60?2.51%,CD3~+CD8~+T cells were 13.84?0.42%,15.71?2.55%,15.25?2.62%,CD3~+CD69~+T cells were 2.48?0.94%,18.02?1.86%,17.00?0.90%,CD4~+CD69~+T cells were 0.78?0.47%,3.21?0.74%,2.91?0.90%,CD8~+CD69~+T cells were 1.68?1.64%,13.67?4.25%,13.06?3.47%.The data showed that both HNX-(35)TK/(35)g E and HNX-(35)TK/(35)g E-ORF2could stimulate cellular immune response,and there was no significant difference between them.Six weeks old BALB/C mice were immunized with DMEM,HNX-(35)TK/(35)g E,HNX-(35)TK/(35)g E-ORF2,respectively.After 14 days,the mice were immunized again with the same virus.After immunization,blood was collected from mice tail vein every week,and the serum was obtained for serology study.Blocking ELISA results showed that the recombinant virus could elicit antibody against PRV-g B in mice.Indirect ELISA,IFA and IHC results showed that the recombinant virus can elicit PCV3-Cap antibody in mice,and the antibody could recognize PCV3-Cap protein expressing Sf-9 cells,PCV3 infected PK-15 cells,lymphoid and lung tissue of PCV3 infected pigs.4.Protective effect of recombinant virus on PRV-HNX infected miceThirty-five days after immunization,all groups were inoculated with PRV-HNX through the foot pad of hind limbs.It was observed that the mice in DMEM inoculation group began to pruritus,ruffled fur on the third day inoculation,and bit the injection site while rotating,all the mice died on the seventh day post inoculation.No mice died in the HNX-(35)TK/(35)g E and HNX-(35)TK/(35)g E-ORF2 immunized groups during the observation period.In the DMEM inoculation group,local perivascular lymphocytes were infiltrated in the brain of mice to form a vascular cuff;lymphocytes and microglia proliferated under the cerebral cortex,and nodules were formed in the local area;diffuse neuronal staining was deepened throughout the brain,degeneration,neurophilic phenomenon appears.HNX-(35)TK/(35)g E and HNX-(35)TK/(35)g E-ORF2 immune group had no obvious pathological changes in brain tissue.This indicates that after expressing PCV3-Cap,the recombinant virus does not its change immune efficacy against pseudorabies virus.In conclusion,in this study,the recombinant PRV expressing PCV3-Cap protein was constructed for the first time.The mice experiment confirmed its safety,and it can significantly activate the cellular immune response and elicit PCV3-Cap specific antibodies.It lays a foundation for the prevention of PR and PCV3 related diseases. |
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