The Role Of Host Protein Sap And SOCS3 In The Replication And Subclinical Infection Of Porcine Circovirus Type 2 With Novel Vaccine Development

Porcine circovirus type 2(PCV2)causes economic losses in the swine industry worldwide.Many host proteins that interacted with PCV2 have been identified by proteomic methods,which are mainly related to virus entry,transport and replication.PCV2 subclinical infection causes weight loss,immune failure or develops into PCVAD.But the mechanism is not completely understood.The interaction between host and pathogen,the immune responses may be the potential factors that contribute to the subclinical mechanism of PCV2.Cytokines,heat shock proteins(Hsps),cell penetrating peptide(CPP)are immuno-regulatory factors that can enhance the innate and adaptive immune responses,thus they could be the potential molecular adjuvants for PCV2 cap subunit vaccine.Thus,in depth understanding the interaction between PCV2 and host proteins,revealing the molecular mechanisms of PCV2 subclinical infection and the development of new subunit vaccine can provide important theoretical basis and application prospects for the control of PCV2.The main research contents in this paper are as follows:1.Study on antiviral effects of Serum amyloid P component against PCV2Immunoprecipitation and mass spectrometry analysis were used to screened out the host proteins interacted with PCV2 Cap protein,which the serum amyloid p component(Sap)protein not only interacted with PCV2 Cap,but also confocal micrology observation result showed co-localization of Sap protein with Cap,and Sap interfered the entry of Cap protein into the nucleus.Over-expressed Sap and soluble Sap were able to inhibit PCV2 replication,RNA interference of Sap also proved the result;in addition,by truncating Sap gene,we found that the functional domain of Sap protein was from the N terminal 155aa to 163aa;the two ligand binding sites 155aa and 157aa were mutated into alanine.We found that the N-terminal mutant 155aa,157aa or mutant 155aa/157aa of the Sap completely lost anti-viral activity.The confocal micrology experiment also showed that the mutant Sap proteins could not prevent Cap from entering the nucleus,thereby losing the effect on inhibiting virus replication.2.Suppressor of cytokine signaling 3 plays an important role in porcine circovirus type 2 subclinical infection by regulating proinflammatory responsesIn this study,peripheral blood mononuclear cells(PBMCs)from PCV2-challenged piglets with no significant clinical symptoms had increased expression of suppressor of cytokine signaling(SOCS)3 but no significant changes in proinflammatory cytokines interleukin(IL)-6 and tumour necrosis factor(TNF)-a,which differed from piglets with significant clinical symptoms.SOCS3 production stimulated by TNF-a in PBMCs from PCV2-infected piglets were significantly lower compared to those from the healthy control group.Elevated SOCS3 inhibited IL-6-and TNF-a-mediated NF-kappa-B inhibitor alpha(IKB-α)degradation in PBMCs and PK-15 cells.SOCS3 was also increased in PCV2-infected PK-15 cells,and IL-6 and TNF-a production induced by PCV2 in PK-15 cells was significantly increased when SOCS3 was silenced by siRNA.SOCS3 interacted with signal transducer and activator of transcription(STAT)3 and TNF-associated receptor-associated factor(TRAF)2,suggesting mechanisms by which SOCS3 inhibits IL-6 and TNF-a signaling.SOCS3 plays an important role in PCV2 subclinical infection by suppressing inflammatory responses in primary immune cells.3.Baculovirus expression of the N-terminus of porcine Heat Shock Protein Gp96 improves the immunogenicity of recombinant PCV2 Capsid proteinRecombinant baculoviruses expressing the PCV2 Cap protein and the Heat shock proteins(Hsps)were constructed and the immune responses were examined in mice.The mouse experiments showed that rBac-Cap/Gp96N increased the titers of specific anti-PCV2 neutralizing antibodies and cell-mediated immunity than other baculoviruses.In the present study,Gp96N and Cap co-expression or Cap alone in Baculovirus system were used to immunize PCV2 and PRRSV negative piglets.The pig experiments showed that the levels of anti-PCV2 antibody,proliferative responses of PBMCs,and IFN-y in the rBac-Cap/Gp96N groups were more markedly increased than those in rBac-Cap group.There were no clear clinical signs of infection following PCV2 challenge in pigs inoculated with recombinant rBac-Cap/Gp96N and rBac-Cap,and the relative daily weight gains were obviously higher than those in the challenge control(CC)group.The pathological lesions,extent of viremia,and viral loads of the vaccinated groups were milder than those in the CC group.Meanwhile,the extent of viremia and viral load present in the rBac-Cap/Gp96N group were significantly lower than those in the rBac-Cap group.These results indicated that porcine Gp96N effectively increased the humoral and cell-mediated immune responses of PCV2 Cap.Gp96N presents an attractive adjuvant or immunotargeting strategy to enhance the protective efficacy of PCV2 subunit vaccines in swine.4.Baculovirus expression of the Interleukin 21 improves the immunogenicity of recombinant PCV2 Capsid proteinThe porcine Interleukin(IL)-21 was amplified with PCR and cloned into pFastBac-Dual with different methods.Enzymes digestion and sequencing analysis were used to identify the recombinant plasmids,the recombinant plasmids were then transformed into competent DH10Bac cells,and were screened and identified by twice blue-white plaque assay and PCR.Sf9 insect cells were transfected with recombinant bacmids and cytopathic effect were observed.Recombinant baculoviruses were obtained and identified by Western blotting,immunofluorescence assay.The recombinant Baculoviruses rBac-Cap/IL-21(expressing Cap and IL-21 individually),rBac-Cap-IL-21(IL-21 was fused with Cap gene),rBac-Cap and rBac-IL-21(expressing IL-21 gene)expressed in Sf9 cells and the immunogenicity of recombinant PCV2 Capsid proteins were tested in mice with twice 3 week-interval immunization.Blood and spleen samples were collected at 35 days post immunization,and the PCV2 ELISA antibody,spleen lymphocyte proliferation,cytokine IL-4 and IFN-y were detected respectively.The results of mice experiment showed that the mice vaccinated with rBac-Cap/IL-21,rBac-Cap-IL-21,rBac-Cap all could produce anti-PCV2 ELISA antibody and neutralization antibody.Among them,rBac-Cap-IL-21 produced the highst anti-PCV2 ELISA antibody.Spleen lymphocytes proliferation test showed that IL-21 could improve Cap proteins to produce the specific lymphocytes.In addition,rBac-Cap-IL-21 induced highest IFN-1 among the five groups IL-4 level were not significantly different among these groups.Mice were challenged with PCV2-SH at 35 days post immunization.Mice were sacrificed and spleens were collected for viral loads detection by quantitative PCR at 7,14,21 days post challenge.The viral loads in the spleens in immunized groups were less than that in the challenge group,and among the immunized groups,rBac-Cap-IL-21 efficiently protect mice from PCV2 infection.It indicated that IL-21 could enchanced the humoral immune response and cell-mediated immune response of Cap protein.Proteins expressed by rBac-Cap-IL-21 among all the groups could offer the best efficiency of protection.5.Study in CPPs and HBM effects on the Baculovirus expressing CapIn order to further improve the efficiency of the expression of Cap protein,promote the secretory expression of recombinant protein,facilitate vaccine antigen preparation.In our study,cell penetrating peptides(CPP)including ppTG20,TAT or Honeybee melittin signal peptide(HBM)genes were fused with N terminal of Cap and cloned into pFast-Bac-Dual(under p10 promoter).Enzymes digestion and sequencing analysis were used to identify the recombinant plasmids,the recombinant plasmids were then transformed into competent DH10Bac cells,and were screened and identified by twice blue-white plaque assay and PCR.Sf9 insect cells were transfected with recombinant bacmids and cytopathic effect were observed.Recombinant baculoviruses were obtained and identified by Western blot,immunofluorescence assay and scanning electron microscope(SEM).Experiment results showed rBac-ppTG20-Cap,rBac-TAT-Cap and rBac-HBM-Cap could be expressed in Sf9 cells efficiently,respectively.CPPs fused with Cap expressed well in the baculovirus system,and did not affect the formation of virus-like particles.HBM did not promote secretory expression of Cap protein.These recombinant baculoviruses expressing PCV2 Cap and the target genes would be potential subunit vaccines against PCV2.6.Establishment and application of a PCR for differential detection of porcine circovirus of type 2a and 2bA differential PCR assay based on the specific subsequences of PCV2a and PCV2b was established.Its sensitivity could reach 9.74 fg/μL and 16.3 fg/μL,respectively.And it had no cross reaction with PPV,PRV,PRRSV,EMCV and PEDV.Total 125 PMWS positive lymph nodes samples were tested by this PCR assay.The results showed that single infection rates of PCV2a or PCV2b and co-infection of PCV2a/PCV2b were 8.0%(10/125),41.6%(52/125)and 50.4%(63/125),respectively.Furthermore,five PCV2 isolates were isolated and sequenced from the lymph nodes sample with PCV2a or PCV2b single infection.The results showed that four of them belonged to PCV2b,one PCV2a,which accorded with the results got with the differential PCR.It indicated that this differential PCR assay is highly specific and sensitive that could be a promising method for rapid differentiation of PCV2a and PCV2b in clinical samples.