Immunomodulatory Effect And Mechanism Of PCV2 Infection On The Pathogenic Process Of PPV

Porcine circovirus type 2(PCV2),as one of the viruses causing immunosuppression,is the main pathogen of porcine circovirus associated disease(PCVAD).Studies have shown that PCV2 infection can invade the immune system,which is mainly manifested by inordinate damage to immune cells,regulate the secretion of most inflammatory cytokines and induce immunosuppress causing the body to be susceptible to other pathogens.Porcine parvovirus(PPV)is an important pathogen causing reproductive disorders in pregnant gilt.The clinical manifestations of PPV infection include abortion,stillbirth,malformation and mummified fetus.In recent years,the infection and pathogenicity rate of PPV increased all the time,and the mixed infection of PCV2 was always detected in PPV-infected pigs,which showed more serious disease symptoms than PPV infection alone causing huge economic losses to the pig industry.The mixed infection ultimately causes huge economic losses to the global pig industry.However,the immunomodulatory effect and mechanism of PCV2 infection on the pathogenic process of PPV is still unclear.In this study,PCV2-infected piglets were further infected with PPV.Then the role of PCV2 in regulating interleukin-10(IL-10),type I interferon-?(IFN-?)and interleukin-12p40(IL-12p40)expression were detected,and the effect of PCV2 on PPV replication were further verified.The study proved the regulatory mechanism of PCV2 Rep in inducing IL-10 expression using the PCV2-infected Porcine alveolar macrophages(PAMs).Then PK-15 and ST cells were infected with PCV2 and PPV,respectively,to investigate the regulatory mechanism of PCV2 infection on PPV-induced IFN-? expression.Finally,PAMs were infected with PCV2 and PPV to study the regulatory mechanism of PCV2 infection on PPV-induced IL-12p40 expression.This study aims to explore the role of PCV2 in the process of PPV infection and pathogenesis and the immune regulation mechanism.These results were as follows:1.PCV2 infection significantly promotes IL-10 expression,inhibits the PPV-induced IFN-?,IFN-? and IL-12p40 expression,and increases the copies number of PPV in serum and target organsThe 28 d old piglets were separately infected with PCV2 or not for 28 d,and then infected with PPV.Blood samples were collected 24 h,48 h after PCV2 infection.ELISA results showed that PCV2 infection could significantly increase the secretion of IL-10(P <0.01).Blood samples were collected 24 h after PPV infection.ELISA results showed that PCV2 infection could inhibit the secretion of PPV-induced IFN-?,IFN-? and IL-12p40(P< 0.01).q PCR results showed that PCV2 infection could inhibit the transcription of ISG15,ISG56,IFIT2 and CXCL10 in peripheral blood monocytes(PBMC)induced by PPV(P <0.01).Furthermore,q PCR results showed that PCV2 infection could significantly increase the numbers of PPV copies in piglet serum when piglets were infected with PPV at 14 dpi and 28 dpi(P < 0.01).Otherwise,the ovary and uterus of piglets were collected after PPV infection for 28 d.The results of western blotting found that PCV2 infection could up-regulate the expression of VP2 in the ovary and uterus(P < 0.01).2.PCV2 Rep activates p38-MAPK signaling pathway to promote IL-10 expression at the late phase of PCV2 infection in PAMsPCV2 infected PAMs,the results of ELISA and q PCR showed that PCV2 infection could induce persistently high expression of IL-10.PCV1,PCV2,PCV2-Cap1 and PCV1-Cap2 infected cells,and the results of ELISA and q PCR showed that PCV2 Cap could induce high expression of IL-10(P < 0.01).PCV1,PCV2,PCV2-Rep1 and PCV1-Rep2 infected PAMs,the results of ELISA showed that when PAMs were infected at 24 h to 48 h,PCV2 and PCV2-Rep1 could significantly induce the secretion of IL-10(P < 0.01);when PAMs were infected at 24 h to 48 h,PCV1-Rep2 could promote the secretion of IL-10(P <0.01);when PAMs were infected at 48 h to 72 h,PCV1-Rep2-induced IL-10 secretion continued to increase(P < 0.01).In addition,the results of q PCR showed that the dynamic changes of IL-10 m RNA expression were basically consistent with protein expression.During r Ad-Rep1 and r Ad-Rep2 respectively infected PAMs,the results of q PCR found that r Ad-Rep2 could significantly upregulate IL-10 transcription(P < 0.05,P < 0.01).Then the results of western blotting showed that r Ad-Rep2 could activate the p38-MAPK signaling pathways.After si RNAs transfected to interfer signal pathways,the results of ELISA found that si-p38 significantly inhibited the secretion of r Ad-Rep2-induced IL-10(P < 0.01).Furthermore,the results of Ch IP showed that r Ad-Rep2 activated the binding of transcription factors NF-?B p50 and Sp1 to the il10 promoter(P < 0.01).PAMs were infected with PCV1,PCV2,PCV2-Rep1 and PCV1-Rep2 for 12 h,24 h and 48 h,respectively.The results of western blotting showed that PCV1-Rep2 upregulated the level of p-p38-MAPK at 48 hpi;the results of Ch IP found that PCV1-Rep2 enhanced the binding of NF-?B p50 and Sp1 to il10 promoter by activating the p38-MAPK signaling pathway at 48 hpi(P < 0.05).Finally,ELISA results showed that si-TDG could significantly inhibit the secretion of Rep-induced IL-10(P < 0.01).The results of q PCR showed that si-TDG significantly inhibited the level of PCV2-induced IL-10 m RNA(P < 0.01).The Ch IP results showed that si-TDG significantly inhibited the binding activities of PCV2-induced NF-?B p50 and Sp1 to il10promoter(P < 0.05).3.PCV2 infection mediating p38-MAPK-USP21-STING signaling pathway inhibits PPV-induced the expression of IFN-?The PK-15 and ST cells were separately infected PCV2 for 72 h,and then challenged with PPV.q PCR results showed that PCV2 infection could inhibit the expression of IFN-?and ISGs m RNA induced by PPV after PPV infection for 8 h(P < 0.01).The results of dual luciferase assay showed that PCV2 infection could inhibit the activities of ifn? promoter induced by PPV after PPV infection for 24 h(P < 0.01).The results of q PCR showed that,PCV2 could significantly promote the PPV copies number after PPV infection for 36 h(P< 0.01).PCV2 infected PK-15 cells for 72 h,and then challenged with PPV.The results of western blotting showed that PCV2 inhibited the levels of p-IRF3,p-TBK1 and the p-IRF3 transfer from the cytoplasm to nucleus induced by PPV.The results of Co-IP found that PCV2 infection could inhibit the level of STING-K63 ubiquitination induced by PPV.Furthermore,PCV2-infected PK-15 cells incubated with c GAMP,the results of q PCR and dual luciferase assay showed that PCV2 could significantly inhibit c GAMP-induced IFN-?transcription and ifn-? promoter activity(P < 0.01);western blotting results found that PCV2 infection could inhibit the c GAMP-induced levels of p-IRF3,p-TBK1 and the p-IRF3 transfer into nucleus;Co-IP found that PCV2 infection could inhibit c GAMP-induced ubiquitination of STING-K63.After si RNAs interfer the signaling pathways,the results of q PCR proved that si-p38 significantly attenuated the inhibition of PCV2 infection in c GAMP-induced IFN-? expression(P < 0.01);Co-IP found that si-p38 could attenuate the inhibition of PCV2 infection in c GAMP-mediated STING-K63 ubiquitination.Through the transfection of si-USP21(ubiquitin specific peptidase 21),the results of q PCR proved that si-USP21 significantly attenuated the inhibition of PCV2 infection in c GAMP-induced IFN-? expression(P < 0.01);Co-IP showed that si-USP21 also could attenuate the inhibition of PCV2 infection in c GAMP-mediated STING-K63 ubiquitination.Finally,si-p38-transfected cells were infected with PCV2 for 24 h ? 72 h.The results of western blotting found that si-p38 could decrease the PCV2-induced p-USP21 level.4.PCV2 Cap and PCV2 Rep inhibit PPV-induced IL-12p40 expression at different stages of infectionPCV2-infected PAMs were further infected with PPV,respectively.The results of ELISA and q PCR showed that PCV2 infection inhibited the expression of IL-12p40 induced by PPV(P < 0.01).Through r Ad-Blank,r Ad-Cap2 and r Ad-Rep2 infected PAMs,respectively.The results of ELISA and q PCR showed that PCV2 Cap and PCV2 Rep significantly inhibited PPV-induced IL-12p40 expression(P < 0.01).PAMs were infected with PPV after PCV1,PCV2,PCV1-Rep2,PCV1-Cap2,PCV2-Rep1 and PCV2-Cap1 infected,the results of q PCR showed that PCV2 Cap significantly inhibited PPV-induced IL-12p40 expression at the early stage of infection(P < 0.01);but PCV2 Cap and PCV2 Rep significantly inhibited PPV-induced IL-12p40 expression at the late stage of infection(P <0.05,P < 0.01).Meanwhile,the results of western blotting showed that PCV2 infection could inhibit PPV-induced p-I?B and the transfer of p65 into nucleus.Otherwise,the results of Ch IP assay showed that PCV2 infection significantly inhibited PPV-induced the binding activity of NF-?B and Sp1 to il12 B promoter(P < 0.01).The dual luciferase assay showed that PCV2 infection significantly inhibited PPV-induced p65 promoter activity(P< 0.01).Cells transfected with si RNAs,the results of q PCR showed that si-Akt and si-p38 significantly attenuated the inhibition of PCV2 infection in PPV-induced IL-12p40 m RNA(P < 0.01);Besides,si-Akt and si-p38 significantly attenuated the inhibition of r Ad-Cap2 in PPV-induced IL-12p40 m RNA(P < 0.01);However,only si-p38 could attenuated the inhibition of r Ad-Rep2 in PPV-induced IL-12p40 m RNA(P < 0.01).Cells transfected with si RNAs,the results of Ch IP assay showed that si-Akt and si-p38 significantly attenuated the inhibition of PCV2 infection in PPV-induced NF-?B and Sp1 to il12 B promoter(P <0.01).Besides,si-Akt and si-p38 significantly attenuated the inhibition of r Ad-Cap2 in PPV-induced NF-?B and Sp1 to il12 B promoter(P < 0.01).However,only si-p38 could attenuated the inhibition of r Ad-Rep2 in PPV-induced NF-?B and Sp1 to il12 B promoter(P< 0.01).This study found that PCV2 infection could inhibit the PPV-induced immune response and promote PPV replication in vivo.Furthermore,the results of cell studies found that PCV2 Rep could activate the p38-MAPK signaling pathway at the late stage of infection and promote the binding of transcription factors NF-k B p50 and Sp1 to il10 promoter promoting the production of IL-10,in which the host protein TDG plays an important role.Otherwise,PCV2 infection increased the level of p-USP21 through the p38-MAPK signaling pathway,which directly inhibited the STING-K63 ubiquitination and the formation of STING-TBK1-IRF3,thereby inhibiting the expression of IFN-?.PCV2 Cap activated PI3K-Akt and p38-MAPK signaling pathways to inhibit PPV-induced IL-12p40 expression at the early stage of infection,while PCV2 Rep activated p38-MAPK signaling pathways at the late stage of infection to inhibit PPV-induced IL-12p40 expression.These results of thesis clarified immunomodulatory effect and mechanism of PCV2 infection on the pathogenic process of PPV,providing a basis for further study on the immune regulation and mechanism of PCV2 infection in the pathogenesis of some pathogens.