Infection Of Porcine Circovirus Type 2 In Porcine Follicles And Its Effect On Proliferation,Apoptosis And Autophagy Of Granulosa Cell

Porcine circovirus type 2(PCV2)is a globally prevalent DNA virus,which cause reproduction related diseases such as abortion and early embryo death in sows.Studies have shown that PCV2 can be detected in porcine ovaries,suggesting that this pathogen may damage the sow ovaries and lead to ovarian dysfunction.Granulosa cells are the largest cell population in ovarian follicles,which play important role in development and maturation of ovarian follicles.Whether PCV2 infects porcine granulosa cells is still unclear.In this study,we detected the PCV2 infection in porcine follicles,and further investigated the effect of PCV2 on functions of porcine ovarian granulosa cells in vitro,including cell proliferation,apoptosis and autophagy.In addition,the effect of autophagy of the porcine granulosa cells on PCV2 replication was explored.The main results are as following:1.Infection of PCV2 in porcine follicular fluid and ovarian granulosa cellsIn this study,the nucleic acid in follicular fluid was extracted,and the infection of PCV2 in porcine follicular fluid was detected by PCR.Meanwhile,the infection of pseudorabies virus(PRV)and porcine parvovirus(PPV)was also detected.The results showed that the PCV2 positive rates were 4.2%(5/120),the positive rates for PRV and PPV were 16.7% and 1.7%,respectively.Primary porcine ovarian granulosa cells were isolated by sedimentation method,and the iosltaed cells were FSH-R positive by indirect immunofluorescence assay(IFA).PCV2 could infect the primary porcine ovarian granulosa cells in vitro,and the antigen was mainly located in the nucleus.In addition,the viral load of PCV2 in cells increased significantly with the development of infection time by q PCR.2.PCV2 inhibits cell proliferation and induces apoptosis and autophagy in porcine ovarian granulosa cellsAfter PCV2 infection,obvious cytopathic effect was observed in porcine ovarian granulosa cells under microscope,displaying shrinkage and roundness.CCK-8 assay showed that PCV2 significantly inhibited the cell proliferation when the ovarian granulosa cells were infected for 72 h or longer.PCV2 infection induced cell apoptosis,resulting in chromatin condensation and nuclear fragmentation using the terminal deoxynucleotidyl transferase-mediated d UTP nick end-labeling(TUNEL)assay.Western blot assay showed that PCV2 induced the phosphorylation of JNK,ERK1/2and P38 proteins,up-regulated the expression of pro-apoptotic proteins BAX,Caspase-3 and Caspase-9,significantly down-regulated the levels of autophagy substrate P62 and autophagy initiation complex ATG13,and promoted the transformation of LC3-Ⅰto LC3-Ⅱ。3.Autophagy of porcine ovarian granulosa cells promotes PCV2 replicationRapamycin or chloroquine was used to induce or inhibit autophagy in porcine ovarian granulosa cells,respectively,followed by PCV2 infection.Nucleic acid load of PCV2 in granulosa cells was detected by q PCR.The results showed that the copy number of PCV2 nucleic acid was significantly increased in ovarian granulosa cells after autophagy induced by rapamycin,which was decreased when the autophagy was inhibited by chloroquine.In conclusion,the present study data indicate that there was PCV2 pathogen in porcine ovarian follicle and PCV2 infects and replicates in porcine ovarian granulosa cells in vitr,causing obvious cytopathic effect and cell dysfunction,while autophagy of the ovarian granulosa cells promotes PCV2 replication.