Damage Of Porcine Circovirus Type 2 To Porcine Luteal Cells And The Mechanism

Porcine circovirus type 2（PCV2）is widely infected in pigs,causing reproductive failure of sow and serious economic losses to pig industry.Studies have shown that PCV2 antigen can be detected in the corpus luteum of sows,but its influence on corpus luteum and its mechanism are not clear.In this study,we first detected the infection of PCV2 in corpus luteum of porcine ovary collected from slaughterhouse and observed its pathological changes by histomorphology.We further isolated the primary corpus luteum cells and study the effects of PCV2 on cell proliferation,apoptosis,hormone secretion and the expression of related signal molecules.The mechanism of PCV2 on corpus luteum was preliminarily explored.This study can provide a theoretical basis for the ovarian dysfunction caused by PCV2 infection in corpus luteum.The main results are as following:（1）PCV2 is widely infected in the luteal tissues of sows and causes tissue damage.The PCV2 pathogen was detected in 116 pig corpus luteum tissues by PCR,and the infection rate was 88.8%（103/116）.Immunohistochemistry assay showed that PCV2 was mainly located in the luteal cells and macrophages of the porcine ovaries;and the histopathological observation showed that PCV2 infection caused nuclear shrinkage and vacuolation of the corpus luteum cells and promoted cell apoptosis.（2）PCV2 causes luteal cell dysfunction.Porcine primary luteal cells with high purity were isolated by collagenase digestion.PCV2 could infect luteal cells in vitro and copies of the viral nucleic acid significantly increased by absolute quantitative PCR.CCK8 assay showed that PCV2 with a titer of 1000 TCID₅₀significantly inhibited the proliferation of luteal cells at 48 h post infection（P<0.01）.Flow cytometry and TUNEL analysis showed that PCV2 infection significantly promoted the apoptosis of luteal cells（P<0.05）.With 100 TCID₅₀virus infection,expression of Caspase-3 gene was significantly up-regulate（P<0.01）,but no significant change in the expression of Bax and Bcl-2;when using 1000 TCID₅₀PCV2,the expression of Bax m RNA was significantly up-regulated（P<0.01）,while the expression of Bcl-2and Caspase-3 showed no change.P38 specific inhibitor could inhibit the increase of Bax and Caspase-3,and the decrease of Bcl-2 induced by PCV2 infection.The protein levels of Bax,Caspase-3,Caspase-9 and p-p38/p38 in luteal cells were significantly increased after PCV2 infection（P<0.05）,and p38 inhibitor could significantly inhibit the increase of Bax and Caspase-3 induced by PCV2 infection.These results indicate that 100 TCID₅₀virus infection can promote luteal cell apoptosis,and p38 MAPK signaling pathway is involved in virus induced luteal cell apoptosis.In addition,PCV2 infection with 1000 TCID₅₀significantly inhibited progesterone secretion in luteal cells,and inhibited the m RNA expression of hormone synthase including St AR,3β-HSD and CYP11A1.All the results showed that PCV2 was widely infected in the luteal tissues of sow and caused tissue damage,which may be caused by apoptosis.PCV2 could replicate in the luteal cells,which inhibited the cell proliferation and progesterone secretion,and promoted the cell apoptosis.