Autophagy Mechanism Of H₂O₂ In Promoting Porcine Circovirus Type 2 Replication In Vitro

Porcine circovirus 2(PCV2)is a small DNA virus with single-stranded circular genome.PCV2 was identified as an essentiall causative agent of post-weaning multisystemic wasting syndrome(PMWS),which is characterized by lymphadenectasis,decreased weight gain,jaundice and dermatitis.PMWS most commonly affects pigs between the ages of 5-18 weeks,with a high mortality rates.However,the morbidity and severity of PCVD varies significantly in different country and different area of the same country,It has been reported that PCV2 replication and PCVD development are linked to co-infection,inmmune stimulation,animal management or other factors,but the mechanism of replication and pathogenesis of PCV2 remains poorly understood.Hydrogen peroxide(H2O2)is a classic compound to induce oxidation.Reactive oxygen species(ROS)is a collective term that describes the chemical species that are formed upon incomplete reduction of oxyen and includes H2O2,O2-,-OH.Autopahgy is a catabolic cellular process conserved in all eukaryotes that involves the degradation and turnover of cytoplasmic components by lysosomal fusion.Viruses could regulate autopagy in infected cells,but detailed mechanisms are elusive and may vary with virus types.On the one hand,autophagy contributes to the recognition of viruses by innate immune system,and helps phagocytes to make an effective phagocytosis and clear extracellular or intracellular viruses.On the other hand,the virus which has the capacity of rapid evolution to respond to the changes in the host cells can make service for its replication through using autophagy.Recent study found that H2O2 and autophagy could promote replication of PCV2,individually,but the exact mechanism is still unclear.This study was conducted to investigate the effect of H2O2 on the promotion of PCV2 replication in PK-15 cells,then we explored the potential mechanism by which H2O2 affects PCV2 replication from the autophagy perspectives,in order to provide a basis for further study of PCV2 replication and pathogenesis.The research contents were as follows:Experiment 1:The effect of H2O2 on regulating porcine circovirus type 2 replication in vitroInfected with PCV2 for 12 h,PK-15 cells were treated with different concentrations of H2O2(0,10 50,100 μM)for 48 h.Cells viability,DNA copies of PCV2 and amount of PCV2-infected cells were measured by MTT,Real-time PCR and immunofluorescence,respectively.The result showed that the viability were not significantly changed after cells were treated with different concentrations of H2O2(0,10,50,100 μM),only significantly decreased in a high concentration of 200 μM and 400 μM.PCV2 DNA copies and PCV2-infected cells were significantly increased in PK-15 cells after treated with H2O2 within the safety concentration for 48h,which indicated that H2O2 could promote the replication of PCV2 in PK-15 cells.In addition,we also studied the effects of PCV2,H2O2 to intracellular redox status.Results showed that GSH decreased significantly and ROS increased significantly after PCV2 infection or H2O2 treatment,and PCV2 induced more aggressively oxidative stress when PK-15 cells co-incubated with H2O2.Experiment 2:Autophagy mechanism of H2O2 in promoting porcine circovirus type 2 replication in vitroIn order to explore the effect of H2O2 regulating autophagy,treated with different concentrations of H2O2(10 50,100 μM)or 100 nM Rapamycin for 48 h,PK-15 cells were analyzed by western blotting using anti-LC3/anti-actin antibody.The ratio of intensity of LC3-II to actin was calculated to present the autophagic level.The result showed that the ratio of intensity of LC3-II/actin was significantly higher than the contol after treated with H2O2.Then,the GFP-LC3 formation and ultrastrucure of PK-15 cells were observed by immunoconfocal microscopy and transmission electron microscopy.The result showed that H2O2 induced formation of GFP-LC3 punctate aggregation autophagic vacuoles in PK-15 cells.In order to explore the autophagic mechanism of H2O2 promoting PCV2 replication,study was implemented with 3-methyladenine(3-MA)and siATG5.Compared with the control group,3-MA or siATG5 significantly decreased the DNA copies,the expression of PCV2 Cap protein and the number of PCV2-infected cells in PK-15 cells.These results indicated that autophagy promoted PCV2 replication induced by H2O2 in PK-15 cells.