The Study Of Sequence Analysis Of PCV3 Cap And Its Effect On Cell Apotosis

Porcine circovirus type 3(PCV3)is a newly emerged virus widely existing in the world.It belongs to the circovirus genus of circoviridae family.Its genome structure is similar to that of porcine circovirus type 2(PCV2).It has no envelope and single negative strand DNA virus.It was first found in the United States in 2016 and has potential risk of disease to pigs.Because PCVs are non envelope viruses,capsid protein is the only structural protein,and many amino acids on the surface of PCVs are the recognition epitopes and key sites of neutralizing antibodies,so Cap plays a key role in the process of virus invasion and interaction between virus and host cells.Previous studies showed that PCV2 b with Cap mutant had higher replication efficiency and induced significant cell death compared with wild-type virus.In addition,the mutation of Cap protein ubiquitination sites(K164A,K179 A and K191A)could significantly improve the replication ability of PCV2 and had stronger tissue pathogenicity.These results indicate that Cap protein is closely related to PCV2 replication and pathogenicity.From the discovery of PCV3 to now,there is no report on isolation and culture of wild-type virus.The reports on PCV3 are mainly focused on detection,but there is little research on the biological function of structural protein and non structural protein of PCV3.As the only structural protein of PCV3,the pathogenicity of PCV3 may be related to it.It is of great significance to study the functional characteristics of PCV3 cap and other non structural proteins in the pathogenesis of PCV3.The purpose of this paper is to study the mechanism and difference of PCV3 on cell damage,related signal pathway and expression of related cytokines,and to systematically evaluate PCV3 Cap protein induced cell death.1.Bioinformatics of PCV3 Cap protein sequenceIn this study,310 brain tissue samples from pig abortions in Hunan Province from 2015 to 2017 were collected and detected.121 PCV3 representative sequences were screened from Gen Bank and compared with the obtained sequences.The loop region sequence of PCV2 Cap was compared and analyzed according to the PCV2 Cap sequence.The inheritance evolution analysis and prediction of PCV3 Cap epitope were carried out,and the mutation in the predicted epitope was analyzed.The results showed that the positive rate of abortion samples was 10%,and it was increasing year by year(7.5%(5 / 66),9.2%(10 / 109)in 2016 and 11.85%(16 / 135)in 2017).PCV3 was positive in many areas of Hunan Province [(Changsha(39 /310),Yiyang(7 / 310),Loudi(24 / 310),Xiangtan(19 / 310),Zhuzhou(12 / 310),Yongzhou(6 / 310),Changde(2 / 310),Shaoyang(8 / 310),Chenzhou(10 / 310),Yueyang(27 / 310),Xiangxi(4 / 310)and Huaihua(8 / 310)],suggesting that PCV3 existed in many areas of Hunan Province The relationship between reproductive disorders in sows needs further study.Sequence analysis of two PCV3 positive strains showed that the amino acid homology of PCV3 Cap sequence from aborted fetus and 121 representative PCV3 strains was 98.60%-100%.Some amino acid mutations were found in Cap(A184S).The results of this study enrich the diversity information of Cap sequence variation of PCV3 strain,and lay a foundation for further study on the influence of PCV3 Cap sequence mutation on virus virulence and antigenicity.2.PCV3 Cap causes cell growth inhibition and cell deathIn order to determine the effect of PCV3 on cell proliferation,we studied the effect of PCV3 on PK-15,Vero and 3D4 / 2 cells using PCV3 rescue virus as materials.The results showed that PCV3 was similar to PCV2,can cause cell growth inhibition.We speculated that PCV3 induced cell growth inhibition may be related to PCV3 Cap protein.Subsequently,we transfected PCV3 Cap to study its effect on cell proliferation,and found that PCV3 Cap can also cause cell growth inhibition.At the same time,48 hours after transfection of PCV3 Cap protein,we found the cell death by microscope observation.We speculate that Cap protein is closely related to the pathogenicity of PCV3.3.Apoptosis and cell cycle changes of PK-15 cells induced by PCV3 CapIn order to determine the effect of PCV3 Cap on cell death,we observed the PK-15 nucleus at 48 hours after PCV3 Cap transfection by Hoechst33527 staining,and found that there were fragmentation and dissolution of the nucleus in the cells.It is suggested that apoptosis may be related to PCV3 Cap.We used annexin-V / PI apoptosis detection kit to detect the apoptosis level of the different cells(PK-15,Vero)at 48 hours after PCV3 Cap recombinant protein transfection.The results showed that PCV3 Cap induced PK-15 apoptosis.To further determine the role of PCV3 Cap in apoptosis,we used caspase-9 inhibitor(ZLEHD-FMK)to treat PK-15 cells,and then the cells transfected by PCV3 Cap were used for apoptosis detection.The results showed that apoptosis induced by PCV3 Cap was achieved by mitochondrial dependent caspase-9 activation.At the same time,it is found that PCV3 Cap can induce PK-15 cell cycle arrest in S phase.It is worth noting that PCV2 cap can also induce PK-15 cell cycle arrest in S phase and is stronger than PCV3 Cap.The results of this study lay a theoretical foundation for revealing the pathogenicity of PCV3,the mechanism of cell damage and related signal pathway,and provide a reference for further study of PCV3 pathogenicity.