Molecular Epidemiology And Replication Regulation Of Porcine Circovirus And Its Mechanism Of Invasion And Transportation

Porcine circovirus type 2(PCV2)is the primary etiological agent of post-weaning multisystemic wasting syndrome(PMWS).It is often mixed with other pathogens to cause serious clinical diseases and huge economic losses to the pig industry.Porcine circovirus type 3(PCV3)is a new type of PCV discovered in recent years,which can cause porcine dermatitis and nephropathy syndrome(PDNS)and reproductive disorders of sows.The emergence of PCV3 poses a new threat to the pig industry.It is necessary to carry out studies on the molecular epidemiology and genetic variation of PCV2 and PCV3 in China.This study first investigated the prevalence of PCV2 and PCV3 in different regions of 9 provinces in China,and determined the complete genome sequences of 66 PCV2 isolates and 4 PCV3 isolates.According to phylogenetic analysis,the three main genotypes of PCV2(PCV2a,PCV2 b and PCV2d)were still widespread in pig farms,of which PCV2 d was the main genotype.Based on the PCV3-ORF2 amino acid sequences analysis,PCV3-CN-JL53/PCV3-CN-LN56 belong to PCV3 a subtype,PCV3-CN-HLJ3 belongs to PCV3 b subtype,and PCV3-CN-0710 belongs to PCV3-IM.This study provides scientific evidence for the molecular epidemiology and genetic diversity of PCV2 and PCV3 in China.The genome of PCV2 is small,which encodes limited protein.The replication of the virus depends on the assistance of host cell-related proteins in vitro.We previously identified that the host protein transcription factor ELF4 interacted with the genome of PCV2.ELF4 has the effect of stimulating the secretion of type I interferon in the innate immune system.To clarify the interaction between ELF4 and PCV2 replication,the eukaryotic expression plasmid pCAGGS-HA-ELF4 was constructed and transiently transfected into PK-15 cells.The results of immunofluorescence assay showed that the protein was mainly localized in the nucleus.Using the IFN-? promoter luciferase reporter plasmid to detect the effect of ELF4 on IFN-? production,the results showed that ELF4 could significantly activate IFN-?-luc in a dose-dependent manner;overexpression of the transcription factor ELF4 could significantly enhance the PCV2 proliferation.The viral titer was measured 72 h after infection.Compared with the control group,overexpression of ELF4 could increase the virus proliferation efficiency by 6.5 times indicating that overexpression of ELF4 could promote virus replication.By designing interfering RNA for ELF4,the result indicated that inhibiting the expression of ELF4 significantly reduced the replication efficiency of PCV2.The above results proved that the transcription factor ELF4 could mediate the production of type I interferon and thereby enhanced the PCV2 proliferation efficiency in vitro.PK-15 cells are the main target cells for PCV2 proliferation in vitro.There are few reports on how PCV2 absorbed and internalized in PK-15 cells.In order to clarify the internalization pathway of PCV2 invading PK-15 cells,we observed the internalization kinetics of PCV2 invading PK-15 at different time points by confocal microscope.The results showed that PCV2 only undergoes adsorption under 4?,while endocytosis did not occur.The endocytosis of PCV2 virus particles endocytosed by PK-15 cells gradually increased with time.The virus particles could be observed to gather around the nucleus at 60 minutes.The drug inhibitors of different endocytosis pathways and overexpression of endocytosis-related proteins were used to explore the specific process of PCV2 binding,internalization and infection of PK-15 cells.The results showed that PCV2 invading PK-15 cells was independent of the acidic environment of the endosome,independent of the endocytosis pathway mediated by clathrin,dynamin and caveolin,the removal of cholesterol in the cell lipid raft could promote virus invasion.