Proteome Analysis Of The Coinfection Of Porcine Circovirus Type 2 And Classical Swine Fever Virus In Vitro

The development of intensive animal production technique and convenient transportation increases the communication between pig herds,brings the opportunity for the breakout and spreading of pathogens and causes coinfection of two or multiple pathogens in pigs.Increasing clinical lines of evidence have shown the coinfection/superinfection of PCV2 and CSFV in pigs,causing serious economic damage to swine industry.Moreover,field trials showed that PCV2 would decrease the protection of CSFV vaccine.However,the interfering mechanism of PCV2 as a DNA virus in the replication of RNA virus CSFV is unknown,as an appropriate coinfection/superinfection model system is still unavailable.In this study,PEG6000 and sucrose density gradient centrifugation were applied to concentrate and purify CSFV virion,which remained infectious to cells,and the titer was 101.5 higher than the original CSFV stock.The purified CSFV virions wereused as antigen to immune New Zealand rabbit,then the specific CSFV serum with high titer were obtained.To efficiently investigate the virus replication and the potential mechanism within host cells during coinfection of PCV2 and CSFV,a model system of PCV2-CSFV coinfection was established.In this model,PCV2 can replicate without CSFV interference,indicating that there was no superinfection exclusion of PCV2 by csfv,but PCV2 infection can reduce CSFV replication in a dose-dependent manner.Viral proteins of PCV2 and CSFV were found to colocalize within the same cells,and CSFV E2 protein can translocate from the cytoplasm to the nucleus.Moreover,there was no difference of PCV2 infection rate and apoptosis induction in PK15 and PK15-CSFV,but CSFV production decreased in a PCV2 dose-dependent manner.However,inactivated virions,viral proteins and genome DNA of PCV2 did not induce apoptosis in cells or decrease CSFV replication,indicating PCV2-induced apoptosis might be the reason for the impairment of CSFV replication in cells,which might explain the serious clinical symptoms associated with PCV2-CSFV coinfection and the failure of the live-attenuated CSFV vaccine in vivo.To further explore the host factors and molecular mechanism involved in PCV2-CSFV coinfection,iTRAQ-based LC-MS/MS proteomic and bioinformatic methods were carried out for analyzing the global changes of cellular samples,including mock-infected PK15(NE),CSFV-infected PK15(SC),and PCV2-infected PK15(SP)and PK15 coinfected with PCV2 and CSFV(PC).LC-MS/MS identified 3932 proteins in triplicate.Comparing with NE,the numbers of up-(fold change>1.200)and down-regulated(fold change<0.833)proteins with statistical significance(P<0.05)were 304 and 198 in SP,60 and 61 in SC,and 196 and 156 in PC,respectively.Dysregulated proteins in proteomic analysis were confirmed using relative quantitative real-time PCR,Western blotting and confocal laser scanning microscopy.Then the hierarchical clustering,GO and KEGG analysis confirmed the dominant role of PCV2 infection in dysregulating protein expression under PCV2-CSFV coinfection.Ingenuity pathways analysis(IPA)of proteins dysregulated in PC/SC showed proteins 14-3-3?,cullin 3,ERK1/2,caspase and NF-?B might play vital roles in the effects of PCV2 infection on CSFV life cycle in vitro.The specifically dysregulated 184 proteins in PCV2-CSFV coinfection were further analyzed by KEGG and IPA.Results showed that proteins of mitochondrial respiratory chain complex ?(Cx ?),Cx ? and Cx ? were down-regulated,suggesting mitochondrial might play an important part during PCV2-CSFV coinfection.Other involved pathways included oxidative phosphorylation,Nrf2-mediated oxidative stress response,tRNA charging,Myc mediated apoptosis signaling and pathways related to molecular metabolism.These data provided references for exploring the cellular pathways and molecular mechanism in the coinfection of PCV2 and CSFV.Taken together,we obtained the concentrated and purified CSFV C-strain and rabbit pAb specifically against CSFV C-strain,and established a coinfection model system of PCV2 and CSFV,with which the effect of PCV2-induced apoptosis on the impairment of CSFV replication was confirmed.Finally,using iTRAQ-based proteomic and bioinformatic analysis,the dominant role of PCV2 and some potential candidate proteins and molecular pathways were confirmed in PCV2-CSFV coinfection.However,the exact functions,effects and molecular mechanisms of these proteins and signaling pathways need further investigation.