Proteomics And Molecular Mechanisms Of Autophagy In PEDV-infected Vero Cells

The new porcine epidemic diarrhea(PED)outbreaks caused by porcine epidemic diarrhea virus(PEDV)variant has been documented all over the world,resulting in devastating economic losses to swine industry worldwide.PED has attracted the attention of an increasing number of researchers.It is particularly important for us to pay more attention to the molecular epidemiological survey and research on pathogenesis.In the present study,three positive monoclonal antibodies(Mc Abs)specific for PEDV were generated and provided basis for the development of clinical diagnosis method and pathogenesis study of PEDV.We performed an i TRAQ-based comparative proteomic analysis of Vero cells infected with virulent and CV777 vaccine strain-like strains of porcine epidemic diarrhea virus.The differential protein expression profiles of Vero cells infected with both PEDV strains were generated,which might accelerate our understanding of the pathogenic mechanisms involved in different PEDV infections.Based on the proteomic analysis,the specific function of autophagy in the process of PEDV infection was investigated,which might facilitate our understanding of the pathogenesis of PEDV infection and provide novel insights into the development of effective therapeutic strategies.The main contents are as follows:1.Generation of McAbs specific for PEDV and its biological characteristic analysisThree positive McAbs specific for PEDV were generated and characterized on PEDV S1 D protein as immunogen.The titers of these three Mc Abs were all above 1:106.The immunoglobulin subtypes of three Mc Abs were all Ig G 2a with ? light chain.They were proved to have good reactivity with S1 protein expressed in baculovirus expression system and native PEDV,by western blot and indirect immunofluorescence assay,respectively.No cross reaction was observed with common viruses causing intestinal diseases,such as transmissible gastroenteritis virus(TGEV)and porcine rotavirus(RV).In addition,these Mc Abs can be applied to immunohistochemical analysis of PEDV infected intestines.2.Development of IFA method to rapidly detect PEDV in intestinesCombining the frozen section technique with hematoxylin-eosin(HE)staining,McAb-based IFA was established to rapidly detect PEDV in intestines.The method can specifically recognize PEDV in intestines,as well as provides a visual pathological damage.One hundred and two intestinal samples were separately detected by our in-house IFA and RT-PCR,and the positive detection rates were 97% and 92% respectively.The overall coincidence rate of the two methods was 91.2%,indicating its potential application for rapid PEDV detection.3.i TRAQ-based comparative proteomic analysis of Vero cells infected with virulent and CV777 vaccine strain-like strains of porcine epidemic diarrhea virusTo dissect out the underlying pathogenic mechanism of virulent PEDV and clarify the differences between virulent and CV777 vaccine strain-like PEDV infections,we performed an i TRAQ-based comparative quantitative proteomic study of Vero cells infected with both PEDV strains.A total of 661 and 474 differentially expressed proteins in Vero cells were identified upon virulent and CV777 vaccine strain-like isolates infection,respectively.The differences regarding cellular responses were mainly involved in protein synthesis,immune regulation,cellular assembly and organization,signal transduction,and apoptosis.Further studies have revealed that the virulent strain could activate NF-?B pathway more intensively than the CV777 vaccine strain-like isolate,and elicit stronger inflammatory cascades as well.In addition,the PEDV virulent strain suppressed protein synthesis of Vero cells through down-regulating m TOR as well as its downstream targets 4EBP1 and p70S6 K activities,and suppressing e IF2 signaling as well.4.PEDV infection can induce autophagy in Vero cellsOur proteomic study indicated that many autophagy relevant proteins were significantly up-regulated,and autophagy key regulator m TOR signaling was down-regulated during PEDV infection.We inferred that autophagy might participate in the process of PEDV infection.We firstly found that PEDV infection promoted the formation of DMVs by TEM.This observation was further supported by the accumulation of GFP-LC3 dots in virus induced syncytia and the increased expression of autophagic marker proteins.These data indicated that autophagy activity might be triggered by PEDV infection.Moreover,UV inactivation test implied that the effective replication of PEDV was required for autophagy induction.Previous reports indicated that autophagosome accumulation might be attributed to their de novo formation or a block in trafficking to lysosomes for maturation.The cellular state of autophagy can be measured by detecting the degradation of SQSTM1(p62)or using lysosome inhibitor,or a tandem reporter construct m RFP-GFP-LC3.Our results from these tests indicated that a complete autophagy process might be triggered to modulate PEDV infection in Vero cells.5.Autophagy facilitates PEDV replicationIn the present study,the concrete effect of autophagy upon PEDV infection was examined by administrating pharmacological regulators.The yield of PEDV was suppressed when autophagy process was inhibited by 3-MA at the early stage,or by CQ at the late stage.Additionally,the induction of autophagy by rapamycin also enhanced the viral replication.The effect of autophagy modulation on PEDV replication was further evaluated by silencing the two essential endogenous genes,Beclin1 and ATG5.The abolishment of their expression reduced PEDV titer.These results suggested that the induced autophagy might benefit PEDV replication.6.Autophagy has a positive correlation with NF-?B signaling pathwayThe role of autophagy in PEDV induced inflammatory responses was firstly investigated by measuring the level of inflammatory cytokines under the circumstances when autophagy was suppressed by silencing the expression of Beclin1 and ATG5 proteins.Data showed that the inflammatory cytokines were significantly down-regulated when autophagy was inhibited in PEDV infected cells,suggesting the potential involvement of autophagy in PEDV-induced inflammation.Further investigation indicated that NF-?B pathway was suppressed when autophagy was inhibited.Likewise,the administration of NF-?B inhibitor abolished the elevated level of LC3-II after PEDV infection.These data indicated a potential positive feedback loop between autophagy and NF-?B signaling pathway might exist during PEDV infection.To exclude the effect of different PEDV titer on cytokine production,TNF-? induction of inflammatory cytokines experiment in autophagy-deficiency cells was carried out.It can be seen that the expression of inflammatory cytokines induced by TNF-? in autophagy-deficient cells was significantly down-regulated,when compared to the transfected cells of negative control.These results also well supported the potential positive relationship between autophagy and NF-?B signaling pathway induced by PEDV infection.