Effect Of Cholesterol 25 Hydroxylase And Magnolol On Replication Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus(PEDV),a member of the alphacoronaviruses(family Coronaviridae,order Nidovirales),is a single-stranded,positive-sense RNA virus.The full genome is about 28 kb in length and composed of 5’UTR,3’UTR and seven open reading frames(ORFs)encoding polyproteins 1a and 1b(PP1a and PP1b),spike(S),nucleocapsid(N),envelope(E),ORF3,and membrane(M)proteins.Small intestinal epithelial cells in vivo primarily are attacked by PEDV which causes an acute watery diarrhea,dehydration,anorexia and vomiting in pigs of all ages.Cholesterol 25-hydroxylase(CH25H)has lately been shown to be a host restriction factor encoding an enzyme that catalyzes the oxidized form of cholesterol to 25-hydroxycholesterol(25HC).Our results showed that overexpression and knockdown assay indicated that CH25H had significant antiviral action against PEDV.Besides,Magnolol was a natural drug extract that inhibited PEDV replication in Vero cells.1.Study on the mechanism of CH25H inhibiting PEDV replicationIn this study,we found that PEDV infection could significantly decrease the expression of CH25H in a time dependent way.And interferon-α treatment cannot change the production of CH25H in Vero cells,which indicated that CH25H is not an interferon-stimulated gene(ISG)in Vero cells.In order to investigate the influence of CH25H on PEDV replication,overexpression and knockdown assay were performed and illustrated that CH25H could inhibit PEDV replication.Furthermore,the enzyme activity products of CH25H,25HC,could significantly inhibit PEDV infection in a dose-dependent way.To study the effect of 25HC on the life cycle of PEDV,we analyzed the function of 25HC on PEDV at the adsorption,internalization,replication and release processes by qRT-PCR.These assays demonstrated that 25HC inhibited PEDV infection by blocking viral entry.At the same time,25HC had a broad-spectrum antiviral effect against PEDV and could significantly repress infection by four strains,including the classic strain(CV777 strain),absent strain(SH strain)and variant strain(YZ strain and MS strain).To investigate catalytic activity whether influenced antiviral effect of CH25H against PEDV.CH25H mutant(CH25H-M),which lacks catalytic activity,was created by site-directed mutagenesis.The overexpression of CH25H-M could also suppress PEDV replication,it indicated that there are other pathways of 25HC to inhibit PEDV infection which is independent of enzyme activity.To further determine the mechanism of CH25H against PEDV,Vero cells overexpressed of CH25H were incubated with PEDV,immunoprecipitates was evaluated by protein mass spectrometry.And DCD was identified and obviously increase PEDV proliferation.Besides,HEK-293T cells were co-transfected with CH25H and DCD.The results elucidated that CH25H significantly degraded DCD in a dose-dependent way and this degradation was independent on catalytic activity.In order to investigate CH25H degrading DCD by which pathway,the apoptosis inhibitor,the lysosome inhibitor and proteasome inhibitor were selected to treat cells.The results illustrated CH25H degraded DCD by lysosomal pathway.Lastly,we also found that CH25H and 25HC performed excellent antiviral activity against porcine transmissible gastroenterit is virus(TGEV),an alphacoronaviruses.This study first revealed that host restriction factor CH25H had antiviral effect against PEDV infections and its mechanism.2.Magnolol effect on the replication of PEDV in Vero cells and its influence mechanismFirstly,to screen 88 drugs of FDA-Approved drugs against PEDV replication.Three drugs were chosen and had antiviral effect against PEDV infection,including Magnolol,(+)-Usniacin and Parthenolide.First of all,a cytotoxicity assay was performed with a Sigma-Aldrich Cell Counting Kit 8 and magnolol and(+)-Usniacin concentration less than 30 μM was the optimal concentration for Vero cells.Next,Treatment with magnolol could significantly inhibit PEDV replication in a dose-dependent method by the detection of Western blotting,qRT-PCR etc.Besides,to investigate whether 25HC had a broad-spectrum antiviral effect against PEDV.Four viral strains of PEDV were used in the experiment,including the classic strain(CV777 strain),absent strain(SH strain)and variant strain(YZ strain and MS strain).The results illustrated that 25HC could significantly repress infection by four strains.Our next experiment was undertaken to determine which step of PEDV infection was inhibited by magnolol.The results demonstrated that magnolol inhibited PEDV infection by blocking viral replication.This study first revealed that magnolol,a natural extract,could inhibit PEDV replication and could provide new insight against PEDV infection in the future.