Mechanism Study Of Bovine Coronavirus-mediated Autophagy Regulating Viral Infection

Bovine coronavirus(BCoV),which can cause diarrhea in newborn calves,respiratory diseases and adult cattle "winter diarrhea",is one of the main pathogens causing calf death,and its long-term circulation in cattle is difficult to prevent and control,which may be related to the ability of BCoV to have persistent infection.Some coronaviruses use autophagy to evade the body’s innate immunity,causing persistent infection of the virus.The pathogenesis of BCoV is currently unclear.Therefore,this experiment aims to explore the effect and molecular mechanism of BCoV infection regulating autophagy on viral replication.(1)Establishment of an in vitro model of BCoV infection of calf primary small intestinal epithelial cells(Bovine primary small intestinal epithelial cells,BIEC).The duodenum samples of newborn calves were collected,the small intestinal cells were separated by the double-enzyme combined digestion method,and the small intestinal epithelial cells were purified by the phasedifference adherence method;the exogenous virus and mycoplasma were detected by RT-PCR;cytokeratin 18 was detected by IFA Carry out BIEC identification.Then,BIEC was infected with BCoV-002 strain for 6h,12 h,24h,48 h,and 72 h to detect BCoV infection by RT-PCR,IFA and Western blot.Results The cobblestone-like samples were isolated and purified,which could express epithelial cytokeratin 18,and BIEC without exogenous pathogenic contamination.The BCoV-002 strain was able to infect BIEC,and the virus proliferated significantly with increasing infection time.(2)To explore the effect of BCoV regulating BIOC autophagy on viral proliferation.Bi EC and HRT-18 cells were infected with a 10 MOI virus,and the expression of autophagy marker protein LC3B-II at different infection time points at 6h,12 h,24h,48 h,and 72 h was detected by Western blot,and the control group was uninfected cells.BIEC 12 h was then treated with autophagy regulator rapamycin(Rap),chloroquine(CQ)and Wortmannin(Wm)to detect the expression of LC3B-II by Western blot to screen for appropriate concentrations of autophagy regulatory drugs.Rap and CQ infected cells with 10 MOI virus for 24 h after treatment with BIEC,and Wm infected cells with 10 MOI virus for 72 h after treating BIEC,and the expression of BCoV-N was detected by Western blot.The results showed that the expression of LC3B-II before the virus infection BIEC 48 h was significantly lower than that of the control group,the phenomenon of virus inhibition of autophagy was most significant at 24 hours of infection,and the expression of LC3B-II after infection 72 h was significantly higher than that of the control group,and the phenomenon of virus promoting autophagy occurred,while when the virus infected HRT-18 cells,it was found that the virus promoted autophagy at different time points of infection.The screened Rap,CQ,and Wm concentrations were 5 μM,10 μM,and 0.01 μM,respectively,and it was found that rap-promoted autophagy had no significant effect on the expression of BCoV-N at 24 h of viral infection,while CQ inhibited autophagy and promoted the expression of BCoV-N;at 72 h of infection,Wm inhibited autophagy and inhibited the expression of BCoV-N.(3)Explore the molecular mechanism of virus regulation of BIEC autophagy.Infect BIEC with a 10 MOI virus and do a transcriptomics analysis,go through the data with KEGG analysis.BIEC was infected with 10 MOI virus,and the expression of m TOR-dependent pathway key proteins m TOR,AKT,AMPK and Erk1/2 and their phosphorylated proteins were detected by Western blot at 24 h and 72 h,and the expression of non-m TOR-dependent pathway protein Beclin1 was detected.Then transfect the BIEC silencing gene with si RNA-Beclin1,detect the expression of Beclin1 and LC3B-II by Western blot,test the expression of BICOV-N at 24 h and72h of infection with Western blot,detect the expression of BCoV-N at 24 h and 72 h of infection by Western blot.Infect BIEC with 10 MOI virus and detect the expression of autophagy tidal integrity marker protein P62 at 24 h and 72 h by Western blot.The results showed that after the virus was infected with BIEC,the interferon genes such as IP10/CXCL10,IRF7,IRF9,Mx A,OAS,IFI16 and the interferon genes such as IL-1A,IL-1B,IL-6,IL-8,IL-33,IL-18 RAP,TNFSF10 and other pro-inflammatory factor genes were upregulated,and the cell membrane gene components such as ITGA8,ITGA10 and ITGB8 were downregulated.Among them,the lysosomal-generating precursor gene FGE and the autophagosome-generating inhibition gene ITPR1 were down-regulated,indicating that the virus may promote autophagosome production and inhibit the production of lysosomals.It was later found that the virus promoted the expression of p-AKT and p-Erk1/2 at 24 h of infection,but did not have a significant effect on the expression of p-m TOR and p-AMPK,and did not have a significant effect on the expression of p-m TOR at 72 h of infection.The virus promotes the expression of Beclin1 at 24 hours of infection,but does not change significantly at 72 hours,indicating that the virus may regulate autophagy through the Beclin1 pathway of non-m TOR-dependent pathways.After that,it was found that the silencing Beclin1 gene could significantly inhibit autophagy,and after silencing,there was no significant change in BCoV-N expression when the virus was infected for 24 hours,and the expression of BCoV-N was significantly reduced at 72 hours of infection,indicating that inhibition of autophagy by silencing Beclin1 could inhibit viral replication.Finally,the expression of P62 was significantly reduced at 24 hours compared with the control group and significantly increased at 72 hours compared with 24 hours,indicating that BCoV induced incomplete autophagy.In this study,an in vitro model of BCoV infection with BIEC was successfully established;inhibition of BIOC autophagosome degradation can promote viral replication,inhibition of autophagosome production can inhibit viral replication;BCoV induces incomplete autophagy of BIEC by promoting Beclin1 during infection,and silence of Beclin1 can inhibit viral replication.This experiment provides a theoretical basis for uncovering the molecular mechanism of BCoV causing persistent infection.