Study On The Mechanism Effect Of Autophagy Induced By Bovine Viral Diarrhea Virus On Virus Infection

Bovine diarrhea virus(BVDV)belong to pestivirus of Flaviviridae.According to cytopathic effect of BVDV on epithelial cells,BVDV is divided into two biotypes: cytopathic(CP)and noncytopathic(NCP).Bovine diarrhea virus(BVDV)is widespread throughout the world.BVDV can infect cattle,deeer,and other ruminants,and can cause complication clinical symptom,especially persistently infected cattle may develop mucosal disease which caused largely economic loss.The pathogenesis of BVDV is complex.PI is related with BVDV escaping innate immune response,but the specific mechanism is not completely clear.Autophagy is an evolutionary ancient pathway that plays an important role in many basic physiological processes of the organism,including immunity,survival,differentiation,and homeostasis.Autophagy regulation is often used to treat and prevent important viral diseases.However,the interaction between BVDV and autophagy remains unclear.Therefore,we evaluated the effects of different biotypes of BVDV on autophagy and the effects of autophagy on viral proliferation,cell survival,apoptosis,and synthesis of type I interferon(IFN-I),which are important for the prevention and control of BVD.In this study,the interaction between BVDV and autophagy is mainly studied in the following aspects: ?.The epidemiological survey of BVDV and screening virus strain in Heilongjiang province dairy farms.In order to screen BVDV strains used in this study,the BVDV antigen was detected in 10591 serum samples from four large-scale dairy farm in different regions of Heilongjiang province in order to screen positive samples,the positive rate of BVDV antigen and susceptible period of cattle were investigated in this study.Viruses were isolated using 27 BVDV positive samples from eight different cattle farms in Heilongjiang,and the 5'-UTR and NS2-3 gene regions of these viruses were used to analyze gene homology and phyletic evolution.The results showed that all 27 Heilongjiang BVDVs were of the cytopathic(CP)biotype and gene 1 type,and a total of four subgenotypes were present among these samples,including BVDV-1b(29.63%,n=8),BVDV-1c(29.63%,n=8),BVDV-1m(7.41%,n=2)and BVDV-1o(33.33%,n=9).The first identification of the BVDV 1o subtype occurred in Holstein cows in China.BVDV-1b was found in 87.5%(7/8)of affected animals and was isolated from all 7 cattle with mucosal disease;BVDV-1c and-1m were found in 100%(10/10)of infected animals and were isolated from asymptomatic dairy cattle.Thus,multiple BVDV sub genotypes coexist in the same dairy cattle farm.Some nucleotide sequences were replaced in the NS2-3 gene of all BVDV isolates,and the FH-2 and YL strains occurred in three successive nucleotide deficiencies.Phylogenetic analysis divided the Heilongjiang BVDVs into three different gene groups(A,B,and C)according to NS2-3 gene;81.48%(22/27)of the BVDVs belong to the A groups.The results of this study demonstrate that multiple BVDV sub genotypes are prevalent and that BVDV-1b,-1c and-1o are mainly a risk to cattle in Heilongjiang province,China.In summary,BVDV-1b was identified as the predominant epidemic strain for use as a strain in this study.?.Construction of MDBK cell autophagy detection systemTo evaluate the interaction between BVDV and autophagy in MDBK cell,the GFP-LC3-MDBK system was constructed by genetic engineering teachnology.GFP-LC3-MDBK cells can stably express green fluorescent protein(GFP)labeled autophagy marker protein,tubulin light chain 3B(LC3B).GFP-LC3-MDBK cells can produce significant punctate aggregation of green fluorescence,and up regulation of LC3B-II expression under starvation conditions.The autophagy defect cell line sh BCN1-MDBK and control-sh BCN1-MDBK cell line were constructed by RNA interference technique.In the case of autophagy induced by starvation,the expression of Beclin1 protein was significantly inhibited in sh BCN1-MDBK cell line compared with that in control group,and the transformation rate of LC3B-II was decreased significantly.Above all of the results suggested that these two cell lines can be used for the evaluation of autophagy research.?.Influence of BVDV infection on autophagy in MDBK cellsTo determine the effect of BVDV on autophagy in MDBK cells,the results of transmission electron microscopy and confocal microscopy showed that the number of autophagic bilayer or monolayer structure and the number of GFP-LC3 green fluorescent spots were significantly increased in CP and NCP BVDV infected MDBK cells compared with the control group(P<0.05).The results of Western blot showed that the conversion rate of LC3B-I to LC3B-II was increased significantly(P<0.05).The above results showed that both type CP and NCP type BVDV infection induced the steady-state autophagy of MDBK cells.In addition,after BVDV infected MDBK cells,chloroquine can significantly enhance the conversion rate of LC3B-II.the colocalization of Lysotracker red and GFP-LC3 can be seen,after BVDV infected GFP-LC3-MDBK cells.These results suggest that BVDV can induce complete autophagy.?.The effect of autophagy on BVDV replicationIn order to determine the effect of autophagy on BVDV replication,autophagy was studied by chemical agents,and the autophagy defect cell lines.The results showed that rapamycin induced autophagy increased the replication of CP and NCP type BVDV(P<0.05);3-MA inhibited autophagy reduced replication of CP and NCP type BVDV(P<0.05).The present results confirm that autophagy induced by BVDV infection promotes replication of CP and NCP type BVDV.?.The influence of autophagy induced by BVDV on apoptosis and IFN-I signaling moleculesTo confirmed the effect of autophagy induced by BVDV on apoptosis and IFN-I signaling molecules,CP and NCP type BVDV infected autophagy deficient MDBK cells,We found that sh BCN1-MDBK compared with the control group the cell survival rate decreased significantly.the cell survival rate decreased significantly in CP group especially after BVDV infection 72h(P<0.05);The apoptosis was detected by Annexin V-FITC apoptosis detection kit,the results showed that BVDV HJ-1 and NY-1 infected-sh BCN1-MDBK group compared with the infected-control-MDBK group,the level of apoptosis was increased,especially BVDV HJ-1 infected-group was more significantly after 72 h(P<0.05);The m RNA molecular level of IFN-alpha,IFN-beta,Mx1 and OAS-1 was significantly increased on BVDV HJ-1 and NY-1 infected-sh BCN1-MDBK compared with the control group.Conclusions: Our studies provide strong evidence that BVDV infection induces autophagy that serves to benefit BVDV replication in MDBK cells and impairs the innate immune response.The findings might help to illustrate the pathogenesis of the persistent infection caused by BVDV.