Autophagy Of Duck Embryo Fibroblast Cells Induced By Duck Hepatitis A Virus Type 1

Duck hepatitis A virus type 1?Avihepatovirus genus,Picornaviridae family,DHAV-1?causes a highly lethal and rapidly spreading acute infectious disease.Autophagy,a highly conserved lysosomal pathway in eukaryotic cells,is responsible for the degradation of intracellular misfolded or redundant proteins,damaged organelles and intracellular pathogens.However,the relationship of DHAV-1 infection and autophagy is uncertain.This present study focused on that the autophagy induction by DHAV-1 infection in the duck embryo fibroblast?DEF?cells and the effect of autophagy on viral replication and proliferation.The results were as follows:1.Autophagy was activated by DHAV-1 infection in DEF cellsDHAV-1-infected DEF cells were harvested for autophagy detection.Autophagy marker protein microtubule-associated protein light chain?LC3?lipidated from LC3-I to LC3-II via Western blotting detection,the formation and structure of autophagosome-like vesicles was observed by transmission electron microscopy?TEM?,the aggregation of pEGFP-C3-LC3 protein into bright spots to form autophagosome was observed by fluorescence microscopy,and the upregulated transcription level of autophagy-related gene5?ATG5?was detected by relative quantitative real-time PCR?qRT-PCR?method.These results suggested that DHAV-1 infection could induce autophagy in DEF cells.DHAV-1-infected DEF cells were further detected the transcription and expression level of autophagic substrate protein SQSTM1/p62 by qRT-PCR and Western blotting methods.The results indicated that DHAV-1 infection declined SQSTM1/p62 protein transcription and expression.The fusion expression plasmid of pmCherry-EGFP-LC3 was transfected into DEF cells to label autophagosome and observed its expression level by fluorescence microscopy.Accompanied by GFP quenching,yellow-labelled autophagosome and red-labelled autolysosome were observed.Co-localization of Lyso-Tracker Red labelled lysosomes and GFP-LC3 labelled lysosomes was observed under fluorescence microscope.These results showed that DHAV-1 infection both promoted the degredation of SQSTM1/p62 and the fusion of autophagosome and lysosome,indicating that DHAV-1 infection induced complete autophagy flux.2.Autophagy depended on DHAV-1 viral protein integrityDEF cells were treated with equivalent replication-competent DHAV-1,heat-and UV-inactivated DHAV-1 respectively,and detected the expression of LC3 by Western blotting.The results showed that the heat-inactivated DHAV-1 did not induce LC3lipidation,while the replication-competent and UV-inactivated DHAV-1 could induce LC3to lipidate from LC3-I into LC3-II,suggesting that autophagy induction was caused by DHAV-1 viral proteins and UV irradiation could not deprive DHAV-1 of its ability to induce autophagy.All of DHAV-1 genes including VP0,VP1,VP3,2A,2B,2C,3A,3B,3C and 3D were eukaryotic expressed and transfected into DEF cells,respectively.The results of Western blotting detection showed that except 2C and 3D proteins,all of DHAV-1structure?VP0,VP1,VP3?and non-structure proteins?2A,2B,3A,3B and 3C?could induce the lipidation of LC3-I to LC3-II.It might explain the reason for autophagy induction by UV-inactivated DHAV-1 infection was that autophagy induction depended on DHAV-1 protein integrity.3.Autophagy affected DHAV-1 proliferation and extracellular releaseAfter dealing with autophagy agonist rapamycin and inhibitor chloroquine respectively,DEF cells were infected with equivalent DHAV-1 and harvested for viral copies detection by qRT-PCR.The results indicated that intracellular and extracellular viral copies were upregulated by rapamycin while down-regulated by chloroquine treatment.By contrasting difference value of intracellular and extracellular viral copies,it was found that the difference value was smallest in the rapamycin treated group while larger in the chloroquine treated group.These results indicated that autophagy promoted DHAV-1replication and extracellular release.To further detect the effect of autophagy inhibition on DHAV-1 proliferation,DEF cells were pretreated with autophagy inhibitor chloroquine and then infected with DHAV-1,and observed cytopathic effect and analysed viral titers by TCID₅₀.It was observed that when chloroquine inhibited autophagy,viral titiers in DHAV-1 infected cells decreased significantly?P<0.05?,which indicates that autophagy inhibition could restrain DHAV-1proliferation.In conclusion,this study demonstrated that DHAV-1 infection could induce complete autophagy and autophagy flux;autophagy activation depended on DHAV-1 viral protein integrity;autophagy induction could promote DHAV-1 proliferation and extracellular release in DEF cells.