The Study On The Mechanism Of Autophagy Induced By Duck Tembusu Virus

Duck Tembusu virus(DTMUV)is a single-stranded positive-stranded RNA virus,which belongs to the flavivirus family.The disease caused by this virus can cause the decrease of egg production in laying ducks,and the infection of ducklings will lead to growth retardation and neurological symptoms,accompanied by death.At present,the disease is prevalent in 17 provinces and cities in China,which has caused economic losses in the duck industry.Autophagy is a highly conserved intracellular catabolism pathway in eukaryotes,which can remove senescent,aggregated,unfolded proteins and damaged organelles.At the same time,a large number of studies have confirmed that autophagy also plays an important role in virus infection,which can regulate virus replication and host immune response,thus affecting the pathogenicity of the virus.In addition to DTMUV,flavivirus also includes a variety of zoonotic viruses,such as West Nile virus(WNV),Japanese encephalitis virus(JEV),dengue virus(DENV)and Zika virus(ZIKV).It has been reported that cellular autophagy plays an important role in flavivirus infection and replication,but the specific interaction between DTMUV and cellular autophagy is unclear.Therefore,this study analyzed the relationship between DTMUV and autophagy,the effect of proteins encoded by DTMUV on autophagy,and the related signal pathways of autophagy induced by DTMUV and its important functional proteins.The results showed that DTMUV could significantly induce the expression of LC3-II and autophagy in DEF cells and BHK-21 cells.The cells were infected with DTMUV after being treated with autophagy inducer rapamycin(RAPA)and autophagy inhibitor 3-MA,respectively.It was found that induced autophagy promoted virus replication,while inhibition of autophagy significantly decreased virus replication.In order to identify the main functional proteins of autophagy induced by DTMUV,three structural proteins(capsid protein C,membrane protein M,envelope protein E)and seven non-structural proteins(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B,NS5)of DTMUV were transfected into DEF cells.The results showed that DTMUV non-structural protein NS3 could induce autophagy,and we verified the induction of autophagy by NS3 protein by western blot,immunofluorescence and transmission electron microscopy.In addition,in order to further clarify the signal pathway of autophagy induced by NS3 protein,we used the specific inhibitors of ERK2 U0126 and si RNA to inhibit the signal pathway mediated by extracellular signal-regulated kinase 2(ERK2),and found that the level of autophagy induced by NS3 protein decreased,indicating that ERK2 is involved in autophagy induced by NS3 protein.Finally,by inhibiting NS3-induced autophagy,compared with the control group,it also proved that NS3-induced autophagy can also promote DTMUV replication.To sum up,NS3 induces autophagy through ERK2 signaling pathway,which further promotes virus replication.The above results laid a foundation for further study of the relationship between DTMUV and autophagy,and deepened the understanding of the pathogenesis of DTMUV.