Study On MiRNA Expression Profile And Its Regulatory Function To DEF Cells Infected By The Duck Enteritis Virus CHv Strain

Duck enteritis virus(DEV)belongs to the herpesviridae family,alpha-herpesvirinae subfamily,Mardivirus genus and Anatid herpesvirus I species.It can cause acute,hot and fatal infectious diseases in ducks,geese and other waterfowls,and it seriously endangers the development of waterfowl breeding industry all over the world.Compared with other herpesviruses,the research on DEV-encoded miRNAs started is late and only at the sequencing identification stage.The regulatory role of miRNAs between virus and host interaction is still unknown.We used DEV CHv(Chinese virulent strain)to infect duck embryo fibroblast(DEF)cells,then analyzed and identified the changes of the miRNA expression profiles.,so as to find the regulatory relationship between miRNA and virus infection.It lays a foundation for further study on the regulatory role of miRNA on DEV replication,immunity and persistent infection.Our research are as follows:A total of 11,462,557 and 11,836,099 high quality smallRNAs(sRNA)were obtained from DEV CHv-infected and uninfected DEF cell samples using deep sequencing.39 viral miRNAs were detected from the DEV CHv infected sample using theRNAhybrid and PITA analysis.Thirty-one miRNAs were known and included in the miRBase,and another 8were predicted as novel DEV CHv-encoded miRNAs.The 8 novel viral miRNAs were confirmed by stem-loop RT-q PCR.Among 39 DEV CHv-encoded miRNA molecules,only 13 miRNAs sequences and 22“seed sequences”of miRNAs were identical in contrast to the VAC-encoded miRNAs.The 39 DEV CHv-encoded miRNAs were distributed throughout the DEV genome like?-herpesviruses.Bioinformatics software predicted that 41 viral genes were targeted by 38 viral miRNAs and 4703 host genes were targeted by 39 viral miRNAs.The viral immediate early genes,ICP4,US1 and UL54,were also targeted by multiple miRNAs,respectively.According to the predicted results,the dev-miR-D8-3p with the highest expression level and its target-gene US1 were selected as the subsequent research objects.Dual luciferase reporter assay(DLRA)showed that dev-miR-D8-3p could target the 3'-UTR of US1 gene.Western Blot analysis showed that dev-miR-D8-3p overexpression couldn't change the expression level of US1 protein.The host miRNA from DEV CHv-infected and uninfected DEF cells were aligned with two reference species(Gallus and Taeniopygia guttata).598 host miRNAs were obtained,386 of which were coexpressed in DEV CHv-infected DEF cells and uninfected cells.There were 38 miRNAs expressing differentially in the infected samples,13 miRNAs were significantly up-regulated and 25 miRNAs were significantly down-regulated.Prediction results showed that 36 of the 38 dysregulated host miRNAs can target the 3'-UTR of 40virus genes,and 36 differentially expressed miRNAs could target 3939 host genes.GO analysis of the host target genes were involved in complex cellular processes including the immune response,metabolic pathway,autophagy and apoptosis.10 differentially expressed host miRNAs were selected as subsequent research objects.The corresponding miRNA mimics and inhibitors were overexpressed in DEF cells and then DEV CHv strain infected the cells.The results showed these differentially expressed host miRNAs have no direct effect on viral replication.Data from High-throughput sequencing showed that miR-30a-5p was significantly down-regulated and the expression of autophagy-related gene Beclin-1 was significantly up-regulated in DEV CHv-infected DEF cells.The above results were also confirmed by fluorescence quantitative PCR.The predict results by bioinformatics software showed that Beclin-1 is one of the target genes of miR-30a-5p.DLRA showed that miR-30a-5p can target the 3'-UTR of Beclin-1 gene.Western Blot assay,cell proliferration assay(MTT),viral half tissue cell infection assay(TCID₅₀),and viral DNA copy number assay showed that overexpression of miR-30a-5p significantly down-regulated the expression level of the Beclin-1 protein,and significantly reduced the ratio of LC3-II/LC3-I,and upregulated the expression level of P62 protein,resulting in decreased autophagy activity of DEF cells and inhibited replication of DEV CHv.Whereas transfection of miR-30a-5p inhibitor contributed to DEV CHv replication by upregulating Beclin-1-mediated autophagy.In summary,this paper firstly identified the miRNA expression profiles of virus and host after the DEV virulent strain DEV CHv-infected DEF cells.39 viral miRNAs and 598host miRNAs were detected from two samples.The conservatism and function of viral miRNA were predicted and identified in this paper.Then,the regulatory relationships of differentially expressed host miRNA to viral infection were also verified.In addition,this study revealed that miR-30a-5p could affect DEV replication by regulating Beclin-1-mediated autophagy.These findings provided a basis for further exploring miRNA regulatory roles in the pathogenesis of DEV infection and contributed to the understanding of the DEV-host interaction at the miRNA level.