Prediction The Target Genes Of Duck Enteritis Virus Encoded MicroRNAs And Preliminary Study The Effect Of Dev-miR-D13-5p On Viral Proliferation

Duck enteritis virus (DEV), also known as duck plague virus (DPV), belonging to a-Herpesvirinae of Herpesviridae, which is a highly infectious and high mortality's virus. MicroRNAs (miRNAs) are a class of single-stranded endogenous RNAs of approximately 22 nucleotides in length that can regulate post-transcriptional gene silencing by base pairing with target mRNAs. A large number of studies have shown that virus-encoded miRNAs play an important role in regulating the virus life cycle, promoting cell survival, immune escape, and tumorigenesis. However, it is little known about DEV-encoded miRNAs for its regulatory mechanism. In this paper, we predicted the target genes of DEV-encoded 33 miRNAs by using bioinformatics, validated part of the target genes of dev-miR-D13-5p and discussed the influence on viral proliferation. The results reported as follows:1. Prediction the target genes of DEV miRNAsThe over 40 viral and 900 host target genes of DEV-encoded 33 miRNAs were predicted through bioinformatics software RNAhybrid, miRanda and Targetscan. Besides, using Cytoscape rendered of miRNAs and mRNA interaction network. To further explore the function of DEV miRNAs, we downloaded the GO (Gene Ontology) annotations of host targets from Ensemble database, generated GO classification annotated map by WEGO, and GO analysis revealed 902 host target genes mapped to 42 GO classifications, including 11 cellular component,10 molecular function and 21 biological process. In addition, DAVID analyzed KEGG pathways of host target genes, which showed that 902 genes only a small part of the host genes occurred in 11 signaling pathways, including enrichment of MAPK. signaling pathway genes most.2. Validation the target genes of dev-miR-D13-5pIn order to explore DEV miRNA whether affects viral proliferation, we choose dev-miR-D13-5p as subsequent object in this paper, because virus replication related DEV UL8(ORF) and UL9 3'UTR showed the possibility of higher in possible target genes by predicted results. First, we successfully built target genes'dual luciferase reporter gene wild type vector pmirGLO-UL8(wt) and mutant vector pmirGLO-UL8(mut), then, we co-transfected mimics and wild type and mutant vectors into COS7 cells. After 48 h, measuring firefly luciferase activity and Renilla luciferase activity, and calculating the ratio of both luciferase activity, results showed that, compared to wild type test group (0.3076±0.0289) and contrast (0.4753±0.0497), the extremely significant difference (P< 0.01), mutant test group (0.4119±0.0147) compared with control group (0.4582±0.0435), no significant difference (P>0.05), wild type test group (0.3076±0.0289) and mutant test group (0.4119±0.0147) compared with significant difference (P< 0.01). Therefore, we judged that UL8 and UL9 were real target genes of dev-miR-D13-5p by utilzing double luciferase reporter gene detection system.3.The effect of dev-miR-D13-5p on target genes and viral proliferationWe successfully built dev-miR-D13-5p target genes'eukaryotic expression vectors pcDNA3.1(+)-UL8(ORF) and pcDNA3.1(+)-UL9(ORF+3'UTR), further to co-transfect dev-miR-D13-5p mimic, NC mimic and UL8, UL9 eukaryotic expression vector into COS7 cells, respectively. After 48 h, we tested mRNA level of targets, and found that dev-miR-D13-5p mimic group compared with NC mimic group, the UL8 mRNA expression of dev-miR-D13-5p mimic group down-regulated by 79.91%, the extremely significant difference (P< 0.01), but the UL9 mRNA expression quantity compared with NC mimic group, no significant difference (P>0.05). The results showed that dev-miR-D13-5p suppresses UL8 mRNA expression level, but dose not significantly affect UL9.To explore the effect of dev-miR-D13-5p on viral proliferation, we cultured DEF cells into 24 well-palte, transfected dev-miR-D13-5p and NC mimic into DEF cells after a period of time, and infected DEV after 12 h. We collected cell viral liquor at 24 h,48 h and 72 h, the quantitative detection of DEV DNA copy number. The results showed that dev-miR-D13-5p mimic group (24 h,5.19×104copies/?L; 48 h,1.54×105copies/?L) compared with NC mimic group (24 h,4.83×104 copies/?L; 48 h,2.28×105copies/?L), differences were not significant (P>0.05). At 72 h, dev-miR-D13-5p mimic group (5.86× 105 copies/?l) and NC mimic group (2.57×106 copies/?L), the significant difference (P< 0.05). Hence, getting dev-miR-D13-5p can inhibit viral proliferation.