Inhibition Of IBDV Replication By Gga-miR-130b And Gga-miR-454 Via Targeting Viral Genome And Cellular SOCS5&6

Infectious Bursal Disease(IBD)is an acute,highly contagious infectious disease that affects chickens across the world.Its infectious agent,Infectious Bursal Disease Virus(IBDV),can cause damage to pro-B or pre-B cells,resulting in immunosuppression,which leads to failure of vaccination and increasing the susceptibility of chickens to other pathogens.MicroRNAs(miRNAs)are a class of single-stranded RNA molecules of approximately 22 nucleotides that regulate the post-translational modification of gene expressions by partly complementing.At present,miRNAs have been reported to be involved in various cell-life activities and play an important role in the host's antiviral response.However,the role of miRNAs in IBDV infection and pathogenesis is still unclear.In this study,we used IBDV to infect DF-1 cells and detected the changes of miRNA expressions in host cells before and after IBDV infection.Based on the high-throughput sequencing results,we chose 8 miRNAs for further analysis.Among them,we found that miR-130b and miR-454 could significantly inhibit the expression of viral protein.In addition,using qRT-PCR,Western Blot,IFA and TCID50 assay,we verified that miR-130b and miR-454 were able to inhibit the replication of IBDV in DF-1 cells.Furthermore,we detected the expression of type I interferons in DF-1 cells after stimulation with poly(I:C)or infection with IBDV,and found that miR-130b and miR-454 significantly up-regulated the mRNA levels of IFN-(3 and transcriptional factors-IRF3 and p65.These results demonstrate that miR-130b and miR-454 can inhibit IBDV replication and enhance the expression of type 1 interferons in host cells.To explore the mechanism underlying the inhibition of IBDV by miRNAs,we used multiple bioinformatics software to predict the targets of miRNAs in the IBDV genome and host cells.The results showed that miR-130b and miR-454 could directly target IBDV genome,and we also identified that cytosolic Suppressors Of Cytokine Signaling 5&6(SOCS5,SOCS6)in host cells are target genes of miR-130b and miR-454 respectively.To verify the effect of miRNAs on their target genes,we used qRT-PCR,Western Blot and dual luciferase reporter gene assays to examine the expression of target genes and found that miR-130b and miR-454 could inhibit the transcription and translation of their target genes.Since the SOCS proteins are the suppressors of the JAK-STAT pathway,we further examined the effect of miRNAs on STATs expression and STAT1 phosphorylation,and found that miR-130b and miR-454 could significantly increase the expression of STATs gene,as well as the phosphorylation of STAT1 at tyrosine701 residue.In conclusion,miR-130b and miR-454 can inhibit viral replication by directly targeting the IBDV genome and its intracellular target SOCS5&6 to enhance host anti-virus response,indicating that miR-130b and miR-454 play important roles in host response to IBDV infection.