Mi Rnaomic Analysis Of PEDV Infected-SIEC And The Effects Of MiR-221-5p And MiR-615 On The Virus Replication And Its Mechanism

Procine epidemic diaherra virus(PEDV)is a single-strand positive-strand RNA virus that causes porcine epidemic diaherra(PED)and belongs to alpha coronavirus of the Coronavirus genus.PEDV was discovered in Europe in 1971,and the classic strain CV777 was isolated in Belgium in 1978.The recurrent of the emerging PEDV mutant strain in 2010 has caused huge economic losses to the world`s pig industry.PED mainly occurs in piglets,causing acute watery diarrhea with a mortality high rate of 80%to 100%.MicroRNAs(miRNAs)are a class of multifunctional small RNAs.Some miRNAs exert antiviral effects by regulating the host's innate immunity during viral infection.In this paper,a PEDV strain was isolated and identified,and the profile of miRNAs in PEDV-infected swine intestinal epithelial cells(SIEC)were analyzed.Then the effects of miR-221-5p and miR-615 on PEDV replication were studied.The results obtained were as follows:1.Isolation and identification of a local strains in PEDV region and analysis of whole gene sequencesSamples from diarrhea piglets were collected for using virus isolation and identification.A PEDV strain was isolated,sequenced and analyzed.The results showed that the viral genome was 28063 nt in length,including 5'(300 nt)and 3'(500 nt)untranslated region;The results of genome-wide evolution analysis suggested that the isolate belongs to the G2 epidemic strain group,and has the closest genetic relationship with the strain isolated in recent years;The insertion or deletion of S gene of the isolate has not been found.The ORF3 gene was 675 nt,and has no deletion or insertion been found.The isolated was named as CH/HBTS/2017.2.miRomic analysis in PEDV-infected SIEC.Target gene prediction of differential miRNAs was conducted.GO and KEEG analysis of these target genes showed that NF-?B,MAPK,TLR and other signaling pathways involved in the process of viral infection was found.The results of KEEG analysis were shown NF-?B pathway was enriched with a highest score and maybe play an important role.A total of 229 mature miRNAs and 232 precursors were identified.Expression analysis showed that CV777 vs.Mock had 102 differentially expressed miRNAs,and NW17 vs.Mock had 97 differentially expressed miRNAs,of which 78 miRNAs appeared simultaneously in these two differentially expressed genes.3.miR-221-5p inhibits viral replication by targeting the viral genome and activating the NF-?B pathway(1)Using the ViTa database to predict miRNAs which targets the viral genome.The results showed that miR-221-5p targets the most conserved region at the 3'UTR among 30 PEDV strains.Overexpression of miR-221-5p mimics in SIEC,Vero and Marc-145 cells inhibited PEDV replication.PEDV replication was inhibited more strongly in Marc-145 cells.(2)The dual luciferase reporter assay suggested that miR-221-5p can bind to the 3' UTR target region of PEDV.Compared with the innate immunodeficiency Vero cells,miR-221-5p exerts a stronger inhibitory effect on viral infection in Marc-145 cells,suggesting that miR-221-5p may affect the innate immunity of cells.miR-221-5p upregulates the expression of IFN-??IFN-? and ISG15.Further studies revealed that miR-221-5p played roles by activating the promoter of NF-?B pathway.Indirect immunofluorescence assay confirmed the increase of nuclear translocation of p65.Western blot further confirmed that the upregulation of p65 protein in the nucleus was due to the decrease of I?B.(3)The target genes of miR-221-5p was further predicted in Marc-145 by Targetscan and five target genes in NF-?B pathway were screened.The dual luciferase reporter gene assay showed that miR-221-5p can bind to the target regions of SOCS1 and NFKBIA.The overexpression of miR-221-5p down-regulated the expression of these two proteins in Marc-145 cells.Overexpression of SOCS1 and NFKBIA can inhibit NF-?B pathway activation during PEDV infection,further research demonstrating that miR-221-5p activated the NF-?B pathway by down-regulating these two negative regulators.4.miR-615 promotes viral replication by targeting IRAK1 to regulate the expression of type ? interferon(IFN-?)(1)Datas from deep sequencing showed that miR-615 was down-regulated in PEDV-infected SIECs.Overexpression of miR-615 promoted PEDV replication in SIEC.(2)Knockdown of miR-615 can inhibit PEDV replication in SIEC.At the same time,the expression of IFN-I and IFN-III was detected,and the results revealed that both IFN-I and IFN-III were down-regulated.In addition,IFN-?1 and IFN-?3 were more significantly down-regulated.(3)Using the miRanda,RNAhybrid and PITA to predict the target genes of miR-615.Results showed that IRAK1 is one of target genes of miR-615 with the highest binding energy.Dual luciferase reporter assay demonstrated that miR-615 can bind to the target gene region of IRAK1.Knockdown of IRAK1 inhibited the expression of IFN-?1 and IFN-?3 and overexpression of IRAK1 upregulated the expression of IFN-?1 and IFN-?3.Moreover,miR-615 can down-regulate the protein level of IRAK1.Co-transfection of miR-615 with IRAK1 revealed that IRAK1 reversed the down-regulation of IFN-?1 and IFN-?3 and the increase in viral replication,which was inhibited by miR-615 transfection.In summary,a PEDV strain was isolated;PEDV altered the profile of miRNAs expression.miR-221-5p and miR-615 has influence on NF-?B pathway by regulating different target genes,thereby affecting PEDV replication.miR-221-5p activates the NF-?B pathway by down-regulating I?B and SOCS1 to inhibit PEDV replication;miR-615 inhibits NF-?B activation by down-regulating IRAK1 to promote PEDV replication.These results show that NF-?B pathway may play an important role in the process of PEDV infection.This study reveals that miRNAs can affect PEDV replication by regulating the NF-?B pathway,and provided a new scientific data for elucidating the pathogenesis of PEDV.