The Effects Of Orf Virus Infection On The Expression Of CircRNAs,mRNAs And MiRNAs In Host Cells

Orf virus(ORFV)is a linear DNA double-stranded virus.It mainly infects goats and sheep,causing huge economic losses to animal husbandry of the world.Currently,the research on ORFV mainly focuses on the function analysis of virulence genes.Reports on the interaction mechanism between ORFV and host cells by high-throughput sequencing are very limited and the research of circRNAs in this field is blank.CircRNAs,mRNAs and miRNAs play important roles in viral infection.Therefore,CircRNA,mRNA and miRNA sequencing were performed in ORFV-infected goat skin fibroblast cells(GSF cells),and differentially expressed circRNAs,mRNAs and miRNAs were analyzed,hoping to provide new clues for pathogenic mechanism of ORFV and the interaction mechanism between ORFV and host cells.The main research contents and findings are as follows:1.We successfully constructed a GSF cell model infected by ORFV,laying foundation for circRNA,mRNA and miRNA research.2.For the first time,circRNA sequencing was applied to the research on interaction between ORFV and host cells.At 6h.p.i,9979 and 10844 circRNAs were predicted in the uninfected group(GSF)and infected group(OV),respectively.More than 98%of the circRNAs were exon circRNAs;151 differentially expressed circRNAs were obtained,of which 59 were up-regulated and 92 were down-regulated;GO and KEGG enrichment analyses were performed on the parental genes of differentially expressed circRNAs.The results indicated that 18 circRNAs were found to be involved in cellular immune response to ORFV infection;Nine differentially expressed circRNAs were selected for qRT-PCR validation,in which circRNA1001,circRNA1684 and circRN3127 were down-regulated,circRNA7880 expression was up-regulated,consistent with high-throughput sequencing results;Furthermore,the circRNA-miRNA regulatory network was successfully constructed.3.Transcriptome sequencing was carried out in GSF cells at 18h.p.i and 30h.p.i..In 18h.p.ivsGSF,619 genes were up-regulated and 110 genes were down-regulated.In 30h.p.ivsGSF,3205 genes were up-regulated and 755 genes were down-regulated.In 30h.p.ivsl8h.p.i,1418 genes were up-regulated and 258 genes were down-regulated.GO enrichment analysis indicated differentially expressed genes were mainly involved in apoptosis,cell cycle,immune response,inflammatory response and defense to viral infection;KEGG enrichment analysis indicated that differentially expressed genes were mainly involved in cell cycle,TNF signaling pathway,p53 signaling pathway,Toll-like receptor signaling pathway,NF-?B signaling pathway,PI3K-Akt signaling pathway and apoptosis,etc.4.The miRNA sequencing was carried out in GSF cells at 18h.p.i and 30h.p.i..In 18h.p.ivsGSF,98 miRNAs were up-regulated and 42 miRNAs were down-regulated;In 30h.p.ivsGSF,154 miRNAs were up-regulated and 67 miRNAs were down-regulated;In 30h.p.ivs18h.p.i,48 miRNAs were up-regulated and 37 miRNAs were down-regulated;The target genes of differentially expressed miRNAs in the three comparison groups were mainly enriched in Wnt signaling pathway,MAPK signaling pathway,NF-?B signaling pathway and TNF signaling pathway.This indicated that differentially expressed miRNAs were involved in host cell immune response to ORFV infection.5.Seven differentially expressed miRNAs(TPM>50)shared in 30h.p.ivsGSF and 18h.p.ivsGSF were selected for qRT-PCR verification.Among these,cfa-let-7a_R+2 was the largetly up-regulated;It had 14 target genes(FPKM>10);After transfection with cfa-let-7a_R+2 mimic for 48 h,qRT-PCR indicated that cfa-let-7a_R+2 significantly down-regulated TEX261,B3GAT3,RNF7,TGFBR3,THBS1 and TIMM17B mRNA expression levels;After transfection with cfa-let-7a_R+2 mimic for 48 h,Western blot indicated that cfa-let-7a_R+2 significantly down-regulated the protein expression level of THBS1;Double luciferase reporter gene assay indicated that cfa-let-7a_R+2 directly targeted wild-type pmirGLO wt-THBS1-3'UTR,inhibiting firefly luciferase activity.Since THBS1 has a function of inducing apoptosis,we speculate that cfa-let-7a_R+2 which is induced after ORFV infection,directly targets THBS1-3'UTR and down-regulates THBS1 mRNA and protein expression,thereby inhibiting cell apoptosis and promoting the replication of ORFV in cells.