Establishment And Correlation Analysis Of MicroRNAs And MRNA Expression Profiles Of SVA Infected PK-15 Cells

Senecavirus A(SVA),the member of the family Piconaviridae,genus Senecavirus,was characterized by vesicular fluid on snout and hoof of sows,sometimes accompanied with fever and diarrhea in piglets.In this study,four SVA strains designated SVA C H-DB-11-2015 ? CH-LX-01-2016 ? CH-ZW-01-2016 and C H-DL-01-2016 respectively were obtained from vesicular fluids and tissues of infected sows and piglet and the complete genome were sequenced and analyzed.PK-15 cells infected by SVA were used to screen the differentially expressed miRNAs and differentially expressed genes by high-throughput sequencing and gene chip technique.GO and KEGG analysis of the differentially expressed miRNAs and differentially expressed genes were carried out to understand the role what they play in innate immunity response to SVA.q RT-PCR was used to determine the transcription level of type I interferon(IFN-I)and signal transduction factors mRNA,Western Blot was used to determined the expression of IFN-? protein,and liquid chip technique was used to determine the concentration of cytokine before and after SVA infection.The main results of this study are as follows:(1)Phylogenetic analysis of SVAPhylogenetic analysis show that that these four isolates and three strains isolated previously in our laboratory were distant from 11-55910-3 and SVV-001,and the similarity were 96.4-96.7% and 94.2-94.4% respectively.The 4 SVA strains and CH-01-2015 were clustered together with three Brazillian isolates,while the remaining two strain that were clustered together with US strains.(2)Two small RNA libraries were sequenced and analyzed by high throughput sequencing.The expression patterns of miRNAs in PK-15 cells were analyzed before and after SVA infection.37 miRNAs were significantly up-regulated and 63 miRNAs were down-regulated.The target genes of 100 differentially expressed miRNAs were predicted,and 11469 transcripts of target genes were obtained.These target genes were mainly enriched in metabolic pathways,B cell receptor signaling pathways and pathways incancers.At the same time,new miRNAs we re predicted,254 new miRNAs were significantly up-regulated and only 4 were down-regulated.(3)mRNA expression profile analysis of PK-15 cell before and after SVA infectionThe results showed that 678 genes were up-regulated and 637 genes were down-regulated in SVA-infected cells.GO analysis of the differentially expressed genes showed that differentially expressed genes were mainly involved in inflammatory response and viral defense response.KEGG analysis showed that differentially expressed genes were mainly enriched in immune-related pathways,such as cytokine-cytokine receptor interaction pathway,Toll-like receptor signaling pathway,intracellular DNA sensing pathway and so on.q RT-PCR was used to determine the transcription level of signal transduction factors mRNA,the results showed that the transcription level of IRF7,IFN-?,STAT1,STAT2,IRF9,OAS,PKR and Mx1 mRNA in the cells infected with SVA were significantly higher than those in the control group at nine time points.(4)miRNA-mRNA association analysisDifferentially expressed miRNAs were identified in PK-15 cells after infection with SVA,and 1315 differentially expressed mRNAs were identified and their correlation analysis was conducted.The results showed that ssc-mi R-296-3p,ssc-mi R-339,ssc-mi R-339-5p,ssc-mi R-4334-3p,ssc-mi R-149,ssc-mi R-1306-5p,ssc-mi R-532-3p and ssc-mi R-744 involved in TLR-and RLR-mediated signaling pathways,JAK-STAT signaling pathway and cytokine-cytokine receptor interactions and other natural immune response process,which indicated that they may play a key role in the pathogenesis of SVA infection.(5)Effects of SVA infection on type I interferon and cytokinesWestern Blot showed that IFN-? was significantly expressed in SVA-infected cells.IFN-?,IL-6 and IL-8 were significantly secreted in the early stage of SVA infection,while IL-1?,IL-4 and IL-12 were secreted since 24 hours post infection(hpi).These results showed that SVA infection can activate the host innate immunity,and induce type I interferon and proinflammatory cytokine expression.