Deep Sequenceing Of Whole Transcriptome Reveals The Cellular Responses To Gcrv Infection And CircRNAs Derived From GCRV

Grass carp hemorrhagic disease caused by grass carp reovirus(GCRV)has seriously affected the development of the grass carp aquaculture industry.China has classified it as a second-class animal disease.Currently,it has failed to find a way which can completely prevent and cure this disease.The RNA sequencing(RNA-Seq)technology can rapidly screen differentially expressed genes,which can analyze the molecular mechanism from the genome level.In order to explore the host cell's response to GCRV infection,we performed RNA-Seq of GCRV-infected CIK cells.We hope to understand the antiviral immunity of cells and the interaction mechanism between GCRV and host cell from the genome level.1.The effect of GCRV infection on the mRNA expression of CIK cellsGCRV-infected CIK cells were subjected to RNA-Seq.Compared with control cells,798 differentially expressed genes were screened out,of which 658 were up-regulated and 140 were down-regulated.GO enrichment analysis showed that the differentially expressed genes were mainly enriched in immune-related items such as innate immune response,inflammatory response,and protein ubiquitination;KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in Immune related signaling pathways such as Toll-like receptor signaling pathways,Toll and Imd signaling pathway and T cell receptor signaling pathway,those differentially expressed genes also enriched in 9 signal transduction pathways such as the tumor necrosis factor signaling pathway.This results showed that GCRV infection not only activated the innate immune response of the cells but also activated the adaptive immune response.2.The effect of GCRV infection on mi RNAs expression of CIK cellssRNA sequencing of GCRV-infected CIK cells yielded 1685 mi RNAs,of which248 were known and 1437 were newly predicted.Compared with control cells,186 differentially expressed mi RNAs were screened out,of which 146 were up-regulated and 40 were down-regulated.1972 genes were obtained through the target gene prediction of differentially expressed mi RNAs,GO and KEGG enrichment analysis showed that most of the target genes are associated with diseases,there are more genes that are involved in human HTLV-I infection and cancer-associated mi RNAs especially.Further analysis of differentially expressed mi RNAs and differentially expressed mRNAs revealed that two up-regulated mi RNAs corresponded to one down-regulated target gene and the eight down-regulated mi RNAs corresponded to 13 up-regulated target genes.Target gene PFKFB4,TG,COL7A1 and ARHGEF4 were regulated by dre-let-7c-5p,Cik-mi R-5272*,dre-mi R-205-5p and Cik-mi R-3547,respectively.All of the four genes belong to gene of enriched Top 20 pathways in KEGG enrichment analysis of target genes of different mi RNAs after GCRV infection.3.GCRV-coded sRNAs analysisThe original sRNA sequencing data of GCRV-infected CIK cells was blasted with the GCRV genome.A large number of virus-derived sRNAs(V-sRNAs)whose size is mainly concentrated at 29-31 bp were obtained.The V-sRNAs distributed on the viral genome positive strand of the dsRNA more than negative strand.Bioinformatics analysis revealed that GCRV may form 50 mi RNAs(V-mi RNAs).The q-PCR detection of five randomly selected V-mi RNAs showed that their expression levels increased with the virus infection.1346 host target genes were predicted from 47 V-mi RNAs,suggesting that the virus can regulate the genes expression of host by encoding V-mi RNAs.GO enrichment analysis showed that the target genes of V-mi RNA were mainly enriched in nuclear,cytoplasm and other items in the cellular component.In the biological processes,the target genes were mainly enriched in transcription ? DNA template?transcription regulation and other items;In the molecular function,the target genes mainly involved the binding of heavy metal ions,ATP binding and other items,those results indicate that V-mi RNAs participates in the control of multiple biological processes of the host.4.The effect of GCRV infection on the expression level of circRNAs in CIK cells3,493 circRNAs were predicted after circRNAs sequencing of GCRV-infected CIK cells.Compared with the control cells,76 differentially expressed circRNAs were obtained,of which 40 circRNAs were up-regulated and 36 were down-regulated.20 circRNAs were selected for PCR sequencing and the PCR sequencing results were basically consistent with high-throughput sequencing results.q-PCR was carried out to detect the expression levels of 8 circRNAs and the q-PCR results were consistent with high-throughput sequencing results.A circ RNA-mi RNA(V-mi RNA)-mRNA regulatory network was constructed,It was found that 9 circRNAs competitively bind 7 mi RNAs and regulate the expression of 17 target genes;5 circRNAs competitively bind 5 viral V-mi RNAs and regulate 10 target genes expression,these 27 target genes are mainly related to metabolism,development,signal transduction and disease occurrence.Those results indicated that host circRNAs can regulate host gene expression through binding to host mi RNAs and viral V-mi RNAs5.GCRV-encoded circRNAs analysisWhether viral transcripts can produce circRNAs by reverse splicing has not been reported today.The circRNAs-sequencing data of GCRV-infected CIK cells was blasted with the GCRV genome,and 32 V-circRNAs which were encoded by GCRV were obtained.PCR product sequencing confirmed that V-circRNAs can generate from GCRV.The junction site analysis of V-circRNAs revealed that there may be a hot spot region in the RNA reverse splicing of GCRV to form V-circ RNA.q-PCR results showed that the expression level of V-circRNAs increased significantly with the infection of the virus.The results of V-circ RNA-mi RNA(V-mi RNA)-mRNA interaction analysis showed that 4 V-circRNAs competitively bind 4 mi RNAs and regulate the expression of 26 target genes;10 V-circRNAs competitively bind 15 viruses V-mi RNAs and regulate the expression of 19 target genes.Those results indicates that V-circRNAs can indirectly regulate target genes through binding to mi RNA/V-mi RNA,and these target genes are mostly related to cell growth,differentiation and apoptosis.V-circ RNA15 and V-circ RNA20 were used to transfect GCRV-infected CIK cells and the results showed that the proportion of late apoptotic cells increased.This results indicates that the virus can produce v-circRNAs by reverse splicing and regulating the expression of host/virus genes.In our study,GCRV-infected CIK cells were subjected to RNA-Seq.The cellular response to GCRV was investigated from the mRNA,mi RNA,and circ RNA levels.It was found that GCRV may produce functional V-mi RNAs and V-circRNAs,indicating circ RNA /V-circ RNA may regulate host cell genes expression through interaction withmi RNA/V-mi RNA.The results provide new clues for understanding the immune response of cells to GCRV infection and interaction between GCRV and the host cells.