Identification Of Cyprinid Herpesvirus 2-Encoded MiRNA Based On RNA-Seq And The Regulation Of Apoptosis By MiR-C12

With the rapid development of the intensive aquaculture industry,various diseases especial viral diseases outbreak and cause heavy economic losses.Since 2011,a novel communicable viral disease caused by CyHV-2 has resulted in high mortality of silver crucian carp(Carassius auratus gibelio)in China.Therefore,strategies to control CyHV-2 infections are required urgently to reduce the serious losses to aquaculture caused by this viral disease.However,the molecular mechanisms of CyHV-2 pathogenesis are still limited.Fish cell line provided a good operation platform for isolation,identification and propagation of fish virus,and also necessary for investigating the interactions between fish and pathogens.miRNA offered a new perspective to study virus-fish interaction.Here,we studied the mechanisms of the interaction between CyHV-2 and Silver crucian carp from the perspective of miRNA and m RNA.Exploring the roles of miRNAs encoded by CyHV-2 and silver crucian carp host will be benefit for the better understanding of molecular mechanisms of CyHV-2 pathogenesis,help to development of antiviral strategies.The main research approaches obtained in this study are listed as follows: 1.Establishment and characterisation of a novel cell line susceptible to cyprinid herpesvirus 2A new cell line(Gi CF)from the caudal fin of C.a.gibelio was established and characterised.The Gi CF cells predominantly consisted of fibroblast-like cells.Chromosome analysis revealed that 35% of the Gi CF cells maintained their normal triploid chromosome number(3n = 156)at passage 30.The cell line was susceptible to infection with CyHV-2 isolated from diseased C.a.gibelio.Morphological changes represented by typical cytopathic effects were induced in the cells by CyHV-2 at 72 – 96 h pi,and infectious CyHV-2 was produced from the Gi CF cells over 20 subcultures from the established cell lines.TUNEL,Anexin ?/PI stainning assay were performed to quantify apoptotic cells at the indicated time points by microscopy in CyHV-2 infected Gi CF cells.The percentage of total apoptotic cells following infection was significantly increased in a time-dependent manner.Taken together,our results indicate that CyHV-2 infection triggers apoptosis in Gi CF cells in a time-dependent manner.2.Integrated analysis of m RNA and s RNA transcriptome in the kidney of Carassius auratus gibelio response to cyprinid herpesvirus 2Here,we used high-throughput sequencing to analyse the miRNA and m RNA expression profiles of the kidney,the control groups(T1K,T2 K,and T3K)and the moribund groups(T3K,T4 K,and T5K).In the m RNA libraries,a total of 573,340,100 raw reads were generated in the uninfected and infected groups.After filtering out the low quality reads,564,633,312 clean reads remained.The clean reads were assembled into 26,664 transcripts with an average length of 744 bp.The length distribution of these transcripts ranged from 201 to 14,287 bp.In the s RNA libraries,a total of 10,714,657 reads were generated in the uninfected and infected groups,with over 90% of the sequences being valid reads.Among them,about 4.02% of the sequences mapping to Rfam,and most of the sequences were within 18 to 26 nucleotides in length.Besides,a total number of 840 known miRNAs and 48 putative novel miRNAs were identified.Then we compared the expression patterns of miRNAs,23 miRNAs were significantly differentially expressed between the uninfected and infected groups.Further,the expressions of 23 miRNAs were validated by quantitative reverse transcription polymerase chain reaction(q RT-PCR),the results showed that the expression patterns were basically the same with the sequencing.Prediction of targets of differentially expressed miRNAs revealed that the miRNAs participated in the regulation of multiple immune-related signaling pathways,including Chemokine signaling pathway,Apoptosis,Jak-STAT signaling pathway and MAPK signaling pathway.3.Identification and function study of CyHV-2 encoded miRNABased on small RNA sequencing and secondary structure predictions,all the s RNA sequences were searched against the CyHV-2 genome,we identified 17 CyHV-2 encoded miRNAs,among which 14 were validated by stem-loop q RT-PCR and eight were validated by northern blotting.In addition,q RT-PCR assays showed that the virus titres increased over time,and reached 107.6 copies/ml at 72 h post infection.PIN1 and NF-?B m RNA levels were significantly downregulated after the infection,MHC-I,IRF3,and MAPK7 expressions were significantly upregulated following infection,whereas RMBX expression showed almost no variation.Furthermore,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of miRNAs-m RNA pairs revealed diverse affected immune signalling pathways,including the RIG-I-like receptor and JAK-STAT pathways.Finally,we presented four genes involved in RIG-I-like pathways,including host gene IRF3,RBMX,PIN1,viral gene ORF4,which are negatively regulated by CyHV-2 encoded miRNA miR-C4.4.miR-C12 Attenuates Virus-Mediated Apoptosis by Targeting Caspase 8In this study,we reveal that miR-C12 inhibits the 3? UTR of the caspase 8 gene by luciferase activity assays.Furthermore,caspase 8 expression was downregulated in cells transfected with miR-C12 during CyHV-2 infection.Additionally,miR-C12 led to a significant decrease in m RNA and protein levels of caspase 8 at 24 h post infection,overexpression of miR-C12 significantly suppressed CyHV-2-induced apoptosis,while silencing of miR-C12 promoted CyHV-2-induced apoptosis.CASP8-si RNA-1 and 50 ?M Z-IETD-FMK efficiently reduced caspase 8 expression compared to the control group,and suppressing caspase 8 by si RNA or Z-IETD-FHM also decreased CyHV-2-induced apoptosis.Furthermore,At 24 h post infection,the CyHV-2 copies in the miR-C12 inhibitor treated cells were significantly decreased compared to the control group,miR-C12 mimics led to a significant increase of CyHV-2 copies compared to the miRNA control group.inhibition of miR-C12 resulted in suppression of CyHV-2 replication,and overexpression of miR-C12 and CASP8-si RNA faciltated CyHV-2 replication.Taken together,our results demonstrate that CyHV-2 induces cell apoptosis,which is at least partially governed by viral miRNAs.