Effect Of Grass Carp Reovirus Infection On Triggering The RNAi Signal Pathway In Grass Carp Cells

Grass carp（Ctenopharyngodon idellus）, member of cyprinid family is a popularcommercial fish species in China, and accounts for about23%of the annual aquculturalproduction in China. Grass carp reovirus （GCRV）, pathogen of haemorrhagic disease, isprevalent, harmful, and leads to high mortality. It’s the one of the most prominent prob-lems of freshwater aquiculture in China. So far, there have been no effective drugs ormeasures to control the viral infection. Therefore, characteritation of GCRV has an impo-rtant significance.RNA interference （RNAi） is a small interfering RNA（siRNA）-mediated gene sile-ncing mechanism. It widely exists in many organisms. Because of its highly specificityand efficiency, RNAi might be a new tool for controlling virul replication.The purpose of this study is to effect of Grass carp reovirus infection on triggeringthe RNAi signal pathway in grass carp cells, the main experiments are as follows:1.To prove whether RNAi exists in Ctenopharyngodon idellus kidney cells（CIK）.Plasmid pEGFP-Nl and in vitro synthesized egfp （fluorescin gene）specificdouble-stranded RNA （EGFP-dsRNA） were co-transfected into CIK cells. The genesilencing effects were monitored through inverted fluorescent microscopy on EGFPprotein expression, real time qRT-PCR analysis on egfp mRNA transcription, andNorthern blot assay on the egfp specific small molecular RNA level from thedegradation of input egfp-dsRNA at48h post transfection. The results showed thatco-transfection of pEGFP-N1and EGFP-dsRNA resulted in significantly reduced levelsof fluorescent signal, a significantly decrease in egfp mRNA quantification, andproduced egfp specific small molecular RNAs, but co-transfection of S10（segment10ofGCRV）-dsRNA and pEGFP-N1demonstrated strong fluorescent signal, hightranscriptional level of the egfp gene, and strong egfp mRNA signal together withnon-detectable level of egfp specific small molecular RNA. These results supported theidea that the RNAi pathway is present in CIK cells.2.To study the effect of GCRV infection on the RNAi signal pathway, threeexperiments were carried out:①To study the interaction of the purified GCRV genomic dsRNA and RNAi,Northern blot analysis was performed to monitor the level of S10specific small molecular RNA in CIK cells transfected with purified GCRV genomic dsRNA orsynthesized S10specific dsRNA（S10-dsRNA） by a DIG-labeled S10-probe. S10specific small molecular RNA was generated in CIK cells transfected with purifiedGCRV genomic dsRNA or S10-dsRNA in Northern blot analysis; while inGCRV-infected CIK cells, no S10specific small molecular RNA could be detected. Thisresult demonstrated that successful GCRV infection could protect its genome integrityfrom generating viral specific siRNA and the purified GCRV genomic dsRNA wassensitive to Dicer digestion.②To study the effect of GCRV infecting CIK on siRNA-mediated gene silencing,pEGFP-N1and the synthetic egfp specific siRNA（EGFP-siRNA） were co-transfectedinto the GCRV-infected CIK cells. The gene silencing effect was directly reflected byinverted fluorescent microscopy on EGFP protein expression at48h post transfection.The transcriptional levels of egfp gene were further determined by qRT-PCR of totalcellular RNA samples to validate the RNAi effect. The result showed thatGCRV-infected CIK cells co-transfected with pEGFP-N1and EGFP-siRNAdemonstrated a strong fluorescent signal similar to those cells transfected withpEGFP-N1only. The amount of egfp mRNA in GCRV-infected cells transfected withpEGFP-N1and EGFP-siRNA was found to be similar to those infected cells transfectedwith pEGFP-N1only, both of which were higher than uninfected cells transfected withpEGFP-N1and EGFP-siRNA. These results indicated that siRNA-mediated genesilencing was inhibited by GCRV infection in CIK cells.③To study the correlation of GCRV replication and Dicer gene transcription level,The infected cells were collected at0,4,8,12,16,20,24,28hrs post infection forextracting total cellular RNA. qRT-PCR was performed to detect the Dicer geneexpression, and the GCRV titration curve through TCID₅₀assay. The result showed thatthe expressions of the Dicer mRNA increased dramatically after20h （P﹤0.05） postinfection, and the TCID₅₀titration assay revealed that viral replication approached thehigher level at20h post infection. Through correlation analysis, correlation coefficientR²was0.9572. This result indicated that Dicer gene expression and viral titer were inhigher correlation, the viral replication could enhance Dicer transcription.Conclusion: RNAi pathway exsits in CIK cells, and GCRV genomic dsRNA issensitive to celluar RNAi pathway, but GCRV infection inhibits the celluar RNAi patway; the Dicer gene transcription level is positively related to the replication level ofGCRV.