| The novel duck reovirus(NDRV)is a segmented ds RNA virus.Due to the damage of immune organ function of infected ducklings caused by NDRV infection,the secondary infection rate and mixed infection rate have been greatly increased,which has attracted widespread attention of waterfowl breeding industry.However,the pathogenesis of NDRV is still in the exploration stage,and there is no effective commercial specific antiviral agent and vaccine for the virus.It is of significance to explore the pathogenesis of NDRV and its interaction with host for preventing NDRV infection and developing new antiviral strategies.micro RNAs(mi RNAs)are a class of single-stranded noncoding small RNA molecules with a length of about 22 nt.At present,they have been found to exist widely in various living organisms,and participate in metabolic activities through the regulation of gene expression at the post-transcriptional level.mi RNAs play a unique role in immune response and virus infection.It has been reported that the expression change of mi RNAs in the host can be caused by the infection of various viruses,but the expression profile characteristics of mi RNAs during the infection of duck embryo fibroblasts(DEF)by NDRV and the biological functions of mi RNAs have not been reported.The purpose of this study is to obtain the expression profile of mi RNAs before and after NDRV infection with DEF,screen the differentially expressed mi RNAs,and further study the mi R-155-1 with significantly increased expression after NDRV infection,to explore its impact on the NDRV infection and the relevant mechanism.1.Obtaining the differential expression mi RNAs spectrum of DEF infected by NDRVThe DEF infected with NDRV N20 strain were used as the exposure group,and the PBS treated cells were used as the control group.The two groups of samples at 36 hpi were collect ed,and 27,069,283 and 25,467,910 clean reads were collected from the exposure cells and the control cells using the small molecule RNA sequencing technology(s RNA-seq).After comp arative screening analysis,there were 26 mi RNAs differentially expressed,of which 19 were accumulated and 7 were dissipated.The GO function enrichment analysis was carried out on t he target genes of differentially expressed mi RNAs.Multiple GO function items were signific antly enriched under the infection of NDRV,such as signal and cell communication biologica l processes.KEGG analysis showed that Fox O signal pathway,autophagy and Toll-like recept or signal pathway showed a large degree of enrichment,which may be related to the infection and life cycle of NDRV.Based on six randomly selected mi RNAs,q PCR verification was per formed,and the results were consistent with the sequencing results.2.Research on mi R-155-1 promoting NDRV replicationThe expression of mi R155-1 was detected at different time points after NDRV infected DEF cells.q PCR analysis showed that the expression of mi R-155-1 gradually increased withi n 36 hours after NDRV infection with DEF.The mi R-155-1 mimic and inhibitor and the corre sponding control were transfected into DEF cells.Then DEF were inoculated with NDRV,an d the expressions of NDRV m RNA and ζC protein were detected to measure mi R-155-1 effec t on the replication of NDRV.The results showed that overexpression of mi R-155-1 significa ntly promoted m RNA and ζC protein expression,while in the inhibitor group,virus copy num ber and virus ζC protein expression decreased.In DEF cells transfected with mi R-155-1 mimi c and inhibitor respectively,NDRV infection was detected,and IFN-α and IFN-β m RNA expr ession level changes were detected by q PCR.Results: the increase of mi R-155-1 led to IFN-αand IFN-β expression decrease,in the inhibitor group of IFN-β showed a promoting effect.3.The target gene SOCS5 participates in the regulation of NDRV replicationIt was predicted by software that SOCS5 was the target of mi R-155-1,and verified by do uble luciferase reporter gene experiment.The results showed that in psi Check2-SOCS5-WT+mi R-155-1mimic/mimic NC transfection group,mi R-155-1mimic inhibited luciferase activit y;in psi Check2-SOCS5-MT+mi R-155-1 mimic/mimic NC group,there was no significant dif ference in luciferase activity,which indicated that the specific region of SOCS5 could comple ment and combine with mi R-155-1.The effect of mi R-155-1 on the expression of SOCS5 was further tested.It was found that the overexpression of mi R-155-1 led to a significant decrease in the level of SOCS5 m RNA and protein.After inhibiting endogenous mi R-155-1,the expre ssion level of SOCS5 increased.These results revealed that mi R-155-1 targeted SOCS5 gene.p CDNA3.1(+)-SOCS5 and pc DNA3.1(+)were transfected into DEF,inoculated with NDRV,and analyzed by q PCR and Western blot.Compared with empty vetor,SOCS5 overexpression mainly reduced the effect of NDRV ζC protein expression level,and the change of virus copy number was not obvious.Further knock-down expression of SOCS5 in the si RNA-SOCS5 treatment group,it resulted in a significant increase in the number of copies of NDRV,and ζC protein expression also increased.pc DNA3.1(+)-SOCS5 and si RNA-SOCS5 were transfected into DEF cells respectively,and then infected with NDRV.IFN-α and IFN-β m RNA expression level changes was detected by q PCR.Results overexpression of SOCS5 caused IFN-α and IFN-β increased expression.Decreased SOCS5 expression mainly caused IFN-β expression level significantly reduced.To sum up,this experiment found that NDRV infection with DEF caused 26 mi RNAs differential expression,of which the significantly increased expression of mi R-155-1 could inhibit the expression of IFN-I mediated by SOCS5 and promoted NDRV infection and replication. |
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