Preliminarily Study On Function Of MicroRNA Targeting Duck Plague Virus ICP4 Gene

Duck plague virus belongs to herpesvirales,Alphaherpesvirinae,triggering acute hematosepsis in birds and being harmful to waterfowl breeding industry.The number of mi RNA encoded by herpesvirus accounts for more than 90% of total viral mi RNAs,and the studies of the mi RNA encoded by most of its members has made great progress.But there is less attention on mi RNA encoded by duck plague virus,which is also a member of herpesvirus.The immediately early gene ICP4 which is important in virus replication is our goal research gene.We predict and screen the candidate mi RNA among the 39 mature mi RNAs encoded by CHv stain we obtained,and studies were performed to learn much more about its function on virus proliferation.Results as follows:1 Screening the candidate mi RNA which targeting ICP4 gene of DPVThe predict results show that mi R-D22-3p,mi R-D22-3p-5p and mi R-D23-5p are complete complementary with ICP4 gene.mi R-D22-3p and mi R-D22-5p are the research object cause they are divided from the one pre-mi RNA.The COS7 cells were co-transfected with mi R-D22-5p mimics or NC mimics,together with pmir GLO-ICP4-WT or pmir GLO-ICP4-MUT5 p.Results show that mi R-D22-5p mimics deeply inhibit the fluorescence of pmir GLO-ICP4-WT comparing with NC mimics but not pmir GLO-ICP4-MUT5 p,indicating that mi R-D22-5p can bind the predicted target bite specifically.The COS7 cells were co-transfected with mi R-D22-3p mimics or NC mimics,together with pmir GLO-ICP4-WT or pmir GLO-ICP4-MUT3 p.However,there is no significant difference between mi R-D22-3p mimics group and other groups.So mi R-D22-5p is the object for further study.2 Effects of mi R-D22-5p on ICP4 gene expressionThe DEFs were transfected with ICP4-EGFP and EGFP-N1 vector,finding that the appearance of recombination fluorescence protein is regular oval distinguished with EGFP-N1 vector.The DEFs were co-transfected with ICP4-EGFP,together with mi R-D22-5p or NC mimics and finding that mi R-D22-5p can decrease the expression of ICP4 m RNA and the number of fluorescence protein in group mi R-D22-5p mimics is less than NC mimics group,illustrating mi R-D22-5p can block the translation of ICP4 protein.3 The influence of mi R-D22-5p on proliferation of duck plague virusThe DEFs were transfected with 50 nmol mi R-D22-5p mimics or NC mimics,then infected with DPV post-transfection,collecting cells and supernatant at 24 h,48h and 72 h post-infection.The virus DNA was extracted and detected using RT-q PCR.Results showed that there is no difference in DNA copy numbers at 24 h and 48 h.The DNA copy numbers of mi R-D22-5p mimics group were significant lower than NC mimics group at 72 h post-infection,indicating that mi R-D22-5p inhibited DPV proliferation.The DEFs were transfected with 50 nmol,100noml,300 nmol mi R-D22-5p mimics or NC mimics and infected with DPV post-transfection,observing the cytopathic effect and detecting the viral DNA copy numbers.Results show that the plaque number is decreasing and the plaque is becoming smaller while the concentration of mi RNA increased from 50 nmol to 300 nmol,which indicate the degree of inhibition of viral proliferation is liner with the concentration of mi RNA.