The Screening And Identification Of Differentially Expressed MiRNAs Of Bovine Ephemeral Fever Virus Infected Host Cells

Bovine ephemeral fever(BEF)is an acute and thermal infection of cattle caused by bovine ephemeral fever virus(BEFV),which can occur and spread rapidly.The high incidence and low death rate of the disease can lead to reduced production of dairy cows,the decline of milk quality,the decrease of breeding ability of bulls,and the lameness of cattle,which has caused great economic losses to the cattle industry.Although using vaccine has some effect,the disease still occurs frequently.Therefore,to research the pathogenic mechanism of BEFV,including the interaction between virus and host,especially the new research hot spots such as miRNAs will provide new targets and development clues for the research on the prevention and control of BEFV.microRNAs(miRNAs)are non-coding small RNAs about 22 bp,which are involved in regulating the expression of protein-coding genes at the post-transcriptional level,and play a very important role in the process of viral infection.miRNAs encoded by the host can affect the replication of the virus and play an important part in the occurrence of regulating antiviral and relevant cancer,but they can also be utilized by viruses to facilitate its replication.The viral miRNAs are mainly encoded by DNA viruses.By regulating the genes of hosts or viruses itself,it can prolong the life of infected cells,escape the immune response of host cells,or restrict the cycle of the cell and so on,which can contribute to proliferation or survival of the viruses.The relationship between miRNAs and vesicular stomatitis virus and rabies virus has been reported,which are in the same family with BEFV,but the interaction between BEFV and miRNA has not been reported.We first performed the high-throughput sequencing of the BEFV infected MDBK cells 12 h,24 h and 48 h,respectively.And we obtained the differentially expressed profiles of miRNAs.Compared with the control samples,there were 106,148 and 90 miRNAs up-regulated in 12 h,24 h and 48 h treated samples,respectively,208,285 and 334 down-regulated.Furthermore,19 miRNAs had significant differences in the expression in all the three time points.In clustering analysis of the targets of differentially expressed miRNAs,we found that they mainly involve in the metabolic process and cellular process,and the cellular component is cell and cell part.The molecular function is bind,and the main enrichment pathway is translation and viral infectious disease.The selected miR-101,miR-139 and miR-2890 were validated by real-time quantitative PCR,and the results showed that BEFV could reduce the expression of miR-101.To verify the effect of miR-101 on BEFV replication,we artificially synthesized miR-101 mimic,transfected into MDBK cells,and infected with BEFV of 1000 TCID50 after 24 hours.After 24 hours of infection,the cell samples were harvested,the BEFV RNA replication was measured by real-time quantitative PCR,and the change in the titers of the BEFV were detected by TCID50.In comparison with the control,the viral RNA replication decreased by 45%and the virus titer declined by 1000 times in the cells transfected with miR-101 mimic,indicating that miR-101 could repress the replication of BEFV.The targets of miR-101 was predicted and the cluster analysis was conducted.According to the classes and distribution of targets and the signaling pathways involved,the mTOR,TTC3,IMPG2 and SMAD2 genes were selected as candidate targets of miR-101 for identification.Using the dual luciferase reporter assay system,miR-101 was found to supress the expression of SMAD2,suggesting that SMAD2 was one of the targets of miR-101.In conclusion,the study utilized high-throughput sequencing technology discovering the expression of miR-101 regulated by BEFV for the first time.miR-101 can also restrain the replication of BEFV and we found one of its targets is SMAD2.