| Porcine Parvovirus (PPV) is a major pathogen of reproductive failure in pigs. PPV can cause diarrhea, dermatitis and respiratory disease, and also play an important role in non-suppurative myocarditis, wasting syndrome and post-weaning multisystemic wasting syndrome. Porcine parvovirus disease exists widely in the world, causing great economic losses to the pig industry. MicroRNA which are encoded by endogenous gene are single stranded RNA molecules of length 19-24 nucleotides, and play a regulatory role in many biological processes by binding to the 3’-UTR of target mRNAs, including virus infection, cell differentiation and apoptosis, tumorigenesis and the balance of the body. At present, it has been reported that cellular miRNA can regulate virus infection, such as Japanese encephalitis virus, porcine reproductive and respiratory syndrome virus, hepatitis C virus, influenza A virus, swine fever virus and so on, but for the induction of miRNA expression has not been reported in PPV infected PK-15 cells.In order to explore the role of miRNA in PPV infection, PPV TCID50 was measured and PK-15 cell were inoculated with PPV at 1 MOI, total RNA were extracted after 24h and subjected to Solexa sequencing to measure the cellular miRNA expression of PK-15 cell before and after infection with PPV. According to the NS1 gene of PPV, a pair of specific primers was designed, and Real-time fluorescent quantitative PCR (RT-PCR) was established. MiRNA targeted PPV genome were predicted and transfected with other immune related miRNA, RT-PCR previously established was introduced to identify miRNA involved in PPV infection.476 miRNA were identified by Solexa sequencing method, of which 240 miRNA belonged to control group,13 miRNA belonged to experimental group. By analyzing expression profiles,182 differentially expressed miRNA were detected, among which 60 miRNA were up-regulated and 122 miRNA were down-regulated. MiR-21 was expressed at the highest level in PK-15 cell, while the most significant difference miRNAs is miR-10b. Some immune related genes like C-JUN, MyD88, TLR7 were predicted, which suggested that these miRNA may be involved in PPV infection. The selected miRNA expression profiles by RT-PCR were consistent with those by deep sequencing. The ideal standard curve can be got while the concentration of the standard plasmid in 1.59×109 to 1.59×104copies/μL with a liner correlation of 0.997 and efficiency of 0.972, the optimal annealing temperature was found to be 59℃, and its specificity and repeatability were good, could be applied to the miRNA function experiment. After target prediction, miR-34a could target NS1 gene of PPV, and the NS1 gene was conserved in PPV. After transfection with miRNA mimics, viral genome DNA were carried out. The results showed that miR-34a inhibited PPV replication, others were not, further study is required to investigate the mechanism.This is the first time to construct small RNA database of PK-15 cells, and construct miRNA expression profiles before and after PPV infection. It is helpful in understanding the mechanism of PPV infection and the mechanism of host antiviral immunity at the miRNA level by analyzing differentially expressed miRNA. At the same time, we confirm that miR-34a inhibits PPV replication, this date may be useful for miRNA-mediated antiviral therapeutic strategies. |
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