Preliminary Study On The Role Of MiRNA In EV71Infection And Proliferation

Hand-foot-mouth disease (HFMD) is a common illness of infants and children. Enterovirus71(EV71) and coxsackie virus A group16(CA16) are the major pathogens of HFMD. Recently, the world wide outbreaks of HFMD are mainly caused by EV71. EV71is a member of Picornaviridae, Enterovirus genus. Although the majority of EV71infectious patients show mild symptoms, some patients may develop aseptic meningitis, encephalitis, acute flaccid paralysis, neurogenic pulmonary edema and severe symptoms of myocarditis and so on. Children with severe symptoms are quickly, easily resulted in death. Because there is no effective antiviral drugs agaist EV71infection, the only thing we can do is to track the general patients and suspected severe symptom patients, and to treat them when they commit symptoms. Thus it is necessary to strengthen the basic research on EV71infection, including molecular biological characteristics of EV71and factors affecting viral replication. Though domestic and foreign research on EV71has made great progresses, fully understanding of the key points of EV71virus replication is still very urgent.Recent studies find that cell-encoding miRNA molecules are closely related to many viruses’replication. MiRNAs may affect viruses’replication on two aspects, on the one hand, miRNAs can impact viral replication by effecting the closely related key molecules, such as the immune response molecules, signal transduction proteins regulating viral replication process, etc. On the other hand, miRNAs can affect viral replication through directly targeting viral mRNAs.MiRNA molecules have universal significance for regulating genes at post-transcriptional level. Futhermore cells can encod many kinds of miRNAs. One miRNA molecule may have multiple potential targets, and one gene may be affected by multiple miRNA molecules, thus a complex regulatory network forms. It suggests that EV71infection process is likely related to the function of miRNA molecules. Therefore we begin our study with change of miRNAs after EV71infection. By bio-informational analysis methods, we analyze the miRNAs and their targets which may affect viral infection and replication, and find the key factor leading to EV71-related diseases.In our study, one EV71strain was isolated from HFMD patients’ feces and the whole genome sequencing was carried out. Meanwhile, a MTT viral titer detecting method better than plaque assay was established. Then we utilized miRNA array to detect the change of miRNA expression level in EV71infected cells, and73miRNAs which up-regulated or down-regulated more than two folds at expression level are found. And analyzing the targets of these miRNAs by bioinformatics, the main targets include cellular structural protein genes and immune molecules related genes are found. In addition, bio-informational software is employed to analyze miRNA molecules directly binding with the EV71-3’-UTR, and25miRNA molecules possibly relating with EV71infection and replication are found. Then the miRNAs possibly relating with EV71infection and replication are cloned into vectors which include30miRNA Array screened miRNAs and10bioinformatics predicted miRNAs, and these vectors form a miRNA expression library. Whereafter miRNA-3620expression vector is selected to validate whether miRNA-3620can effect with EV71-3’-UTR to repress EV71infection and replication.