MiRNAs Regulating The Pathogenesy Of Viral Myocarditis And Screening Recombined Coxsackievirus CVB3Attenuated Strain

CVB is the first pathogen which cause viral myocarditis in human, especially CVB3. Viral myocarditis is a commonly infectious disease in children which causes sudden, acute heart failure and dilated cardiomyopathy, and adults are sporadic.The pathogenesis is mostly destruction of virus and immune, inflammatory response caused by virus infection. There is no effective preventive measures and specific drug therapy in clinical use at present. It is very imperative to explore CVB3infection prevention and treatment. So it is needed to research the pathogen and pathogenesis deeply for developing effective preventive and cure medicine. For this reason, we carried out two parts of work as below.The first part, we investigated that microRNA changes and the roles in regulating the pathogenesy of viral myocarditis. Objective:To evaluate the role of miRNA involving the pathogenesis of viral myocarditis caused by CVB3utilizing animal model. Methods:we detected miRNA expression profiling by microarray utilizing mouse model on day4post infection with CVB3. We validated differntially expressed miRNAs by real-time PCR. We predicted target genes using miRNA target prediction databases, and assessed them by mRNA microarray and qRT-PCR measurement. Through analyzing the target function of differntially expressed miRNAs, we initially explored the regulating role of miRNAs in viral myocarditis. Results:We found five differntially expressed miRNAs. These miRNAs invove in regulating several important innate immune and antival pathways, such as Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, MAPK signaling pathway, JAK-STAT signaling pathway, Natural killer cell mediated cytotoxicity. Conclusion: miRNAs regulate the pathogenesy of viral myocarditis. This study may provide a new perspective for deeper undestanding pathogenesis of viral myocarditis and discovering novel therapies against CVB3infection.The second part, we explored the stability of inserting exogenous miRNA in CVB3UTR. Objective:we inserted miRNA complementary series in5’UTR and3’UTR in CVB3genome for exploiting an recombined CVB3plasmid using CVB3full lengenth cDNA plasmid for searching appropriate CVB3mocular vaccine.Methods:Utilizing molecularcloning technique and microRNA operating technique, dsmiRNAs anealled using single and three series miRNA were inserted in5’UTR and3’UTR. We transfected recombined clones to Hela cells for observing if they could recover complete virus particles. When obtained virus, we infected them to TE671with miR-133. We extracted RNA in cell fliud, reversed RNA to cDNA and cloned it to T vector for sequencing to identify the integrity and mutation. Results: The5’recombined plasmids had identifical sequence with target sequences. They could recover complete virus particals after being transfected into Hela. TE671cells didn’t apper cytopathic effect when infected by recombined viruses, which determined endogenous miR-133recogonized the miR-133complementary strand and inhibited CVB3repetition, translation. TE671cells appered cytopathic effect when infected by recombined viruses passaged three times in Hela cells. We extracted virus RNA, sequenced and found miR-133complementary sequences was deleted.We did test again and got the same results. So we contined to search constructing point and completed this construction by many cloning steps. The3’recombind plamids could also recover complete virus particals after being transfected into Hela. At present, we infect TE671using recombined virus, and passage it some times for observing inserting stability at the same time. Conclusion:We inserted miR-133complementary sequence in5’UTR. Because5’UTR has many functional regions, this insert influenced CVB3reproduction and caused genome destablity. The recombined virus occurred reversion. Virus cutted the exogenous sequences to maintain genome envolutionary stability. Therefore, researchers should avoid this site in5’UTR when construcing the recombined CVB3. At present,3’UTR inserting has feasibility in theory and actual resuts.