50 ml of adenovirus Ad-A20 or the control Ad-LacZ 2 days before CVB3 infection. Infected mice receiving saline treatment were used as control. Tissue Histopathology and Myocarditis Grading Seven days following CVB3-infection, the heart tissues were collected, sectioned and stained with hematoxylin and eosin. MedChemExpress 2353-45-9 Sections were examined by two independent investigators in a blinded manner, and the severity of myocarditis was assessed by previously described 04 scale, in which 0 = no inflammation; 1 = one to five distinct mononuclear inflammatory foci with involvement of 5% or less of the cross-sectional area; 2 = more than five distinct mononuclear inflammatory foci, or involvement of over 5% but not over 20% of the cross-sectional area; 3 = diffuse mononuclear inflammation involving over 20% of the area, without necrosis; and 4 = diffuse inflammation with necrosis. Materials and Methods Ethics Statement All experiments carried out in this study were strictly performed in a manner to minimize suffering of laboratory mice. All animal procedures were performed according to the Guide for the Care and Use of Medical Laboratory Animals and with the ethical approval of the Shanghai Medical Laboratory Animal Care and Use Committee as well as the Ethical Committee of Fudan University. Quantization of Viral Burden in Heart Tissues Viral titers in the heart were determined as previously described. Hearts were collected, weighed and homogenized in 400 ml 10%FBS-DMEM using a sterile glass mortar and pestle. After centrifugation at 14,0006g to remove cellular debris, 100 ml of the supernatant serially diluted in 10-fold increments, and incubated on confluent HeLa cell monolayer for 1 h at 37uC and 5% CO2 to allow virus attachment, and then incubated for 5 days to allow plaque formation. Viral titers were expressed as the mean lg TCID50/100 mg tissue6SEM. Mice and Virus Specific pathogen free Male BALB/c mice, 6 weeks of age were purchased from Shanghai Experimental Animal 
Centre of Chinese Academy of Sciences. CVB3 was maintained by passage through HeLa cells. Viral titer was routinely determined prior to infection by a 50% tissue culture infectious dose assay of HeLa cell monolayer. Cytokine Assays Levels of TNF-a, IL-1b, IL-6, MCP-1 of cell culture supernatants and heart homogenates were determined by enzyme-linked immunosorbent assay following the manufacturers’ instructions. Preparation of Adenovirus Adenoviruses encoding A20 and the control were created using the Virapower adenovirus expression system according to the manufacturer’s instructions. The resulting PCR product was digested with restriction enzyme BamH I and Xho I, then inserted into pENTR3C to yield entry vector pENTR-A20.The insert and junctions were sequenced to verify the absence of mutations. Site specific recombination between pENTR-A20 and the adenoviral destination vector were established with LR clonase II. Digest the recombinant plasmid pAd-CMV-A20 and the control pAd-CMV-LacZ with Pac I. Transfect 1 mg of Pac I-digested pAd-DEST expression plasmid into 293A cells. Harvest culture supernatants of 293A cells when visible regions of cytopathic effect were observed. This stock was used to infect 293A cells to generate a higher titer viral stock. Adenovirus using in vivo were purified by cesium chloride banding. Virus titers were determined by a plaque assay using serial dilution. Purification of Neonatal Murine Cardiac Myocytes Cardiac myocytes from neonatal mice within 72 h of bir