Other results between the groups were compared by ANOVA analysis. lethal dose (3??LD50) of highly virulent SG-MCMV. Specific antibodies and IFN- secreting splenocytes were detected by immunoblotting and ELISPOT, respectively. Protective abilities in mice provided by the vaccines were evaluated by residual virus titers in organs, survival rate and weight loss. Results These DNA vaccines, especially m04, M84 and IE-1, could effectively reduce the virus loads in salivary glands and spleens of mice, but they couldnt GsMTx4 completely clear the residual virus. Survival rates of 100% in GsMTx4 mice after a lethal dose of MCMV infection could be reached by more than one dose of M84 vaccine or two doses of m04 or IE-1 vaccine. Immunization with PLCG2 M55 or M105 DNA at four doses offered mice only 62.5% survival rate after the lethal challenge. Conclusions The study demonstrated that DNA vaccines could effectively afford mice protection against infection with a highly virulent MCMV and that the protection offered by induced CD8+ T cell immunity might be superior to that by gB-specific antibodies. These results are valuable references for development and application of HCMV vaccines. passaged for virulence enhancement and isolated from salivary glands of the infected mice, was referred to as SG-MCMV (salivary gland-derived MCMV). The high-virulence SG-MCMV stock was prepared through 14 times of passages and was used in challenge experiments. Challenge was performed with 3 LD50 virus stock. Plasmid DNAs and peptide Plasmids pcDNA3.1/m04, pcDNA3.1/M84, pcDNA3.1/M105, pcDNA3.1/IE-1 and pcDNA3.1/M55 were constructed by cloning the PCR products of m04, M84, M105, IE-1and M55 gene from the MCMV smith strain into the plasmid expression vector pcDNA3.1/myc-His B (Invitrogen, CA), which encoded gp34, p65, DNA helicase, pp89 and glycoprotein B (gB) proteins, respectively. PCR amplifications for m04, M84, M105 and M55 genes were carried out using the following paired sense and antisense primers: 5AGaagcttATGTCTCTCGTATGTCGGC3 (containing Hind III site) and 5GCctcgagGGTTAGTTACTCTTAAGCGGT3 (containing Xho I site) for m04, 5GCaagcttCATGTCGGTCAACGTTTACT3 (containing Hind III site) and 5GCtctagaGGCTCTGTCTGTTTGTCTATG3 (containing Xba I site) for M84, 5GCgaattcGTTGATCATGGAGAAGAG3 (containing EcoR I site) and 5GCtctagaGTCAGAAAACCAGAGTG3 (containing Xba I site) for M105, 5GTaagcttGATCGCTGAACAACGCTC3 (containing Hind III site) and 5GAggatccTCCTCGCAGCGTCTCCAAT3 (containing BamH I site) for M55. As for IE-1, its ORF has a four-exon structure, in which three exons encoded pp89 protein . We had to construct the continuous IE-1 gene by overlap-PCR with the three pairs of primers: 5TAggatccGAGATGGAGCCCGCCGCAC3 (containing BamH I site) as IE-1 sense, 5GGCGACATGAGCTGGCACCTTGTCTGATGGGTAGAC3 as Exon 2 antisense, 5GTGCCAGCTCATGTCGCC3 as Exon 3 sense, 5ACAACAGAACGCTCCTCACTGCAGCATGCTTGATGG3 as Exon 3 antisense, 5GAGGAGCGTTCTGTTGTC3 as Exon 4 sense, and 5CGgaattcGGGCTTGTGGATTCACTTCT3 (containing EcoR I site) as IE-1 antisense. All the plasmids were propagated in E. coli XL1-blue bacteria and purified using NucleoBond Xtra Maxi purification kits (Macherey-Nagel, Germany). As the H-2d restricted epitope for gB has not been reported anywhere thus far, we only obtained epitope peptides of the other four MCMV GsMTx4 proteins. The peptide 243-YGPSLYRRF-251 for gp34 protein , 297-AYAGLFTPL-305 for p65 protein , 207-TYWPVVSDI-215 for DNA helicase , and 168-YPHFMPTNL-176 for pp89 protein  were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., and were used for IFN- ELISPOT assay. Immunization electroporation was carried out according to the method described by Aihara and Miyazaki . Mice were immunized with plasmid DNA dissolved in 100?l of Tris-EDTA buffer at a dosage of 100?g by injection into the left and right quadriceps muscles, 50?g each. After the injection, a pair of electrode needles with 5?mm apart was inserted into the muscle to cover the DNA injection sites and electric pulses were delivered using an electric pulse generator (Electro Square Porator T830 M; BTX, San Diego, CA). Three pulses of 100?V each, followed by three pulses of the opposite polarity, were delivered to each injection site at a rate of one pulse per second. Each pulse lasted for 50?ms. The non-immunized mice were set up as controls. Mice were immunized 1?~?4 times, at an interval of 2?weeks. Specific antibody assay Serum samples of mice were collected 13?days after each immunization and stored at ?20C. Titers of IgG Abs against the respective viral proteins were measured by using immunoblotting as previously described . Confluent 3T3 cells were infected with MCMV (m.o.i?=?1) for 1.5?hours at 37C. Unadsorbed virions were removed and infection medium was added..