The Establishment Of Murine Congenital Infection Model By MCMV And The Observation Of The Nervous System Infection

ObjectivesTo establish murine congenital infection model by MCMV and observe the pathological changes and infection status of brain tissue.MethodsThe preparation of MCMV strain and the determination of infectious titer of the virus: Take the BALB/c fetal mice out of the uteruses of pregnant mice in the second or third trimester. Prepare murine embryonic fibroblasts (MEF). Amplify MCMV RM461 strain using MEF, and detect infectious titer of the virus by plaque assay. Dilute the viral suspension to working titer (1×10~3PFU/μL), and store it for use.2)The construction of MCMV congenital infection model: After anesthesia, mice that were pregnant 11～13.5 days (E11～13.5d) were intra-amniotic injected one uterus by one with virus (MCMV RM461 suspension, 1μL, 1×10~3PFU). The control group of the same period were intra-amniotic injected with culture medium DMEM (1μL). Carefully reset the uteruses and close the abdomen. After 5 days of separated feeding, kill the pregnant mice, take the fetus out of the uterus, anesthetize and kill them. Make frozen sections of these fetal brains.3)The detection of the pathological changes and the viral antigen of the fetal brains: Some sections stained using conventional H & E method, observe the pathological changes under light microscope. Detect MCMV early antigen in the brain tissue by immunohistochemisty staining and immunofluorescence assay. Results1) MCMV RM461 virus was successfully amplified. The infectious titre of it was 3.27×107 PFU/mL.2)Murine model of MCMV congenital infection were established successfully. The survival rate of infected group and control one were 71.9% and 76.7% respectively. Compared with the control group, intra-amniotic inoculation of MCMV does not affect the rate of fetal survival, fetal absorption, fetal death and the average weight of the heads, but decrease their average weight of the bodies.3)The pathological changes of the brain tissue: There were no structural abnormalities in brains of control mice. While in the infection group, dysplasia could be observed in the encephalocoeles and brain folds. Edema was present in the nerve cells, and inflammatory cells including lymphocytes and mononcytes infiltration was observed in encephalocoele zone, subencephalocoele zone, and the cerebral cortex area.4)The detection of the viral antigen: Through enzyme immunohistochemistry assay, there are many MCMV infected cells in brain-ventricular zone, brain subependymal zone, cerebral cortex and hippocampus area in the infection group. Similar findings were observed by immunofluorescence method.ConclusionsBy intra-amniotic injection of MCMV RM461 virus suspension, murine model of MCMV congenital infection can be successfully established. This model could be used to study the mechanisms of encephalodysplasia caused by congenital CMV infection in vivo.