Effect Of Human Cytomegalovirus Primary Persistent Infection On The Learning And Memory Capability In BALB/c Mice

Objective Establishing the model of human cytomegalovirus primary infection in BALB/c mice, We aimed to know the stage which cognitive impairment was showed by investigating the effect of HCMV persistent infection on the learning and memory capability in mice and the pathological change in brains of mice. This would provide a new proof for the relativity between HCMV persistent infection and Alzheimer's Disease.Methods Firstly, plaque assay was performed for purifying HCMV AD169 for three times, and then the viruses were kept in passage in HF fibroblasts. Thirty-five specific-pathogen-free BALB/c mice of 6~8 weeks old were divided into three groups, 15 mice in HCMV infected group, 10 mice in each control group. Mice in HCMV infected group were inoculated with 1.8×107 PFU of HCMV intraperitoneally respectively, mice in inactivated HCMV group were inoculated with1.8×107 PFU of HCMV which were inactivated at 56℃for 30min, and mice in HF control group were inoculated with 1×105 of HF cells. Animals were housed and maintained in microisolator cages for three months, the behavior and weight of mice was observed during the period. Then all mice were tested in the Morris Water Maze, step-down passive avoidance task and autonomic activities for determining the change of its behavior. Since then all the mice were sacrificed and each brain was harvested for the following experiments: (1)Virus isolation: Brains of mice were ground on ice, 1:10 dilutions of undiluted clarified homogenates from the brain were inoculated into HF cells and then were observed by inverted-phase microscopy for cytopathologic characteristics of HCMV infection. Results of virus isolation were identified by indirect immunofluorescent assay, polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR). (2) Extracted DNA and mRNA from the brain, HCMV UL83 and UL54 gene and transcripts were analyzed by PCR and RT-PCR. (3) Slices of brain were embedded in paraffin for detecting the expression of HCMV UL83 gene by indirect immunofluorescent assay. (4) Tissue sections of brain were stained by HE method for observing the pathological damage. (5) Ultrastructure and virus particles of neuron were observed by electron microscope.Results (1) Three mice of HCMV infected group were depressed and torpid, while activities, drinking and feeding decreased. No obvious abnormalities in two control groups were observed. There was no significant differences from the weight in three groups(P>0.05).(2) The number of autonomic activities decreases in HCMV infected group compared with two control groups. No significant differences in three groups(P>0.05).(3)①During the place navigation test, the latency in HCMV infected group was much longer than that of two control groups through training days (P<0.05), but the differences between two control groups were not significant (P>0.05). There was significant differences in three groups at different time.On the 1th day, there is no significant differences(P>0.05). On the 2th ~3th day, the differences between HCMV infected group and inactivated HCMV group were significant (P<0.05). On the 4th ~6th day, the differences were significant (P<0.05) in three groups.②During the probe trial test, the number of crossings platform location decreased in HCMV infected group compared with the control groups(P<0.05), and the time of first crossings platform location of HCMV infected group was also longer than that of two control groups(P<0.05). (4) In step-down passive avoidance task one mouse of HCMV infected group was dead in training days. In the learning period the frequency of mistakes were higher in HCMV infected group compared with two control groups(P<0.05). After 24h, the frequency of mistakes decreased in each group, while the differences were significant in three groups (P<0.05). The latency in HCMV infected group was significantly shortened than that of two control groups (P<0.05).(5) The state of infection was identified. Isolates exhibiting HCMV cytopathologic characteristics in eleven mice of HCMV infected group, HCMV UL83 gene and transcripts was detected by PCR and PT-PCR, HCMV pp65 antigen was also observed in cell slide by indirect immunofluorescent assay. The results of viral culture were negative in three mice,but we could detect HCMV UL83 and UL54 gene in the brains of these mice by PCR. Virus isolation of the dead mouse was not done, the result of PCR to detect HCMV UL83 and UL54 gene in the brain was positive, and the result of RT- PCR was negative. The results of each control group were negative by the detection mentioned above. Therefore the state of infection in HCMV infected group was persistent infection. (6) In HCMV infected mice, a lot of neuron in the cerebral cortex and hippocampi were swollen and stained slightly by HE stain, the quantity of neuron was also decreased. HCMV-specific intracytoplasmic inclusion bodies and Herpes virus like particles could be observed by electron microscope. No obvious abnormalities were observed in two control groups.Conclusion For the first time, we established a model of human cytomegalovirus primary persistent infection in central nervous system of mouse.Through the study , we come to the conclusion as follows: (1)Mice in HCMV infected group have't obvious abnormalities after three months. The differences from average weight of mice were not significant compared with control groups.(2) The state of infection in HCMV infected group was persistent infection, and pathologic changes also could be found in the cerebral cortex and hippocampi of these mice.(3) HCMV persistent infection can damage at some extent the learning and memory capability of mice, but the frequency of autonomic activities of theirs were not obviously affected.