Studies On The Correlation Between The Load Of Latent UL83 Gene And The Risk Of Reactivation In Mouse With HCMV Latent Infection

Objective: On the basis of establishment of a model for human cytomegalovirus (HCMV )latency in mice which were primary infected by different doses HCMV ,the study aimed at observing the latent sites and variation of the load of UL83 gene in corresponding sites in latency and reactivation, investigating the relationship between the burden of primary infection and the load of UL83 gene in latent infection,discussing the relationship between the risk of reactivation and the load of UL83 gene in latent infection. Based on the studies above ,to provide a new idea for diagnosis and therapy and prevention of HCMV disease in immunocompromised patients. Methods: Firstly, to determine the dose of HCMV resulting in latent infection in mice, and then establish a model for HCMV latency in mice. Sixty 6-8 week SPF BALB/c mice were divided into 5 groups, six female and six male mice in each group. The mice in group A were subjected to the intraperitoneal injection of 3.5 X 105 PFU/ml HCMV 1.0 ml per mouse. The mice in group B and group C were respectively infected in 3.5X 105 PFU/ml HCMV 0.75ml and 0.5 ml per mouse by intraperitoneal injection. Group D and Group E were normal control groups and the mice were respectively subjected to the intraperitoneal injection of DEME and HF 1.0 ml per mouse . After fourteen monthes, half of mice in per group were killed. The lungs and the brains and the blood leukocytes of mice were collected to be assayed as follows: (1) Human fetal fibroblasts(HF) grown as monolayers in cell culture were inoculated with lung or brain homogenate or blood leukocytes for virus isolation.(2)To look for HCMV like virus particle by electron microscopes.(3) The UL83 gene was detected qualitatively bypolymerase chain reaction(PCR).(4) The exsistence of UL83 gene was detected by in suit nucleic acid hybridization assay.(5) The expression of pp65 was assayed by immunofluorescent test.(6)The load of UL83 gene was measured by fluorescence quantitative real-time PCR. At the same time, the cyclophosphamide - a kind of immunosuppressant were injected into the intraperitoneum of the remained mice (150mg/kg) every six days for three times. Six days after the last injection, all mice were killed . The lungs and the brains and the blood leukocytes were collected to be assayed as test above . Results: HCMV can establish latency in mice ,which can be induced by using of immunosuppressant , moreover, UL83 gene is a marker gene in latency and reactivation.The lung and the brain and the blood leukocyte are the latent sites for HCMV, but the lung is the major site for latent HCMV. The load of latent UL83 in each latent site is remarkably different. There was 8513 31 copies/ 106cells UL83 gene in lung ,which is the highest.There were 250 + 18 copies/ 106cells and 739+28 copies/106cells UL83 gene in brain and blood leukocyte respetively. The difference is obviously (P<0.01) . The load of latent UL83 gene and the risk of recurrence in group A is the highest, as is level of primary infection of mice . The load of latent UL83 gene and the risk of recurrence in group B is lower than those in group A. The load of latent UL83 gene and the risk of recurrence in group C is the lowest. Our research showed that prolonged high-level virus replication during primary infection in mice led to a high load of latent viral DNA and a high incidence of recurrence when individual was immunosuppress. Correlative coefficent and P value were 10.02 (P< 0.05) ,8.65 (P< 0.05 ) .Conclusion: The level of primary infection defined the load of latent UL83 gene and the latter determined the latent sites and the risk of recurrence. A high load of latent UL83 gene can lead to a high incidence of recurrence from latency. The load of latent UL83 gene in HCMV latent infection mice had positive correlation with the risk of recurrence.