Pathological Old Mouse Model Of Human Cytomegalovirus Latency

Objective For the first time, we established the pathological old mouse model of Human Cytomegalovirus latency, providing a platform for the research on the latency and reactivation of HCMV, at the same time, we aimed to know whether HCMV IE and UL83 gene could be the latency-specific gene which could prove the existence of a molecular latency. Methods On the cell and molecular level ,we quantitated the infectivity of viral suspension. (1) We have improved the method for detecting low-level persistent infectivity by combining infection of permissive indicator cells in culture and sensitive detection of immediate-early RNA in the infected cells by reverse transcriptase PCR.(l) Measured infective virus particles in virion suspension by plaque assay;(2)Extracted DNA from virion suspension and quantified viral genomes copies by fluorescence quantitated real-time PCR;(3)HF cell monolayers were inoculated with 50 l 0.01 to 10PFU of HCMV and scored daily for 30 days for viral cytopathiceffect(CPE);(4)Extracted mRNA from infected cells and analyzed HCMVimmediate-early RNA by reverse transcriptase PCR.( 2 ) 6 ~ 8 weeks old ,female,specific-pathogen-free BALB/c mice were divided into two groups, 12 mice in each group,and were injected with 1 X 107 PFU/mK 1 X 105PFU/ml of HCMV intraperitoneally respectively. Animals surviving acute infection were maintained for 1 years in isolation cages and analyzed for the replicating virus particles in tissue and the degree of pathologic damage. Then we could ascertain the optimal dose of HCMV in order to establish latency. Based on the results of these advance experiments, 12 BALB/c mice were infected with 1 X 105PFU/ml of HCMV intraperitoneally in order to establish HCMV latency(A group). At the same time, 12 mice which were infected intraperitoneally with 1ml DMEM were maintained as negative controls(B group).! year later, half of each groups(6 mice) were sacrificed ,and the other half were injected with 150mg/kg cyclophosphamide ,6 days apart with one dose, totally three doses, 6 days after the last dose, the mice were sacrificed. All the serum of mice were extracted for detection of anti-HCMV immunoglobulin. Brain and lung of each mouse were harvested for the following experiments:( 1) Virus isolation: Tissue samples obtained from mice were minced on ice,l:10 dilutions of undiluted clarified homogenates from each organ were overlaid on confluent HF monolayers and then were observed by inverted-phase microscopy for cytopathic effects characteristic of HCMV infection(4~ 8 weeks). By the method mentioned in advance experiment, we detected the productive virus particles in tisses.(2)Tissue section of brain and lung were stained by HE method for observing the pathological damage.(3)HCMV pp65 were assayed by immunofluorescent test with poly-clonal antibodies of pp65.(4)HCMV IE and UL83 gene were detected by in situ hybridizaiton assay.(5) Uninfected and latently infected mice were then screened for the presence of HCMV IE and UL83 DNA and RNA by analysis of nucleic acid extracted from various tissues.(6)Ultrastructure and virus particles were observed by electron microscope.(7) Virus DNA load were quantitated by fluorescence quantitative real-time PCR. Results (1) The infectivity of a virionsuspension was first quantitated as PFU and was found to be 3.5 107PFU/ml, viral genomes in 1ml virion suspension was 8.75 X 109copies/ml which means the genome-to-PFU ratio is 250, that is ,1 PFU of HCMV is the equivalent of 250 viral genomes. To quantitate the sensitivity of the assay, dilutions of virus were prepared so that inocula contained between 0.01 and 10 PFU of HCMV. Inocula containing an average of 1 PFU per well infected 90% of cells with HF monolayers. With the sensitivity of the RT-PCR to detect the HCMV IE transcript, infectivity was detected with an inoculum virus dose of 0.01 PFU, that is, within 2.5 viral genomes.(H)We could find productive virus particles and pathologic damage in tissue of mice that were infected with lX107PFU/ml HCMV, while all the resul...