Study On Relativity Between Human Cytomegalovirus Infection Of Mouse Embryo Fibroblast In Vitro And Multiplicity Of Infection

Objective To research HCMV proliferate infection of mouse embryo fibroblast (MF) cells in vitro. To discuss the relativity between HCMV proliferate infection of MF and multiplicity of infection(MOI). To establish experimental supports for the reseach of the molecular mechanism of HCMV cross-species infection.Methods HCMV AD169 was cultured in human embryo fibroblast (HF) cells for proliferation.The virus was harvested and the virus titre was detected by plaque-forming units (PFU) assay. The MF and HF cells were infected with HCMV AD169 at an MOI of 0.1,1,10 and 100 PFU per cell respectively. The HCMV specific CPE was observed daily by microscope. The cells and the supernatant of the cultures were collected 1,2,3,5,7,10and14 d postinfetion for the following experiments:(1) determining the virus titre of the supernatant of the cultures by PFU essay; (2) detecting the transcriptional level of HCMV IE and UL83 mRNA by half quantity reverse transcriptase polymerase chain reaction (RT-PCR); (3) detecting the HCMV UL83 of the cell cultures 14 d postinfection by polymerase chain reaction (PCR); (4) observing the herpers virus-like particles and the ultrastructure in MF and HF cells by Electron microscope.Results On the 10th and 7th day, the HCMV specific CPE such as celluar swelling could be observed in HF cells infected with HCMV AD169 at an MOI of 0.1 and 1 PFU per cell, while there was no CPE in MF cells at the same MOI. On the 7th and 5th day, the specific CPE could be observed both in MF and HF cells infected with virus at an MOI of 10 and 100 PFU per cell, on the 7th and 5th day in MF cells and on the 5th and 3rd day respectively. The infection increased with the time prolonged until extensive cytopathic cells were observed. The supernatant of the MF cultures was detected in different time by PFU assay at an MOI of 0.1 and 1 PFU per cell.The virus titre was fairly low on the 1st and 2nd day while no plaque could be detected at another 5 time points. The virus titre was 5.8×101PFUs/ml on the 1st day, 1.5×102 PFUs/ml on the 2nd day, 2.1×105 PFUs/ml on the 5th day and it reached the peak of 1.2×107 PFUs/ml on the 10th day in MF at an MOI of 100 PFU per cell. Then the titre began to descend until the cells were deceased completely and was 3.4×105 PFUs/ml on the 14th day. The results were accordance with the specific CPE by microscope. At an MOI of 10 PFU per cell , the similar changing tendency could be found , only in the 14th day, the MF cells deceased partly. In the control group of HF, the virus could be detected in every quantity by PFU assay and the titre increased with the time prolonged until the cells began to perish. The results were similar between the group of HF and MF at an MOI of 10 and 100 PFU per cell. With every quantity, the cultures on the 14th day were all positive by PCR. By RT-PCR, the results were negative at an MOI of 0.1 and 1 PFU per cell while positive at an MOI of 10 and 100 PFU per cell. The transcriptional level increased progressively up to the peak and then began to decrease. The herpers virus-like particles could be observer in the intracellular only at an MOI of 10 and 100 PFU per cell by transmission electron microscope.Conclusion (1)HCMV AD169 has the ability to proliferately infect MF in vitro through PFU assays. (2)Ascertaining the positive relativity between proliferate infection and MOI by PFU assays and molecular biology methods.