Infection Of Mouse Embryo Fibroblasts With Human Cytomegalovirus Mediated By Heparan Sulfate In Vitro

Objective To research the productive infection of mouse embryo fibroblasts with human cytomegalovirus(HCMV) in vitro and to research the relationship between cell surface heparan sulfate and the adsorption of HCMV to mouse embryo fibroblasts. Methods (1) Firstly, plaque assay was carried out to examine the infective virus quantity of HCMV AD169 in viral suspension. Secondly, MF and HF cell monolayers were inoculated with the virus at an MOI of 100 PFU per cell and observed daily for characteristic cytopathic effect(CPE) of HCMV. Until most of the cells showed extensive cytopathic effects, the cells were harvested to detect HCMV UL83 and IE gene and their corresponding transcripts by PCR,RT-PCR and to detect intracellular virus particles by transmission electron microscope.Thirdly, MF and HF cell monolayers were inoculated with the virus.Then the supernate of cell cultures was taken respectively on the 1st,2nd,3rd,5th,7th,10th and 14th day and infective virus particles in the samples were measured by plaque assay.(2) The various concentration heparin and heparinase together with the appropriate amount of virus were respectively added to MF and HF cell monolayers and plaque assay was carried out to detect the quantity of virus which had adsorbed and infected the cells in HCMV infected group,heparin group and heparinase group. RT-PCR was carried out to examine the expression of HCMV IE mRNA in HCMV infected group, heparin group and heparinase group.Result (1)The MF cell monolayers showed the same viral cytopathic effect as HF cells after being inoculated with the virus. Both the UL83 and IE gene and their corresponding gene transcripts could be detected in the infected MF cells. And herpesvirus-like particles could be observed in the cytoplasm and nucleus of MF cells by transmission electron microscope. MF and HF cell monolayers were inoculated with the virus and observed daily for two weeks.The results showed that the characteristic CPE of HCMV could be observed on the 4th or 5th day , extensive CPE were showed on the 7th day, and on the 10th day all the cells showed CPE and the pathological cells began to perish on the 12th ~14th day. At the same time, the supernate of cell cultures was detected by plaque assay. The results showed that the infective virus quantity in cell culture supernate increased from the 3rd day, tended to peak on the 10th day, and decreased on the 14th day.(2)The inhibition test of HCMV plaque formation with heparin and heparinase showed the PFU decreased obviously in heparin group and heparinase group compared with HCMV infected group, and the higher the concentration of heparin and heparinase, the stronger the preventive effect. The expression of HCMV IE mRNA in the heparin and heparinase groups was significantly lower than that of HCMV infected group(P<0.01).Conclusion The productive infection of mouse embryo fibroblasts with human cytomegalovirus was confirmed in vitro. Through the reseach of heparin, the analog of heparan sulface, and heparinase,the inhibitor of cell sulface heparan sulfate, it was confirmed that the binding of human cytomegalovirus to mouse embryo fibroblasts was related with cell sulface heparan sulfate proteoglycan.It was also proved that heparin and heparinase could reduce the interaction between virus and cells.