Real-time Dynamic Monitoring Of HCMV Replication In MRC-5 Cells By Using Real-time Cell Assay (RTCA)

Objective The replication curves of HCMV in MRC-5 cells was analyzed by RTCA technique, the purpose of this study was to evaluate the value of RTCA technique, in monitoring the quantitative changes of virus titers and determining the neutralization test of HCMV.Methods Dynamic processes were observated for the growth index of MRC-5 cells at different stages. The most optimal concentration of cells to study HCMV virus proliferation were determined. At the same time, applying plaque forming assay indirect immunofluorescence assay and RT-PCR as traditional methods to compare and validate the results of RTCA technique.(1) The optimal concentration of cells were obtained through the following experiment:Firstly, 100μl/well containing 10% fetal calf serum nutrient solution was added to obtain the background value in 8-well E-plate. Secondly 300μl of MRC-5 cell suspension were added to each well to reach the final cell concentrations of 100,000/well,50,000/well and 25,000/well. Thirdly, each dilution were duplicate, and the instrument was placed in CO2 incubator for continuous observation for 7 days Then the growth curve of different concentrations of MRC-5 cells were obtained, cell impedance was converted to Cell Index (CI) and visual dynamics curves by software.(2) By using the parameter of the optimal cell concentration of 50,000 cells/well, MRC-5 cells were seeded into 8-well E-plate. After 24h, the nutrient solution were discarded, then 10-fold serial dilutions of HCMV AD 169 at the final concentration of 100,000PFU/well,10,000PFU/well, 1,000PFU/well and 100PFU/well were added to the 8-well E-plate. MRC-5 cells was infected.The control group was added 300μl of maintenance medium. Under the condition of 37℃,5% CO2, the incubation sustained for 1.5h. After that, the virus suspension were removed,300μl /well containing 2% fetal calf serum DMEM maintenance medium were added, and each virus dilution was set up for triplicates. Then the 8-well E-plate was put back into the real-time cell analyzer for providing every 30min monitored the CI value, a total of 200h monitoring period.(3) RTCA technique was compared with the HCMV plaque formation test,and the accuracy, repeatability and stability of both detection methods were analyzed. Also, the results of indirect immunofluorescence test and RT-PCR were set as a result of verification. (4) Collecting the serum of HCMV infection in the mouse model, determining the serum HCMV neutralizing antibody titers. At the same time, applicating the traditional fixed-diluted serum virus neutralization test as a method to compare and validate the results of science.Results (1)RTCA recorded the CPE values of the MRC-5 cell induced by HCMV completely. The optimal cell concentration was obtained as 50,000/well. At the final concentration of 50,000/well the MRC-5 cells had grown for 20h to enter the logarithmic growth phase, about to reach the plateau, and cells formed a uniform monolayer, and the time of the cells platform was able to meet the time of CPE observation in HCMV-infected cells (2)The RTCA system showed a significant negative correlation with time continuation with the detection of cell index and the virus titer (r=-0.977,P<0.005).(3)The results of RTCA monitoring HCMV CPE on MRC-5 cells and those of virus plaque forming unit (PFU) had a good correlation. In indirect immunofluorescence experiments and RT-PCR, with the increasing time in HCMV-infected cells, IE1 and pp65 expression in the nucleus increased much more, copies of the virus and the apple green fluorescence positive signal also showed more and more, which proved the accuracy of the RTCA CI values, stability and empty plaque formation accuracy of the results.(4)In the RTCA to analysis neutralizing antibody test, HCMV virus infection in mice and serum antibody titers CI value results is consistent with the traditional 96-well microtiter plates by microscopic examination.Conclusion RTCA technique was successfully established to monitor the growth status of the cells in real time, which.might reflect the cell’s adhesion and spreading. The RTCA system also could timely monitor the HCMV titer by interpreting the cell index and also be used to detect neutralizing antibodies.All the data are solid and reliable. This method would be helpful for future basic experiments on creating a new experimental technique and method.