Abstract
BackgroundHuman herpesvirus 6 (HHV-6A and HHV-6B) infection of cell cultures can be measured by different methods, including immunofluorescence microscopy, flow cytometry, or quantification of virus DNA by qPCR. These methods are reliable and sensitive but require long processing times and can be costly. Another method used in the field relies on the identification of enlarged cells in the culture; this method requires little sample processing and is relatively fast. However, visual inspection of cell cultures can be subjective and it can be difficult to establish clear criteria to decide if a cell is enlarged. To overcome these issues, we explored a method to monitor HHV-6B infections based on the systematic and objective measurement of the size of cells using an imaging-based automated cell counter.ResultsThe size of cells in non-infected and HHV-6B-infected cultures was measured at different times post-infection. The relatively narrow size distribution observed for non-infected cultures contrasted with the broader distributions observed in infected cultures. The average size of cultures shifted towards higher values after infection, and the differences were significant for cultures infected with relatively high doses of virus and/or screened at longer times post-infection. Correlation analysis showed that the trend observed for average size was similar to the trend observed for two other methods to measure infection: amount of virus DNA in supernatant and the percentage of cells expressing a viral antigen. In order to determine the performance of the size-based method in differentiating non-infected and infected cells, receiver operating characteristic (ROC) curves were used to analyze the data. Analysis using size of individual cells showed a moderate performance in detecting infected cells (area under the curve (AUC) ~ 0.80-0.87), while analysis using the average size of cells showed a very good performance in detecting infected cultures (AUC ~ 0.99).ConclusionsThe size-based method proved to be useful in monitoring HHV-6B infections for cultures where a substantial fraction of cells were infected and when monitored at longer times post-infection, with the advantage of being relatively fast and easy. It is a convenient method for monitoring virus production in-vitro and bulk infection of cells.
Highlights
Human herpesvirus 6 (HHV-6A and HHV-6B) infection of cell cultures can be measured by different methods, including immunofluorescence microscopy, flow cytometry, or quantification of virus DNA by qPCR
HSB-2 cells (CCL-120.1TM, American Type Culture Collection, Manassas, VA) were infected by mixing fresh non-infected cells with a cell pellet of HSB-2 cells infected with Human herpesvirus 6A (HHV-6A) strain GS at a ratio 5:1, and cultured in Iscove’s Modified Dulbecco’s medium supplemented with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and 5% fetal bovine serum (FBS) at a density of 0.5 × 106 cells/ mL; cultures were inspected every 3 – 4 days and a sample of the cell suspension was analyzed in the cell counter each time
Characterization of the cytopathic effect induced by Human herpesvirus 6B (HHV6B) infection in SupT1 cells In order to verify that the infection with HHV-6B virus induced the characteristic cytopathic effect in SupT1 cells, i.e. the occurrence of enlarged cells, cells were infected with HHV-6B strain Z29 at a ratio of 300 copies DNA per cell and the cultures were monitored daily for up to 7 days
Summary
Human herpesvirus 6 (HHV-6A and HHV-6B) infection of cell cultures can be measured by different methods, including immunofluorescence microscopy, flow cytometry, or quantification of virus DNA by qPCR. These methods are reliable and sensitive but require long processing times and can be costly. An alternative is the analysis of cells stained with the fluorescent-labeled antibodies by flow cytometry [9,10,11] This method circumvents the subjectivity of the microscopy methods, still requires multiple incubations and washing steps and adequate instrumentation. They require specialized equipment, reagents, and sample processing, and do not provide information about the actual number or frequency of infected cells or the amount of infectious virus produced, and so represent only an indirect measure of the infection
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