The online version of this article (https://doi.org/10.1186/s12985-017-0917-z) contains supplementary material, which is available to authorized users.
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. 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.
The 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).
The 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.
Additional file 1: Effect of heat-inactivation and UV-inactivation of HHV-6B in the average size of cells. Control: non-infected SupT1; HHV-6B: live, heat-inactivated, UV-inactivated. Statistical significant differences observed only between control and live virus. (PDF 76 kb)12985_2017_917_MOESM1_ESM.pdf
Additional file 2: A. Phase-contrast microscopy images (20×) of SupT1.CIITA, MOLT-3 and Jurkat E6 cells non-infected or infected with HHV-6B strain Z29 (4 dpi); the right panel shows size histograms of non-infected cells (clear bars) and infected cells (gray bars) for each of the aforementioned cell lines; also shown are the non-linear fits for each sample (dashed lines = non-infected; solid lines = infected). B. Graphs summarizing data on average size of non-infected and infected cells in infections of SupT1.CIITA, MOLT-3 and Jurkat E6 with HHV-6B strain Z29 (all n = 3) and non-infected HSB-2 and HSB-2 cells infected with HHV-6A strain GS (n = 17). (PDF 6263 kb)12985_2017_917_MOESM2_ESM.pdf
Additional file 3: ROC curves for size data analyzed as percentage of cells above the cutoff at 4 dpi (A) and 7 dpi (B). (PDF 197 kb)12985_2017_917_MOESM3_ESM.pdf
Additional file 4: Alternative approaches for measuring size of cells. SupT1 were infected with various doses of HHV-6B strain Z29 and data collected at 7 dpi. A. Data collected using the imaging-based Cellometer Auto T4 cell counter: size distribution of non-infected (light gray) and infected cultures (dark gray). B. Data collected in a LSRII flow cytometer: forward-scatter area histograms of non-infected (light gray) and infected cultures (dark gray). (PDF 423 kb)12985_2017_917_MOESM4_ESM.pdf
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- Evaluation of a method to measure HHV-6B infection in vitro based on cell size
Lawrence J. Stern
- BioMed Central
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