Department of Pathology, University of Massachusetts Medical School, 55 Lake Ave North, Worcester, MA, 01655, USA.
Department of Pathology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Ave North, Worcester, MA, 01655, USA.
Virol J. 2018 Jan 5;15(1):4. doi: 10.1186/s12985-017-0917-z.
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.
人疱疹病毒 6（HHV-6A 和 HHV-6B）可通过不同方法感染细胞培养物，包括免疫荧光显微镜、流式细胞术或通过 qPCR 定量病毒 DNA。这些方法可靠且敏感，但需要较长的处理时间且成本较高。该领域使用的另一种方法依赖于鉴定培养物中的细胞增大；这种方法需要很少的样品处理，相对较快。然而，细胞培养物的肉眼观察可能具有主观性，并且难以建立明确的标准来决定细胞是否增大。为了克服这些问题，我们探索了一种基于使用基于成像的自动细胞计数器系统和客观测量细胞大小的方法来监测 HHV-6B 感染。
在感染后不同时间测量非感染和 HHV-6B 感染培养物中细胞的大小。非感染培养物中观察到的相对较窄的尺寸分布与感染培养物中观察到的较宽分布形成对比。感染后培养物的平均尺寸向更高值移动，对于感染相对高剂量病毒的培养物和/或在感染后较长时间筛选的培养物，差异显著。相关分析表明，观察到的平均大小趋势与另两种测量感染的方法的趋势相似：上清液中病毒 DNA 的量和表达病毒抗原的细胞百分比。为了确定基于大小的方法在区分未感染和感染细胞方面的性能，使用接收者操作特征（ROC）曲线分析数据。使用单个细胞的大小进行分析显示出检测感染细胞的中等性能（曲线下面积（AUC）0.80-0.87），而使用细胞平均大小进行分析显示出检测感染培养物的非常好的性能（AUC0.99）。
基于大小的方法被证明在监测感染细胞比例较大的 HHV-6B 感染以及在感染后较长时间监测时非常有用，具有相对较快且易于操作的优点。它是一种用于监测体外病毒产生和细胞批量感染的方便方法。
