Lin Yipu, Gu Yan, McCauley John W
Mill Hill Laboratory, The Francis Crick Institute.
Mill Hill Laboratory, The Francis Crick Institute;
J Vis Exp. 2016 Dec 14(118):54897. doi: 10.3791/54897.
The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.
微量中和（MN）试验是一种用于测量流感病毒感染性和抑制病毒复制的标准技术。在本研究中，我们展示了一种基于成像的MN试验方案，以量化病毒之间真正的抗原关系。与依赖可见噬斑的典型蚀斑减少试验不同，该试验对每个孔中的整个感染细胞群体进行定量。该方案根据细胞系的选择来匹配病毒类型或亚型，以实现最大感染性，这在成像和图像处理过程中增强了样本对比度。定量滴定的引入定义了中和时输入病毒的量，并使不同实验的结果具有可比性。配备平板扫描仪和免费可下载软件的成像设置使该方法具有高通量、成本效益高、用户友好且易于在大多数实验室中部署的特点。我们的研究表明，改进后的MN试验对当前流行的甲型（H1N1）pdm09、甲型（H3N2）和乙型流感病毒效果良好，不受甲型（H3N2）病毒神经氨酸酶（NA）中氨基酸取代的显著影响。它对于那些在培养细胞中生长至低血凝素滴度和/或经历流产感染而无法形成蚀斑的病毒的特性鉴定特别有用。
