High-throughput virus quantification using cytopathic effect area analysis

The useful part

Adaptation of these methods to high-throughput formats (96-, 384- or 1,536-well plates) is challenging due to both assay automation constraints and the lack of well surface area available for reliable analysis. Here, we present a scalable alternative to traditional methods that uses whole-well image thresholding to quantify infectious virions by measuring virus-induced cytopathic effect (CPE) via cell lysis and detachment. The CPE area assay is best positioned as a practical high-throughput preliminary screening tool, effectively quantifying samples within the assay range and flagging samples above or below the concentration thresholds.

How it works

• To improve analysis efficiency and reduce user bias in CPE area selection, we evaluated an nnU-Net model for automated image segmentation against images segmented by manually defined brightness thresholds.
• The model achieved strong correlation with manual thresholding (R 2 = 1.00), showing minimal differences in the identified CPE area and thus demonstrating that nnU-Net accurately reproduces manual analysis.
• Paul Morris Traditional infectivity-based virion quantification methods, such as 50% tissue culture infectious dose (TCID 50) and plaque assays, are typically performed in 6-, 12-, 24-, or 48-well cell...
• This approach provides two complementary pipelines: manual thresholding, which tolerates adjustments of hyperparameters, and fully automated segmentation via nnU-Net, which streamlines analysis and enhances...
• This flexible CPE area assay enables accurate and automated quantification in high-throughput screening formats, thereby greatly accelerating a routine laboratory task while decreasing subjectivity and bias.