Validation of the Filovirus Plaque Assay for Use in Preclinical Studies
USAMRIID Frederick United States
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A plaque assay for quantitating filoviruses in virus stocks, prepared viral challenge inocula and samples from research animals has recently been fully characterized and standardized for use across multiple institutions performing BSL-4 studies. After standardization studies were completed, Good Laboratory Practices GLP-compliant plaque assay method validation studies to demonstrate suitability for reliable and reproducible measurement of Marburg Virus Angola MARV variant and Ebola virus Kikwit EBOV variant commenced at USAMRIID. Validation parameters tested included accuracy, precision, linearity, robustness, stability of the virus stocks and system suitability. The MARV and EBOV assays were confirmed to be accurate to 0.5 log10 PFUmL. Repeatability precision, intermediate precision, and reproducibility precision were sufficient to return viral titers with a CV of 30 , deemed acceptable variation for a cell-based bioassay. Intraclass correlation statistical techniques for evaluation of the assays precision when the same plaques were quantitated by 2 analysts returned values passing acceptance criteria, indicating high agreement between analysts. The assay was shown to be accurate and specific when run on NHP serum and plasma samples diluted in plaque assay medium, with negligible matrix effects. Virus stocks demonstrated stability for freeze-thaw cycles typical of normal usage during assay retests. The results demonstrated that the EBOV and MARV plaque assays are accurate, precise and robust for filovirus titration in samples associated with the performance of GLP animal model studies.