Validation of the Filovirus Plaque Assay for Use in Preclinical Studies

Abstract

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 PFU/mL. 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.

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Document Details

Document Type
Technical Report
Publication Date
Sep 02, 2016
Accession Number
AD1009282

Entities

People

  • Amy C. Shurtleff
  • Brian Audet
  • Holly A. Bloomfield
  • Michelle J. Richards
  • Shannon Mort
  • Sina Bavari
  • Steven A. Orr
  • Thomas Whitaker

Organizations

  • United States Army Medical Research Institute of Infectious Diseases

Tags

Communities of Interest

  • Biomedical

DTIC Thesaurus Topics

  • Accuracy
  • Biomedical Research
  • Culture Techniques
  • Data Sets
  • Diseases And Disorders
  • Ebola Virus
  • Electronic Mail
  • Experimental Design
  • Indicator Dyes
  • Infectious Diseases
  • Information Science
  • Medical Personnel
  • Proteins
  • Standards
  • Tissue Culture
  • Virion
  • Viruses

Fields of Study

  • Biology

Readers

  • Mathematics or Statistics
  • Virology (or Medical Virology).