Validation of the Filovirus Plaque Assay for Use in Preclinical Studies

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 Biosafety Level 4 (BSL-4) studies. After standardization studies were...

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Main Authors:	Shurtleff, Amy C., Bloomfield, Holly A., Mort, Shannon, Orr, Steven A., Audet, Brian, Whitaker, Thomas, Richards, Michelle J., Bavari, Sina
Format:	Online
Language:	English
Published:	MDPI 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848606/

id	pubmed-4848606
recordtype	oai_dc
spelling	pubmed-48486062016-05-04 Validation of the Filovirus Plaque Assay for Use in Preclinical Studies Shurtleff, Amy C. Bloomfield, Holly A. Mort, Shannon Orr, Steven A. Audet, Brian Whitaker, Thomas Richards, Michelle J. Bavari, Sina Article 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 Biosafety Level 4 (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 the Marburg Virus Angola (MARV) variant and Ebola Virus Kikwit (EBOV) variant commenced at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). The 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 coefficient of variation (%CV) of ≤30%, deemed acceptable variation for a cell-based bioassay. Intraclass correlation statistical techniques for the evaluation of the assay’s precision when the same plaques were quantitated by two analysts returned values passing the acceptance criteria, indicating high agreement between analysts. The assay was shown to be accurate and specific when run on Nonhuman Primates (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. MDPI 2016-04-21 /pmc/articles/PMC4848606/ /pubmed/27110807 http://dx.doi.org/10.3390/v8040113 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Shurtleff, Amy C. Bloomfield, Holly A. Mort, Shannon Orr, Steven A. Audet, Brian Whitaker, Thomas Richards, Michelle J. Bavari, Sina
spellingShingle	Shurtleff, Amy C. Bloomfield, Holly A. Mort, Shannon Orr, Steven A. Audet, Brian Whitaker, Thomas Richards, Michelle J. Bavari, Sina Validation of the Filovirus Plaque Assay for Use in Preclinical Studies
author_facet	Shurtleff, Amy C. Bloomfield, Holly A. Mort, Shannon Orr, Steven A. Audet, Brian Whitaker, Thomas Richards, Michelle J. Bavari, Sina
author_sort	Shurtleff, Amy C.
title	Validation of the Filovirus Plaque Assay for Use in Preclinical Studies
title_short	Validation of the Filovirus Plaque Assay for Use in Preclinical Studies
title_full	Validation of the Filovirus Plaque Assay for Use in Preclinical Studies
title_fullStr	Validation of the Filovirus Plaque Assay for Use in Preclinical Studies
title_full_unstemmed	Validation of the Filovirus Plaque Assay for Use in Preclinical Studies
title_sort	validation of the filovirus plaque assay for use in preclinical studies
description	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 Biosafety Level 4 (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 the Marburg Virus Angola (MARV) variant and Ebola Virus Kikwit (EBOV) variant commenced at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). The 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 coefficient of variation (%CV) of ≤30%, deemed acceptable variation for a cell-based bioassay. Intraclass correlation statistical techniques for the evaluation of the assay’s precision when the same plaques were quantitated by two analysts returned values passing the acceptance criteria, indicating high agreement between analysts. The assay was shown to be accurate and specific when run on Nonhuman Primates (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.
publisher	MDPI
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848606/
_version_	1613572311854612480

Validation of the Filovirus Plaque Assay for Use in Preclinical Studies

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