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Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies
Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA
Virology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA
Center for Aerobiological Studies, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA
Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, TX 78227, USA
The FANG Assay Working Group is an interagency group is composed of the following institutes as well as those referenced with the authors above: Susan Garges and Aparna Kolhekar, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA; Christine Bruce, Health Protection Agency, Colindale, London NW9 5EQ, UK; Sophie Smither, Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK; Rebecca Kurnat, Chemical Biological Medical Systems Joint Vaccine Acquisition Program, Fort Detrick, MD 21702, USA.
* Author to whom correspondence should be addressed.
Received: 12 October 2012; in revised form: 10 November 2012 / Accepted: 20 November 2012 / Published: 6 December 2012
Abstract: The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines.
Keywords: plaque assay; filovirus; Ebola; ebolavirus; marburgvirus; Marburg virus; Vero
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Shurtleff, A.C.; Biggins, J.E.; Keeney, A.E.; Zumbrun, E.E.; Bloomfield, H.A.; Kuehne, A.; Audet, J.L.; Alfson, K.J.; Griffiths, A.; Olinger, G.G.; Bavari, S. Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies. Viruses 2012, 4, 3511-3530.
Shurtleff AC, Biggins JE, Keeney AE, Zumbrun EE, Bloomfield HA, Kuehne A, Audet JL, Alfson KJ, Griffiths A, Olinger GG, Bavari S. Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies. Viruses. 2012; 4(12):3511-3530.
Shurtleff, Amy C.; Biggins, Julia E.; Keeney, Ashley E.; Zumbrun, Elizabeth E.; Bloomfield, Holly A.; Kuehne, Ana; Audet, Jennifer L.; Alfson, Kendra J.; Griffiths, Anthony; Olinger, Gene G.; Bavari, Sina. 2012. "Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies." Viruses 4, no. 12: 3511-3530.