title: Standardization of the filovirus plaque assay for use in preclinical studies.
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.
authors.affiliation: Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA. amy.c.shurtleff.ctr@us.army.mil
authors.forename: Amy C
authors.initials: AC
authors.lastname: Shurtleff
authors.forename: Julia E
authors.initials: JE
authors.lastname: Biggins
authors.forename: Ashley E
authors.initials: AE
authors.lastname: Keeney
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Zumbrun
authors.forename: Holly A
authors.initials: HA
authors.lastname: Bloomfield
authors.forename: Ana
authors.initials: A
authors.lastname: Kuehne
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Audet
authors.forename: Kendra J
authors.initials: KJ
authors.lastname: Alfson
authors.forename: Anthony
authors.initials: A
authors.lastname: Griffiths
authors.forename: Gene G
authors.initials: GG
authors.lastname: Olinger
authors.forename: Sina
authors.initials: S
authors.lastname: Bavari
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D029043:Ebolavirus / Q000254:growth & development / Q000302:isolation & purification*; D029024:Marburgvirus / Q000254:growth & development / Q000302:isolation & purification*; D014709:Vero Cells; D019562:Viral Load / Q000379:methods* / Q000592:standards*; D010948:Viral Plaque Assay / Q000379:methods* / Q000592:standards*