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 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.
Molecular and Translational Sciences Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA. amy.c.shurtleff.ctr@mail.mil.
Amy C
AC
Shurtleff
Center for Aerobiological Studies, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA. holly.a.bloomfield.civ@mail.mil.
Holly A
HA
Bloomfield
Nonclinical Development Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA. shannon.m.mort.ctr@mail.mil.
Shannon
S
Mort
Nonclinical Development Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA. sorr@southernresearch.org.
Steven A
SA
Orr
Cell Culture Laboratory, Virology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA. brian.e.audet.civ@mail.mil.
Brian
B
Audet
Cell Culture Laboratory, Virology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA. thomas.j.whitaker26.ctr@mail.mil.
Thomas
T
Whitaker
Cell Culture Laboratory, Virology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA. michelle.j.richards.civ@mail.mil.
Michelle J
MJ
Richards
Molecular and Translational Sciences Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA. sina.bavari.civ@mail.mil.
Sina
S
Bavari
D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D029043:Ebolavirus / Q000502:physiology; D016563:Filoviridae / Q000502:physiology*; D029024:Marburgvirus / Q000502:physiology; D011786:Quality Control; D015203:Reproducibility of Results; D010948:Viral Plaque Assay / Q000592:standards*
Ebola; GLP compliant; Marburg virus; Marburgvirus; Vero E6 cells; animal rule; ebolavirus; filovirus; plaque assay; validation