The Experts below are selected from a list of 165 Experts worldwide ranked by ideXlab platform
Gene G. Olinger - One of the best experts on this subject based on the ideXlab platform.
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Standardization of the filovirus Plaque Assay for use in preclinical studies
Viruses, 2012Co-Authors: Amy C. Shurtleff, Ashley E. Keeney, Elizabeth E. Zumbrun, Holly A. Bloomfield, Ana Kuehne, Jennifer L. Audet, Julia E Biggins, Kendra J. Alfson, Anthony Griffiths, Gene G. OlingerAbstract: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.
Daniela Pilz - One of the best experts on this subject based on the ideXlab platform.
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wild rabies virus detection by Plaque Assay from naturally infected brains in different species
Veterinary Microbiology, 2004Co-Authors: Tereza C Cardoso, Daniela PilzAbstract:A simple, sensitive and specific Plaque Assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degrees C. The parameters affecting Plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degrees C in 5% CO(2) atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described Plaque Assay would be a new valuable tool in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied.
Amy C. Shurtleff - One of the best experts on this subject based on the ideXlab platform.
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Validation of the Filovirus Plaque Assay for Use in Preclinical Studies.
Viruses, 2016Co-Authors: Amy C. Shurtleff, Holly A. Bloomfield, Shannon Mort, Steven A. Orr, Brian Audet, Thomas Whitaker, Michelle J. Richards, Sina BavariAbstract: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.
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Standardization of the filovirus Plaque Assay for use in preclinical studies
Viruses, 2012Co-Authors: Amy C. Shurtleff, Ashley E. Keeney, Elizabeth E. Zumbrun, Holly A. Bloomfield, Ana Kuehne, Jennifer L. Audet, Julia E Biggins, Kendra J. Alfson, Anthony Griffiths, Gene G. OlingerAbstract: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.
Tereza C Cardoso - One of the best experts on this subject based on the ideXlab platform.
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wild rabies virus detection by Plaque Assay from naturally infected brains in different species
Veterinary Microbiology, 2004Co-Authors: Tereza C Cardoso, Daniela PilzAbstract:A simple, sensitive and specific Plaque Assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degrees C. The parameters affecting Plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degrees C in 5% CO(2) atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described Plaque Assay would be a new valuable tool in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied.
Ashley E. Keeney - One of the best experts on this subject based on the ideXlab platform.
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Standardization of the filovirus Plaque Assay for use in preclinical studies
Viruses, 2012Co-Authors: Amy C. Shurtleff, Ashley E. Keeney, Elizabeth E. Zumbrun, Holly A. Bloomfield, Ana Kuehne, Jennifer L. Audet, Julia E Biggins, Kendra J. Alfson, Anthony Griffiths, Gene G. OlingerAbstract: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.