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Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID₅₀ Assays.

ABSTRACT: Ongoing efforts to develop effective therapies against filoviruses rely, to different extents, on quantifying the amount of viable virus in samples by plaque, TCID₅₀, and focus assays. Unfortunately, these techniques have inherent variance, and laboratory-specific preferences make direct comparison of data difficult. Additionally, human errors such as operator errors and subjective bias can further compound the differences in outcomes. To overcome these biases, we developed a computer-based automated image-processing method for a focus assay based on the open-source CellProfiler software platform, which enables high-throughput screening of many treatment samples at one time. We compared virus titers calculated using this platform to plaque and TCID₅₀ assays using common stocks of virus for 3 major Filovirus species, Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus with each assay performed by multiple operators on multiple days. We show that plaque assays give comparable findings that differ by less than 3-fold. Focus-forming unit (FFU) and TCID₅₀ assays differ by 10-fold or less from the plaque assays due a higher (FFU) and lower (TCID₅₀) sensitivity. However, reproducibility and accuracy of each assay differs significantly with Neutral Red Agarose Overlay plaque assays and TCID₅₀ with the lowest reproducibility due to subjective analysis and operator error. Both crystal violet methylcellulose overlay plaque assay and focus assays perform best for accuracy and the focus assay performs best for speed and throughput.

SUBMITTER: Keiser PT

PROVIDER: S-EPMC7826780 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID<sub>50</sub> Assays.

Keiser Patrick T PT Anantpadma Manu M Staples Hilary H Carrion Ricardo R Davey Robert A RA

Microorganisms 20210112 1

Ongoing efforts to develop effective therapies against filoviruses rely, to different extents, on quantifying the amount of viable virus in samples by plaque, TCID<sub>50</sub>, and focus assays. Unfortunately, these techniques have inherent variance, and laboratory-specific preferences make direct comparison of data difficult. Additionally, human errors such as operator errors and subjective bias can further compound the differences in outcomes. To overcome these biases, we developed a computer ...[more]

PMID: 33445537

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Project description:BackgroundConventional assays to titrate polioviruses usually test serial dilutions inoculated into replicate cell cultures to determine a 50% cytopathic endpoint, a process that is both time-consuming and laborious. Such a method is still used to measure potency of live Oral Poliovirus Vaccine during vaccine development and production and in some clinical trials. However, the conventional method is not suited to identify and titrate virus in the large numbers of fecal samples generated during clinical trials. Determining titers of each of the three Sabin strains co-existing in Oral Poliovirus Vaccine presents an additional challenge.ResultsA new assay using quantitative multiplex polymerase chain reaction as an endpoint instead of cytopathic effect was developed to overcome these limitations. In the multiplex polymerase chain reaction-based titration assay, cell cultures were infected with serial dilutions of test samples, lysed after two-day incubation, and subjected to a quantitative multiplex one-step reverse-transcriptase polymerase chain reaction. All three serotypes of poliovirus were identified in single samples and titers calculated. The multiplex polymerase chain reaction-based titration assay was reproducible, robust and sensitive. Its lower limits of titration for three Sabin strains were 1-5 cell culture 50% infectious doses per ml. We prepared different combinations of three Sabin strains and compared titers obtained with conventional and multiplex polymerase chain reaction-based titration assays. Results of the two assays correlated well and showed similar results and sensitivity. Multiplex polymerase chain reaction-based titration assay was completed in two to 3 days instead of 10 days for the conventional assay.ConclusionsThe multiplex polymerase chain reaction-based titration (MPBT) is the first quantitative assay that identifies and titrates each of several different infectious viruses simultaneously in a mixture. It is suitable to identify and titrate polioviruses rapidly during the vaccine manufacturing process as a quality control test, in large clinical trials of vaccines, and for environmental surveillance of polioviruses. The MPBT assay can be automated for high-throughput implementation and applied for other viruses including those with no cytopathic effect.

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Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID₅₀ Assays.

Publications

Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID<sub>50</sub> Assays.

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Dataset Information

Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID50 Assays.

Publications

Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID<sub>50</sub> Assays.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID₅₀ Assays.