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Updated: Feb 21, 2026
Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems
Published on: November 4, 2014
In vitro infectivity of oncolytic Newcastle Disease Virus: Correlation between plaque and fluorescent focus assays
Benjamin S Rush1, Melissa L Coughlin1, Gautam Sanyal1
1Analytical Sciences, Biopharmaceutical Development, MedImmune LLC, One MedImmune Way, Gaithersburg, MD 20878, United States.
Abstract:
Newcastle Disease Virus (NDV) is an avian paramyxovirus that has no significant pathogenicity in humans. Cancer cells with impaired immune defense mechanisms are susceptible to infection and lysis by NDV. A recombinant construct of a lentogenic form of NDV (rNDV) containing an insertion of granulocyte macrophage colony stimulating factor (GMCSF) transgene was earlier reported and shown to have acceptably low avian pathogenicity as well as oncolytic potential. Reliable measurement of infectious titer is key to determining the effectiveness of virus preparations to infect and lyse cells. We report here a comparative evaluation of two infectious titer assays as applied to rNDV: plaque assay and fluorescent focus assay (FFA). Optimization of assay conditions for both titer methods has produced concordant results spanning several orders of magnitude. While plaque formation is the gold standard measure of virus titer, FFA provides higher throughput and faster turn-around. FFA has been further evaluated on two different instrument platforms, for automated versus manual foci recognition and counting, with equivalent results. These results point to amenability of FFA to transfer between different laboratories and analysts, without introducing significant subjectivity in data analysis.
Insights
Newcastle Disease Virus (NDV) is a promising oncolytic virus for cancer therapy. Fluorescent focus assay (FFA) offers a faster, high-throughput alternative to plaque assays for measuring rNDV infectious titer.
Area of Science:
- Virology
- Oncology
- Biotechnology
Background:
- Newcastle Disease Virus (NDV) exhibits oncolytic potential against cancer cells.
- A recombinant NDV (rNDV) expressing GMCSF has shown promise for cancer therapy.
- Accurate measurement of infectious virus titer is critical for evaluating oncolytic virus efficacy.
Purpose of the Study:
- To comparatively evaluate plaque assay and fluorescent focus assay (FFA) for determining rNDV infectious titer.
- To assess the reliability and throughput of FFA for rNDV titration.
- To investigate the reproducibility of FFA across different instrument platforms.
Main Methods:
- Optimization of plaque assay and FFA conditions for rNDV.
- Comparative analysis of results obtained from both assays.
- Evaluation of FFA on automated and manual instrument platforms for foci counting.
Main Results:
- Optimized plaque assay and FFA yielded concordant results for rNDV titer across several orders of magnitude.
- FFA demonstrated higher throughput and faster results compared to plaque assay.
- FFA showed equivalent results on automated and manual platforms, indicating low subjectivity.
Conclusions:
- FFA is a reliable, high-throughput, and reproducible method for titrating rNDV.
- FFA is suitable for inter-laboratory transfer and consistent data analysis.
- FFA can be effectively utilized for assessing oncolytic virus preparations.
