High-Throughput Determination of Infectious Virus Titers by Kinetic Measurement of Infection-Induced Changes in Cell Morphology

Infectivity assays are the key analytical technology for the development and manufacturing of virus-based therapeutics. Here, we introduce a novel assay format that utilizes label-free bright-field images to determine the kinetics of infection-dependent changes in cell morphology. In particular, cel...

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Bibliographic Details
Published inInternational journal of molecular sciences Vol. 25; no. 15; p. 8076
Main Authors Hotter, Dominik, Kunzelmann, Marco, Kiefer, Franziska, Leukhardt, Chiara, Fackler, Carolin, Jäger, Stefan, Solzin, Johannes
Format Journal Article
LanguageEnglish
Published Switzerland MDPI AG 01.08.2024
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Summary:Infectivity assays are the key analytical technology for the development and manufacturing of virus-based therapeutics. Here, we introduce a novel assay format that utilizes label-free bright-field images to determine the kinetics of infection-dependent changes in cell morphology. In particular, cell rounding is directly proportional to the amount of infectious virus applied, enabling rapid determination of viral titers in relation to a standard curve. Our kinetic infectious virus titer (KIT) assay is stability-indicating and, due to its sensitive readout method, provides results within 24 h post-infection. Compared to traditional infectivity assays, which depend on a single readout of an infection endpoint, cumulated analysis of kinetic data by a fit model results in precise results (CV < 20%) based on only three wells per sample. This approach allows for a high throughput with ~400 samples processed by a single operator per week. We demonstrate the applicability of the KIT assay for the genetically engineered oncolytic VSV-GP, Newcastle disease virus (NDV), and parapoxvirus ovis (ORFV), but it can potentially be extended to a wide range of viruses that induce morphological changes upon infection. The versatility of this assay, combined with its independence from specific instruments or software, makes it a promising solution to overcome the analytical bottleneck in infectivity assays within the pharmaceutical industry and as a routine method in academic research.
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ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms25158076