Efficient Selection of Recombinant Fluorescent Vaccinia Virus Strains and Rapid Virus Titer Determination by Using a Multi-Well Plate Imaging System

Abstract

Engineered vaccinia virus (VACV) strains are used extensively as vectors for the development of novel cancer vaccines and cancer therapeutics. In this study, we describe for the first time a high-throughput approach for both fluorescent rVACV generation and rapid viral titer measurement with the multi-well plate imaging system, IncuCyte®S3. The isolation of a single, well-defined plaque is critical for the generation of novel recombinant vaccinia virus (rVACV) strains. Unfortunately, current methods of rVACV engineering via plaque isolation are time-consuming and laborious. Here, we present a modified fluorescent viral plaque screening and selection strategy that allows one to generally obtain novel fluorescent rVACV strains in six days, with a minimum of just four days. The standard plaque assay requires chemicals for fixing and staining cells. Manual plaque counting based on visual inspection of the cell culture plates is time-consuming. Here, we developed a fluorescence-based plaque assay for quantifying the vaccinia virus that does not require a cell staining step. This approach is less toxic to researchers and is reproducible; it is thus an improvement over the traditional assay. Lastly, plaque counting by virtue of a fluorescence-based image is very convenient, as it can be performed directly on the computer.

The Multiplicity of Infection of Recombinant Vaccinia Virus Expressing the T7 RNA Polymerase Determines the Rescue Efficiency of Vesicular Stomatitis Virus

“…Fluorescence-based plaque assay . The efficiencies of rescue were quantified by plaque-formation assay ( van Remmerden et al, 2012 ; Ye et al, 2021 ). The plaque assay is the gold standard for determining the infectious titer of VSV.…”