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Why Measurement of Viral Infectivity Matters and How to Improve It

TheCyte

LumaCyte's blog site for all news, information, featured stories, article reviews, and tech notes related to single cell analysis across many applications including Vaccine R&D and biomanufacturing, viral infectivity, bioreactor monitoring, neutralization assays, oncolytic vaccines, adventitious agent testing, phenotypic drug discovery, label-free biomarkers, anti-viral testing, gene therapy, CAR-T therapy, label-free single cell discovery and sorting.

Why Measurement of Viral Infectivity Matters and How to Improve It

Keya Rodrigues

Posted by Colin Hebert, VP Biotechnology & Keya Rodrigues, Quality Assurance Bioengineer 

Viral quantification involves the counting of viruses or viral molecules in a known volume to determine their concentration. It plays an essential role in studies carried out in the fields of recombinant protein production, viral vaccine production and infectious disease. Methods used in viral quantification can generally be grouped into 3 categories: the first uses cells to measure viral infectivity, the second analyzes viral proteins or gene expression levels and final group directly counts viral particles. While each technique provides information, the measurement of infectious particles, as opposed to total particles, is essential as it directly relates to vaccine efficacy. 

Methods in the first group include the viral plaque assay, fluorescent focus assay (FFA) and endpoint dilution assay (TCID50). Each of these assays rely on numerous viral dilutions added to cells to measure infectivity. In the plaque assay, an overlay medium is used to limit the spread of virus, resulting in areas of cell clearing or plaques that are then counted to determine the number of plaque forming units (PFU) per mL. The FFA uses fluorescently labeled antibodies that can detect infected host cells and/or viral particles prior to the formation of plaques, while the end-point dilution assay monitors the viral or cytopathic effects (CPE) on cells to quantify infectivity. These often subjective methods are well accepted but are not without their limitations and challenges. Specifically, they require a significant amount of time and labor as well as skilled operators, decreasing the efficiency of research and development efforts while at the same time driving up costs. They can also be difficult to reproduce and standardize and may require costly antibody labels, such as the FFA. 

Some of the techniques that fall into the second group are Quantitative Polymerase Chain Reaction (qPCR) and Enzyme-Linked Immunosorbent Assay (ELISA) based methods. In qPCR, the amount of viral DNA or RNA can be measured using virus specific probes or primers. ELISA relies upon the detection of viral particles and/or viral antigens binding to antibodies. These methods generally have good reproducibility and are somewhat less time consuming but are limited by their reliance on viral specific molecules, which can be difficult, expensive, and time consuming to produce. Finally, both qPCR and ELISA are not generally considered a measure of viral infectivity as the presence of viral molecules does not indicate actively infectious particles. 

Methods for directly counting viral particles include Transmission Electron Microscopy (TEM) and the Virus Counter®, which allow the user to directly count viruses in biological samples. However, the former method requires expensive equipment, skilled operators, and often extensive sample preparation while the latter has limited dynamic range and requires expensive reagent kits for some viruses. Most importantly, they count total viral particles, only a fraction of which may be infectious. Should the number of infectious particles change due to biological or physical conditions that might reduce infectivity, this will not be captured by a technique that only measures total viral particles. 

LumaCyte’s Laser Force Cytology technology falls into the first category as it measures viral infectivity in a novel way using a combination of optical and fluidic forces to characterize the properties of individual cells and their response to viral infection. Using this unique technology, LumaCyte’s Radiance instrument overcomes some of the challenges that current viral quantification methods face by enabling easier sample preparation, shortening detection time, providing high quality objective data to reduce subjectivity, ultimately lowering costs and improving efficiency of viral infectivity measurements.