Viral Infectivity Assays

High-throughput, automated, and scalable viral infectious titer assays are needed: Accurate assessment of viral infectious titer is essential for evaluating process parameters during viral vaccine development. The conventional assays, such as plaque-forming units (PFU) and tissue culture infectious dose 50% (TCID₅₀), are endpoint assays. They rely on the detection of virus-induced cytopathic effects (CPE), often requiring labelling techniques to visualize infection. They are inherently time-consuming—often taking several days—labor-intensive, low-throughput, and subject to variability due to the subjective interpretation of results. As high-throughput bioreactor systems become more prevalent in vaccine manufacturing, these traditional methods increasingly limit the speed of development and process optimization. To address these challenges, there is a growing need for rapid, automated, and scalable infectious titer assays. Agilent addresses this need by offering real-time, label-free, high-throughput TCID₅₀ solutions through platforms such as the BioTek Cytation 5 with automated imaging analysis and the impedance-based xCELLigence RTCA systems for real-time, objective CPE monitoring. To further increase throughput, the Agilent HT BioSpa system combines an automated incubator with an RTCA HT system for effortless maintenance of cell culture and quantification of virus concentration using a 384-well plate format.

Stabilization of the Cytopathic Property of Viruses

The Virus Reduction Neutralization Test (VRNT) is a quantitative assay used to assess the ability of antibodies—typically from serum samples or therapeutic candidates—to neutralize viruses by reducing their infectivity. In this assay, antibodies are incubated with a defined quantity of virus. If neutralization occurs, the number of infectious particles capable of inducing cytopathic effects (CPE) is reduced. The consistency and reliability of the VRNT depend heavily on the stability of the virus’s infectious properties. Many viruses lose viability over time when held in solution without host cells or after repeated freeze-thaw cycles. To preserve infectivity and ensure reproducible CPE, virus stocks should not be refrozen once thawed. Instead, prepare single-use aliquots of the virus in suspension that are appropriately sized for the intended experiment. For each newly prepared and cryopreserved virus batch, re-establish the infectious dose to ensure accurate and consistent assay performance.

Selection of permissive cell line and assay condition optimization

The sensitivity and specificity of viral titer assays can vary greatly depending on the virus-host system. Developing an infectious titer assay for different virus types requires extensive optimization and validation. Key factors influencing assay performance include the selection of a permissive cell line, cell culture conditions (such as growth medium and seeding density), and the timing of virus inoculation relative to cell seeding. These parameters can significantly impact viral replication and detection. In some cell lines, variability in viral antigen expression or cytopathic appearance can lead to inconsistent results. For example, in plaque assays, the use of Vero cells—particularly low-passage Vero E6 cells—is critical, as they influence plaque morphology, size, and clarity. Due to the improved automation, throughput, and hands-off time, Agilent Cytation 5 and xCELLigence RTCA systems are suitable for assay condition optimization. Ultimately, the choice of assay must align with the specific goals of the study, and careful evaluation is essential to ensure that the selected method is appropriate for the intended application.