A Flow Cytometry Based Assay for the Quantification of Viruses Using Changes in Infected Cell Granularity

Abstract

A need exists for rapid, low-cost, and accurate infectious viral quantification method in the bio-pharmaceutical field for the production of vaccines and virus-based therapeutics. Two of the most common and traditionally employed methods to quantify infectious viruses are plaque assays and cell culture infectious dose 50 (CCID50); both are relatively inexpensive, however they can be time consuming and demonstrate significant variability, between experiments and operators. More recently developed methods use fluorescence to quantify viral components (e.g. DNA, protein, mRNA, etc), which allows for earlier enumeration, but cost and complexity are the major tradeoffs with respect to these techniques. Presented in this thesis is a novel method to quantify infectious viruses that is aimed at resolving the limitations of the aforementioned techniques; namely, marrying the traditionally mutually exclusive parameters of rapidness, economics, and variability. The proposed method seeks to quantify the infectious virions through the use of post-infection physiological changes within the cell using flow cytometry. To validate this method, a complementary Vero cell line was infected with a replication-deficient herpes simplex virus type 2 mutant and monitored over a 72 hour period for changes in intracellular complexity, also known as granularity, using flow cytometry. Granularity can be measured using flow cytometry by recording the amount of light that bent at approximately 90° from the incident beam. This bending of light is most commonly caused by light being reflected or refracted against internal cellular structures such as proteins or vesicles. It was found that, between 16-20 hours post infection (hpi), the percentage of the infected cell population displaying an increased degree of granularity could be correlated with the viral titers obtained through a traditional plaque assay with R2 values greater than 0.9 using a semi-logarithmic scale. To further demonstrate that this as a universal method, the technique was applied to Japanese quail muscles fibroblast cells (QT-35) infected with a highly attenuated canarypox virus (ALVAC). A similar increase in granularity corresponding to infection was detected in the infected population, thereby giving credence to the utility of this viral enumeration technique across a potentially broad virus-host cell range. Given the high level of correlation between the proposed and traditional methods of viral quantification, the use of flow cytometry could deliver cost savings and increase the throughput for use in the bio-pharmaceutical industry.