Virus quantification is of great importance for commercial and academic laboratories involved in research or production of viral vaccines, recombinant proteins, viral antigens, or antiviral agents. The most commonly used methods for virus quantification include the plaque titer assay, 50% tissue culture infectious dose (TCID50), fluorescent focus assay (FFA), and transmission electron microscopy (TEM).
While more modern methods, including quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assays (ELISA) are becoming more frequently used, there remain significant drawbacks associated with all of these assays.
For example, plaque titer assays require between 4 to 10 days to provide a measure of infectious counts. Likewise, while qPCR may be able to provide results within a single day, it is laborious, requires a skilled operator, and is sensitive to contamination. In general, there is still a need for new analytical methods that can rapidly quantify viral concentration to reduce costs and alleviate bottlenecks associated with current assays.
InDevR has developed a personalized flow cytometer, the Virus Counter®, for rapid virus quantification in liquid samples. This instrument and assay represent a time and cost-effective alternative for the determination of virus concentrations. The assay uses two dyes—one specific for proteins and one specific for nucleic acids—to stain virus samples.
Samples prepared with these universal reagents are quantified by the Virus Counter instrument, which measures the concentration of virus particles (vp/mL) based on the number of particles producing simultaneous events on each of the two distinct fluorescence channels and the measured sample flow rate.
Virus Counter analysis requires ~10 minutes per sample. Figure 1 is a screenshot from the Virus Counter’s InCyt software showing fluorescence signals for the protein (red) and nucleic acid (blue) channels for an influenza A/California/4/2009 (H1N1) sample. The larger upper window shows a 3 ms segment of data and the smaller rectangle box in the lower portion of Figure 1 shows 75 ms of elapsed data. The upper display includes the nucleic acid and protein threshold values (indicated by the overlapping horizontal lines) used to discriminate virus events from background noise.
The Virus Counter has been used to quantify a wide range of viruses, matrix purity levels, and virus concentrations. Some of the most commonly analyzed viruses include adenovirus, baculovirus, coronavirus, cytomegalovirus (CMV), dengue virus, herpes simplex virus (HSV), influenza A (H1N1), influenza B (Flu B), parainfluenza, respiratory syncytial virus (RSV), and rubella.