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The Virus Counter is an instrument for rapid quantification of viruses in liquid samples. It is a specialized flow cytometer that uses high-sensitivity fluorescence detection to give a direct measurement of the concentration of virus particles in a fraction of the time required for traditional plaque assays.
History and Development
The Virus Counter’s roots can be traced back to work done in Professor Kathy Rowlen’s lab at the University of Colorado in Boulder as part of the Ph.D. thesis project of Matthew Ferris. The Single Nanometric Particle Enumerator (SNaPE) was developed in 2001 and evaluated using respiratory viruses. The SNaPE research instrument was based on the principle of fluorescence detection from single stained nucleic acids aggregates. While the quantification results from this instrument correlated well with expected virus concentration, the values tended to be significantly higher than those obtained by standard plaque titer methods typically used for virus quantification. The measured concentrations were similar in magnitude to quantitative PCR results, both being substantially higher than plaque titer values. While this early, simple version of the instrument provided proof of concept using several viruses from commercial and collaborator sources, it was not ideal for commercial application.
The next step of the Virus Counter’s evolution also took place in the Rowlen’s lab at UC-Boulder as Carrie Stoffel’s Ph.D. project. By 2004 Carrie had added a second detection channel to the SNaPE instrument and improved data analysis substantially to increase specificity. The principle of detection was based on staining proteins and nucleic acids with fluorescent dyes within a liquid sample, where the protein stain emits in the red region of the visible spectrum and the nucleic acid stain emits in the yellow region of the visible spectrum. As stained particles pass through the probe region and interact with a laser, emissions are detected and recorded for analysis. This dual channel system established the concept of measuring intact virions through detection of co-localized protein and nucleic acids. Results from baculovirus analyzed with this dual-channel virus counter system were lower in magnitude than the SNaPE results, being more similar in magnitude and correlated with infectious assay results. This system was validated using several viruses from commercial sources as well as real-world samples from collaborators.
A commercial version of the Virus Counter is now available from InDevR Inc. The instrument is representative of the next generation of “personal” flow cytometers designed exclusively for a niche application, in this case virus quantification. Similar to virus concentration determined by transmission electron microscopy, the Virus Counter provides a measurement of the total number of virus particles per mL. The Virus Counter has been successfully demonstrated to readily quantify a diverse range of viruses, including influenza, adenovirus, dengue, baculovirus, respiratory syncytial, parainfluenza, rubella, cytomegalovirus, and herpes simplex virus.
The ability to rapidly quantify viruses has high impact in:
- Vaccine Research, Development, and Manufacturing
- Antiviral Research
- Viral Vector Research and Protein Expression
- Bioprocess Optimization
- Viral Antigen Characterization
- Viral Diagnostic Standard Characterization
- Viral-Based Gene Therapy Research
- Ferris, M. M.; Maurer, T. T.; Rowlen, K. L. (2006). "Detection and Enumeration of Single Nanometric Particles: A Confocal Optical Design for Fluorescence Flow Cytometry". Review of Scientific Instruments 73 (6). doi:10.1063/1.1476714.
- Ferris, M. M.; McCabe, M. O.; Doan, L. G.; Rowlen, K. L. (2002). "Rapid Enumeration of Respiratory Viruses". Analytical Chemistry 74 (8): 2863–2871. doi:10.1021/ac011183q. PMID 11985317.
- Ferris, M. M.; Stoffel, C. L.; Maurer, T. T.; Rowlen, K. L. (2002). "Quantitative Intercomparison of Transmission Electron Microscopy, Flow Cytometry and Epifluorescence Microscopy for Nanometric Particle Analysiss". Analytical Biochemistry 304 (2): 249–256. doi:10.1006/abio.2002.5616. PMID 12009703.
- Stoffel, C.L.; Rowlen, K. L.; Rowlen, KL (2005). "Design and Characterization of a Compact Dual Channel Virus Counter". Cytometry 65 (A): 140–147. doi:10.1002/cyto.a.20145. PMID 15830378.
- Stoffel, C.L.; Rowlen, K. L. (2005). "Data Analysis for a Dual-Channel Virus Counter". Analytical Chemistry 77 (7): 2243–2246. doi:10.1021/ac048626l. PMID 15801759.
- Stoffel, C.L.; Finch, R.; Christensen, K.; Edwards, D.; Rowlen, K. L. (2005). "Dual-Channel Virus Counter". American Biotechnology Laboratory 37 (22): 24–25.