Identification of Viruses Using Microfluidic Protein Profiling and Bayesian Classification Julia A. Fruetel,* Jason A. A. West, † Bert J. Debusschere, Kyle Hukari, † Todd W. Lane, Habib N. Najm, Jose Ortega, Ronald F. Renzi, Isaac Shokair, and Victoria A. VanderNoot Sandia National Laboratories, Livermore California 94551-0969 We present a rapid method for the identification of viruses using microfluidic chip gel electrophoresis (CGE) of high- copy number proteins to generate unique protein profiles. Viral proteins are solubilized by heating at 95 °C in borate buffer containing detergent (5 min), then labeled with fluorescamine dye (10 s), and analyzed using the µChem- Lab CGE system (5 min). Analyses of closely related T2 and T4 bacteriophage demonstrate sufficient assay sen- sitivity and peak resolution to distinguish the two phage. CGE analyses of four additional viruses—MS2 bacterio- phage, Epstein—Barr, respiratory syncytial, and vaccinia viruses—demonstrate reproducible and visually distinct protein profiles. To evaluate the suitability of the method for unique identification of viruses, we employed a Baye- sian classification approach. Using a subset of 126 replicate electropherograms of the six viruses and phage for training purposes, successful classification with non- training data was 66/69 or 95% with no false positives. The classification method is based on a single attribute (elution time), although other attributes such as peak width, peak amplitude, or peak shape could be incorpo- rated and may improve performance further. The encour- aging results suggest a rapid and simple way to identify viruses without requiring specialty reagents such as PCR probes and antibodies. Virus isolation in cell cultures has long served as a “gold standard” method for virus identification. 1,2 Advantages of this method include the ability to isolate a wide variety of viruses, the sample provides an isolate for additional studies, and increased sensitivity over rapid antigen tests. Disadvantages are the long incubation periods required (days to weeks), technical expertise needed to read and interpret the cytopathic effect, and the cost and maintenance of a variety of cell culture types. Nonculture methods such as antigen detection by immunofluorescence show generally poorer sensitivity compared to cell culture, require expertise to read the results, and are not available for all viruses. Molecular methods such as PCR, although very sensitive, highly specific, and considerably faster than cell culture, suffer from high cost and sensitivity to polymerase inhibitors. Moreover, the higher mutation rates in viruses, especially retroviruses, 3 may be prob- lematic and result in false negatives when using dedicated PCR primers. 4 Both PCR and viral antigen detection are useful for viruses that do not proliferate in standard cell cultures. A significant drawback to all these methods is that they require specialty reagents that depend on the virus to be detected, such as specialized cell culture lines, antibodies, and PCR primers. Biosensors utilizing surface plasmon resonance and quartz-crystal microbalance have shown promise in detection of viral samples. 5 However, these methods require incorporation of recognition elements such as affinity ligands which limit their usefulness in situations where the infectious agent may be unknown, such as early in a disease outbreak or for environmental monitoring of a potential bioterrorist attack. Protein profiling is a technique broadly applicable to character- izing microorganisms and has been described predominantly in the mass spectrometry literature for identifying bacterial and viral proteins, 6-10 although capillary electrophoresis methods have also been described. 11,12 Using MALDI-TOF, for example, small acid- soluble proteins (SASPs) were found to be useful for identifying five Bacillus species. 13 Protein profiling is very appealing for diagnostics, as it is an approach broadly applicable to a variety of organisms, including viruses, and does not require specialty reagents. Currently this approach is both labor and equipment intensive, however, typically requiring 2D gel separation of proteins followed by mass spec- trometric analysis of the manually excised protein gel bands. We have developed a microfluidic protein profiling approach using protein solubilization coupled with microfluidic chip gel * To whom correspondence should be addressed. Julia A. Fruetel, Ph.D. Sandia National Laboratories P.O. Box 969 MS 9292 Livermore, CA 94551-0969. Phone: 925-294-2724. Fax: 925-294-3020. E-mail: jfruet@sandia.gov. † Current address: Arcxis Biotechnologies, 6920 Koll Center Parkway, Suite 215, Pleasanton, CA 94566. (1) Hsiung, G. D. Yale J. Biol. Med 1984, 57, 727–733. (2) Leland, D. S.; Ginocchio, C. C. Clin. Microbiol. Rev. 2007, 20, 49–78. (3) Svarovskaia, E. S.; Cheslock, S. R.; Zhang, W. H.; Hu, W. S.; Pathak, V. K. Front. Biosci. 2003, 8, D117–D134. (4) Clem, A. L.; Sims, J.; Telang, S.; Eaton, J. W.; Chesney, J. Virol. J. 2007, 4, 65–75. (5) Amano, Y.; Cheng, Q. Anal. Bioanal. Chem. 2005, 381, 156–164. (6) Krishnamurthy, T.; Rajamani, U.; Ross, P. L.; Eng, J.; Davis, M.; Lee, T. D.; Stahl, D. S.; Yates, J. ACS Symp. Ser. 2000, 745, 67–97. (7) Jarman, K. H.; Cebula, S. T.; Saenz, A. J.; Petersen, C. E.; Valentine, N. B.; Kingsley, M. T.; Wahl, K. L. Anal. Chem. 2000, 72, 1217–1223. 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