1. Edgewood Chemical Biological Center Detection of Viruses by the Integrated Virus Detection System (IVDS) Dr. Charles Wick R&T Directorate U.S. Army Edgewood Chemical Biological Center Aberdeen Proving Ground, MD 21010 Charles.h.wick@us.army.mil; telephone: (410) 436-3321

2. Edgewood Chemical Biological Center Integrated Virus Detection System (IVDS) Introduction to IVDS Results of other viruses Honey Bee Results Outline

3. Edgewood Chemical Biological Center Integrated Virus Detection System Concept Instrument and Sample Data Fate curves Viruses in mixtures Honey Bee Results References Rapid Virus Detection

4. Edgewood Chemical Biological Center Gathers wide ranged information about virus threats Not limited by reagents Not limited by prior knowledge Detection of multiple viruses in one analysis. Detects unknown and modified viruses Detects empty virus capsules Commercialized/COTS Integrated Virus Detection System

5. Edgewood Chemical Biological Center Virus Sample Concentrate (10 -4 mechanical Advantage) Counting means Automatic recording of virus concentration and size Fast Virus Counting Platform Utilizes “Virus Window” Counts intact virus particles without PCR, antibodies or reagents Detection of all viruses types – enables detection of all viruses, including unknowns. Multiple viruses counted in same sample in a few minutes. Accumulate over time ( additional advantage) Integrated Virus Detection System

6. Edgewood Chemical Biological Center Sample Analysis Graph of Concentration vs. Virus Size Detailed Data Virus Size Distribution Table 1 2 3 Integrated Virus Detection System

7. Edgewood Chemical Biological Center Percent intact for 70C: 90% at 1 minute 50% at 3 minutes 20% at 10 minutes MS2 stability vs temperature at 70 o C Integrated Virus Detection System 2001 ILIR Review: Temperature Fate Curve

8. Edgewood Chemical Biological Center Kilham Rat Virus KRV Ultrafiltration 100K UF, Buffer washed Salt peak removed revealing Parvovirus at 22.5 nm Unexpected contamination viruses at 34.6, 49.6 and 71nm (confirmed by other means). Multiple viruses counted in single sample. Integrated Virus Detection System

9. Edgewood Chemical Biological Center Mouse Hepatitis Virus A59 MHV-A59 Neat Coated Coronavirus at 73.7 nm Uncoated virus at 51.4 nm Delicate viruses detected directly from source material. Integrated Virus Detection System

10. Edgewood Chemical Biological Center IVDS Bee Test 2_2 Grind 2 g bees (~20 bees + 30 ml AA) Pre-filter Centrifuge 20,000 rpm/60 min (~40,000 x g) Decant and filter through 0.2 um hollow fiber cartridge UF 300K 26 ml reduced to 3 ml Avg. of 5 scans each

11. Edgewood Chemical Biological Center IVDS Bee Test 3 Grind 2 g bees (~20 bees + 30 ml AA) Pre-filter Centrifuge 20,000 rpm/60 min (~40,000 x g) UF 300K 30 ml reduced to 4 ml Avg. of 5 scans each

12. Edgewood Chemical Biological Center IVDS Bee Test 4 Grind 2 g bees (~20 bees + 30 ml AA) Pre-filter Centrifuge 20,000 rpm/60 min (~40,000 x g) UF 300K 30 ml reduced to 4.5 ml Avg. of 5 scans each

13. Edgewood Chemical Biological Center IVDS Bee Test 5

14. Edgewood Chemical Biological Center IVDS Bee Test 6 CA #1

15. Edgewood Chemical Biological Center IVDS Bee Test 7

16. Edgewood Chemical Biological Center IVDS Bee Test 8

17. Edgewood Chemical Biological Center IVDS Bee Test 9

18. Edgewood Chemical Biological Center IVDS Bee Test 10 CA #2

19. Edgewood Chemical Biological Center Honey Bee Summary Detected, Comments Yard #1, Sample F #131.1, 38.5nm -Test 2 Yard #1, Sample F #2 [31.1nm] -Test 3 Yard #1, Sample F #3 31.1nm, 38.5nm - Test 4 Aussie #1, 38.5nm -Test 5 Location #1, CA; 3/14/07; PA-Key; CAF3B; 1 Frame Bee 25, 33.4nm -Test 6 Aussie #2, 27.9nm, [37.2nm] - Test 7 Aussie #3, 26.9nm, [37.2nm] - Test 8 Aussie #4, 27.9nm, [37.2nm] - Test 9 Location #2, CA; 3/18/07; Strong hive 3; 10-20 frame, 27.9nm - Test 10 [low concentration]

20. Edgewood Chemical Biological Center Work to date indicates the following: Detect viruses in 5 minutes* Determines presence of Threat Determines absence of Threat Gives Concentration and Size Can be used in combination with other technologies. * Depending on sample source and need for preparation. Integrated Virus Detection System

21. Edgewood Chemical Biological Center References C.H. Wick, I. Elashvili, M. F. Stanford, P.E. McCubbin, S. V. Deshpande, Mass Spectrometry and Integrated Virus Detection System Characterization of MS2 Bacteriophage. Toxicology Mechanisms and Methods, Accepted for publication, (2006). Kuzmanovic, D. A., I. Elashivili, C. H. Wick, C. O'Connell, and S. Krueger..Structural Analysis and Quantification of RNA in Bacteriophage MS2-like Viruses in Solution by Small Angle X-ray Scattering. 2005, Journal of Applied Environmental and Microbiology, accepted 2006. Kuzmanovic, D. A., I. Elashivili, C. H. Wick, C. O'Connell, and S. Krueger. The MS2 Coat Protein Shell is Likely Assembled Under Tension: a Novel Role ofr the MS2 Bacteriophage A Protein as Revealed by Small-angle Neutron Scattering, J. Molecular Biology, 355, 1095-1111, (2006). Wick, C.H., P.E. McCubbin, R.E. Jabbour, S.V. Deshpande, Detecting Bacteria by Direct Counting of Structural Protein Units by IVDS and Mass Spectrometry. Toxicology Mechanisms and Methods, 16: 485-493, (2006). Wick, C.H., Kuzmanovic, D.A, Effects of Sample Impurities on the Analysis of MS2 bacteriophage by Small Angle Neutron Scattering, ECBC TR-441, (2005). Kuzmanovic, D.A., I. Elashvili, C. H. Wick, C. O’Connell and S. Krueger, Bacteriophage MS2: Molecular Weight and Spatial Distribution of the Protein and RNA Components by Small- Angle Neutron Scattering and Virus Counting, Structure, Vol. 11, 1339-1348, (2003). Integrated Virus Detection System

22. Edgewood Chemical Biological Center Integrated Virus Detection System Patents Wick, C.H., 6, 491, 872 Method and system for detecting and recording submicron sized particles Wick, C.H., 6, 485, 686 Method and apparatus for counting submicron sized particles Wick, C.H., 6, 238, 866 Detector for nucleic acid typing and methods for using the same. Wick, C.H., 6, 051, 189 System and method for detection, identification and monitoring of submicron-sized particles Others pending

23. Edgewood Chemical Biological Center Point of Contact Charles Wick Phone: 410-436-3321 Fax: 410-436-6783