edgewood chemical biological center integrated virus detection system (ivds) introduction to ivds...
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Edgewood Chemical Biological Center
Integrated Virus Detection System (IVDS)
• Introduction to IVDS
• Results of other viruses
• Honey Bee Results
Outline
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
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
Edgewood Chemical Biological Center
MS2 on GEMMA
0
200
400
600
800
1000
1200
1400
1600
1800
2000
10.6
11.3
12.2
13.1
14.1
15.1
16.3
17.5
18.8
20.2
21.7
23.3
25.0
26.9
28.9
31.1
33.4
35.9
38.5
41.4
44.5
47.8
51.4
55.2
59.4
63.8
68.5
73.7
79.1
85.1
91.4
Diameter (nm)
GEMMA Counts
Statistics
Median (nm) 23.3Mean (nm) 23.1Geometric Mean (nm)22.9Mode 23.3Standard Deviation3.1Geo. Standard Deviation1.1Skewness -0.1Coefficient of variation (%)13.6
Total Concentration (#/ml)1.13x105
DiametersCounts10.6410.96.711.35.311.87.912.24.112.63.113.13.913.63.814.128.914.623.715.17.515.76.116.3916.8317.5318.1318.86.519.55.520.214.320.940.321.7175.122.5930.923.3196824.1123925.0105.725.918.926.912.827.910.528.98.430.09.431.15.632.21233.4534.6435.9437.2238.5140.0241.4242.9344.5046.14.347.84.749.6151.4153.3155.2357.3359.4561.54
63.8066.1168.50.671.00.473.7076.4079.1082.0185.1088.2091.4194.70
MS2 on GEMMA
0
200
400
600
800
1000
1200
1400
1600
1800
2000
10.6
11.3
12.2
13.1
14.1
15.1
16.3
17.5
18.8
20.2
21.7
23.3
25.0
26.9
28.9
31.1
33.4
35.9
38.5
41.4
44.5
47.8
51.4
55.2
59.4
63.8
68.5
73.7
79.1
85.1
91.4
Diameter (nm)
GEMMA Counts
Virus Counting
Graph of Concentration vs. Virus Size
Detailed Data
Virus Size Distribution Table
1
2
3
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
Edgewood Chemical Biological Center
MS2 on GEMMA
0
200
400
600
800
1000
1200
1400
1600
1800
2000
10.6
11.3
12.2
13.1
14.1
15.1
16.3
17.5
18.8
20.2
21.7
23.3
25.0
26.9
28.9
31.1
33.4
35.9
38.5
41.4
44.5
47.8
51.4
55.2
59.4
63.8
68.5
73.7
79.1
85.1
91.4
Diameter (nm)
GE
MM
A C
ou
nts
Statistics
Median (nm) 23.3Mean (nm) 23.1Geometric Mean (nm) 22.9Mode 23.3Standard Deviation 3.1Geo. Standard Deviation 1.1Skewness -0.1Coefficient of variation (%) 13.6
Total Concentration (#/ml) 1.13x105
Diameters Counts10.6 410.9 6.711.3 5.311.8 7.912.2 4.112.6 3.113.1 3.913.6 3.814.1 28.914.6 23.715.1 7.515.7 6.116.3 916.8 317.5 318.1 318.8 6.519.5 5.520.2 14.320.9 40.321.7 175.122.5 930.923.3 196824.1 123925.0 105.725.9 18.926.9 12.827.9 10.528.9 8.430.0 9.431.1 5.632.2 1233.4 534.6 435.9 437.2 238.5 140.0 241.4 242.9 344.5 046.1 4.347.8 4.749.6 151.4 153.3 155.2 357.3 359.4 561.5 4
63.8 066.1 168.5 0.671.0 0.473.7 076.4 079.1 082.0 185.1 088.2 091.4 194.7 0
MS2 on GEMMA
0
200
400
600
800
1000
1200
1400
1600
1800
2000
10.6
11.3
12.2
13.1
14.1
15.1
16.3
17.5
18.8
20.2
21.7
23.3
25.0
26.9
28.9
31.1
33.4
35.9
38.5
41.4
44.5
47.8
51.4
55.2
59.4
63.8
68.5
73.7
79.1
85.1
91.4
Diameter (nm)
GE
MM
A C
ou
nts
Sample Analysis
Graph of Concentration vs. Virus Size
Detailed Data
Virus Size Distribution Table
1
2
3
Integrated Virus Detection System
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 70oC
Integrated Virus Detection System
2001 ILIR Review: Temperature Fate Curve
Edgewood Chemical Biological Center
Kilham Rat Virus
CRL KRV (lot 091991) 100K UF 22 Jan 04
0.0
100.0
200.0
300.0
400.0
500.0
600.0
700.0
0 25 50 75 100
Channel Particle Midpoint (nm)
Co
un
ts
13.6 nm
22.5 nm
34.6 nm
49.6 nm
71 nm
20 scan accumulation
Unexpected contamination viruses at 34.6, 49.6 and 71nm (confirmed by other means).
Multiple viruses counted in single sample.
Integrated Virus Detection System
Edgewood Chemical Biological CenterMouse Hepatitis Virus A59
CRL MHV-A59 Neat (lot 120998)
91.4
73.7 nm
51.4 nm
0.0
100.0
200.0
300.0
400.0
500.0
600.0
0 25 50 75 100 125 150
Channel Particle Midpoint (nm)
Co
un
ts
10 scan accumulation
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
Edgewood Chemical Biological Center
Bee Test 2_2 Yard #1, Sample #2, Good Hive
Dilute 1:10 AA
31.1 nm38.5 nm
0.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
0 10 20 30 40 50 60 70 80 90 100
Channel Particle Midpoint (nm)
Co
un
tsIVDS Bee Test 2_2
Avg. of 5 scans each
Edgewood Chemical Biological Center
IVDS Bee Test 3
Bee Test 3 Yard #1, Sample #3, Failing Hive
Dilute 1:10 AA
31.1
0.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
0 10 20 30 40 50 60 70 80 90 100
Channel Particle Midpoint (nm)
Co
un
ts
Avg. of 5 scans each
Edgewood Chemical Biological Center
IVDS Bee Test 4
Bee Test 4Yard #1, Sample #1, Failed Hive
Dilute 1:10 AA
31.1 nm38.5 nm
0.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
0 10 20 30 40 50 60 70 80 90 100
Channel Particle Midpoint (nm)
Co
un
ts
Avg. of 5 scans each
Edgewood Chemical Biological Center
IVDS Bee Test 5
Bee Test 5Aussie #1
38.5
0.0
50.0
100.0
150.0
200.0
250.0
300.0
350.0
400.0
450.0
500.0
0 10 20 30 40 50 60 70 80 90 100
Channel Particle Midpoint (nm)
Co
un
ts
Coffee grinder - ~20 bees + 30 ml AAGrind 30-40 s, 2 timesPre-filter through #934 glass filter by gravityCentrifuge in 50 ml tubes at 20,000 rpm/60 min (~40,000 x g)UF 300K 30 ml reduced to 3.8 mlDilute 1:10 AAAvg of 5 scans each
Edgewood Chemical Biological Center
IVDS Bee Test 6
Bee Test 6
25
33.4
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
10000.0
0 10 20 30 40 50 60 70 80 90 100
Channel Particle Midpoint (nm)
Co
un
ts Le Grand, CA; 3/14/2007; PA-Key; CAF3B; 1 Frame BeeBlender - ~60 bees (6 g) + 100 ml AA, 30-40 s, 2 timesPre-filter by gravityCentrifuge in 50 ml tubes at 20,000 rpm/60 min (~40,000 x g)300K Da UF wash, 30 ml washed w/400 ml DI, 5 ml retainedDilute 1:10 for IVDSAverage of 5 scans each
CA #1
Edgewood Chemical Biological Center
IVDS Bee Test 7
Bee Test 7
37.2
27.9
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
10000.0
0 10 20 30 40 50 60 70 80 90 100
Channel Particle Midpoint (nm)
Co
un
ts
Aussie #2Blender - ~60 bees (6 g) + 100 ml AA, 30-40 s, 2 timesPre-filter by gravityCentrifuge in 50 ml tubes at 20,000 rpm/60 min (~40,000 x g)300K Da UF wash, 30 ml washed w/400 ml DI, 5 ml retainedDilute 1:10 for IVDSAverage of 5 scans each
Edgewood Chemical Biological Center
IVDS Bee Test 8
Bee Test 8
37.2
26.9
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
10000.0
0 10 20 30 40 50 60 70 80 90 100
Channel Particle Midpoint (nm)
Co
un
ts
Aussie #3Blender - ~60 bees (6 g) + 100 ml AA, 30-40 s, 2 timesPre-filter by gravityCentrifuge in 50 ml tubes at 20,000 rpm/60 min (~40,000 x g)300K Da UF wash, 30 ml washed w/400 ml DI, 5 ml retainedDilute 1:10 for IVDSAverage of 5 scans each
Edgewood Chemical Biological Center
IVDS Bee Test 9
Bee Test 9
37.2
27.9
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
10000.0
0 10 20 30 40 50 60 70 80 90 100
Channel Particle Midpoint (nm)
Co
un
ts
Aussie #4Blender - ~60 bees (6 g) + 100 ml AA, 30-40 s, 2 timesPre-filter by gravityCentrifuge in 50 ml tubes at 20,000 rpm/60 min (~40,000 x g)300K Da UF wash, 30 ml washed w/400 ml DI, 4.5 ml retainedDilute 1:10 for IVDSAverage of 5 scans each
Edgewood Chemical Biological Center
IVDS Bee Test 10
Bee Test 10
27.9
0.0
2000.0
4000.0
6000.0
8000.0
10000.0
12000.0
14000.0
16000.0
18000.0
20000.0
0 10 20 30 40 50 60 70 80 90 100
Channel Particle Midpoint (nm)
Co
un
ts
Lohman; Strong hive 3; 10-20 frames; from 3/18/2007Blender - ~60 bees (6 g) + 100 ml AA, 30-40 s, 2 timesPre-filter by gravityCentrifuge in 50 ml tubes at 20,000 rpm/60 min (~40,000 x g)300K Da UF wash, 30 ml washed w/400 ml DI, 4.5 ml retainedDilute 1:10 for IVDSAverage of 5 scans each
CA #2
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]
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
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
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