edgewood chemical biological center detection of viruses by the integrated virus detection system...

Edgewood Chemical Biological Center

Detection of Viruses by the Integrated Detection of Viruses by the Integrated Virus Detection System (IVDS)Virus Detection System (IVDS)

Dr. Charles Wick

R&T Directorate U.S. Army Edgewood Chemical Biological Center

Aberdeen Proving Ground, MD [email protected]; telephone: (410) 436-3321


Integrated Virus Detection System (IVDS)

• Introduction to IVDS

• Results of other viruses

• Honey Bee Results

Outline


Integrated Virus Detection System

• Concept

• Instrument and Sample Data

• Fate curves

• Viruses in mixtures

• Honey Bee Results

• References

Rapid Virus Detection


• 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



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)



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



Percent intact for 70C:

90% at 1 minute

50% at 3 minutes

20% at 10 minutes

MS2 stability vs temperature at 70oC


2001 ILIR Review: Temperature Fate Curve


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

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.


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


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.



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


Co

un

tsIVDS Bee Test 2_2

Grind 2 g bees (~20 bees + 30 ml AA)Pre-filterCentrifuge 20,000 rpm/60 min (~40,000 x g)Decant and filter through 0.2 um hollow fiber cartridgeUF 300K 26 ml reduced to 3 mlAvg. of 5 scans each


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


Co

un

ts

Grind 2 g bees (~20 bees + 30 ml AA)Pre-filterCentrifuge 20,000 rpm/60 min (~40,000 x g)UF 300K 30 ml reduced to 4 mlAvg. of 5 scans each


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


Co

un

ts

Grind 2 g bees (~20 bees + 30 ml AA)Pre-filterCentrifuge 20,000 rpm/60 min (~40,000 x g)UF 300K 30 ml reduced to 4.5 mlAvg. of 5 scans each


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


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


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


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


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


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


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


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


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


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


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


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


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]


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.



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).




• 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


Point of Contact

Charles Wick

Phone: 410-436-3321Fax: 410-436-6783

edgewood chemical biological center detection of viruses by the integrated virus detection system...

Documents

rapid virus detection

virus window

virus threats

nm uncoated virus

size fast virus

intact virus particles

reagents detection

modified viruses