Detection of Bovine High Consequence Agents

on an Electronic Microarray Platform

Kimberley Burton HughesCanadian Food Inspection Agency

Lethbridge Laboratory

Bovine High Consequence Pathogen Assays

• Component 1: – Foot and Mouth Disease Virus

• Component 2: – FMD Differentials

• Vesicular Stomatitis• Exotic Malignant Catarrhal Fever • Rinderpest• Blue Tongue• BVD• BHV• Parapox complex

Clinical Symptoms:FeverBlisters in mouthExcessive salivationBlisters on feetWeight loss

FMD Strain Identification (with strain specific probes)

- Vaccine selection - Epidemiology

FMDPositive

+

Bovine HC Pathogen Assays: Overview Suspect case Detect and Serotype FMD

FMD ELECTRONIC MICROARRAY A

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ASIA

SAT1

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Neg Control

Ser

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FMD Isolates

Neg Control

Detection probes

FMD ELECTRONIC MICROARRAY A

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Neg Control

Detection probes

Heat map depicting fluorescent intensities of 2 detection (A) and 12 serotyping (B) probes against 23 isolates that represent all 7 serotypes.

Bovine HC Multiplex Testing for 7 FMD differentials

FMDNegative

FMD Strain ID Strategy

- 3 highly variable sites were identified within VP 1 coding region

- Probes were designed at the 3 sites for reference strains of each serotype

Reference strains selected: - WRLFMD recommended vaccine strains - major genetic lineages or topotypes (genetic lineages that fall within geographical boundries)

1 2 3VP 1

Example

Reference Strain

A B C D E F

Site 1

Site 2

Site 3

Unknown strain

Site 1

Site 2

Site 3

Referencestrains

Pro

bes

FMD O Strain Identification

*WRLFMD recommended vaccine strains**Samuel and Knowles, J. Gen Virol. 2001

Reference Strain Topotype**

*O Manisa Middle East - South Asia

*O TAW 10/97 Cathay

*O BFS 1860 Euro - South America

O GHA 5/93 West Africa

O MAL 98 East Africa - 2

O MYA 98 South-East Asia

Strains Tested

Pro

bes

Bovine High Consequence Pathogen Assays

• Component 1: – Foot and Mouth Disease Virus

• Component 2: – FMD Differentials

• Vesicular Stomatitis• Exotic Malignant Catarrhal Fever • Rinderpest• Blue Tongue• BVD• BHV• Parapox complex

Bovine HC Multiplex

PCR

BVD BHV VSV (NJ)

VSV (IND) RPV BTV

MC

FV

OR

F

BTV

Virus Amplicon(bp)

BVD 277

BHV ~432

VSV 270

RPV 235

MCFV 405

PPV 84

BTV 342

Entero 195

Ent

ero Clinical Neg Oral

NT

C

Bovine HC Electronic Microarray Results (PN)

Virus Strains

BVDV

BHV

VSV

RPV

MC

FV

BTV

OR

FV

En

tero

N

TC

1 2 NJ IND

Pro

bes

RPV Titration Series

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RPV-P001 NDV HP VC22

Probes

Po

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(P

N R

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Sensitivity Bovine HC Assay

Virus PCR NC400

RP 0.1 0.1

VSV 2-3x103 2-3x103

BTV 100 100

BVD 31 31

BHV 68 6.8x102

Limit of Detection (TCID50/ml)103 102 101104 100 10-1 10-2 10-3

RPV 235 bp

TCID50/ml:

NC400 Results

Clinical Samples TestedVirus No. of

SamplesNo. of

AnimalsSample Type

VSV 24 2 NJ, 2 IND oral/nasal swabs

RPV 16 4 oral/nasal swabs

MCFV 24 2 Buffy coat

BTV 7 1 whole blood

BVD 6 (spiked) 3 oral/nasal swabs

BHV 4 (spiked) 2 oral/nasal swabs

Negative 36 31 oral/nasal swabs

VSV Oral Swabs Animal #855

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BVDV Com BVDV1 BVDV2 IBR VSV RPV MCF PPV BTVa BTVb Entero Neg

Flu

ore

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dpi -5

dpi 1

dpi 2

dpi 3

dpi 4

dpi 5

dpi 6

dpi 7

dpi 8

dpi 9

dpi 10

dpi 11

NTC

Pos

Neg

Strong, specific reaction starting at 2 dpi.

No cross reaction to other probes in assay.

Clinical Sample Testing Results

Advantages of electronic microarray: - highly multiplexed, type and subtype - robust platform - faster - higher throughput - simplifed data analysis

VSV Oral Swabs Animal #855

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VSV Neg

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dpi 3

dpi 4

dpi 5

dpi 6

dpi 7

dpi 8

dpi 9

dpi 10

dpi 11

NTC

PCR

NC400

RT-PCR*

DPI -5 1 2 3 4 5 6 7 8 9 10 11

Slide Array

- - + + + + + - - + - -

- - + + + + + - - + - -

- - - + - inc - - - inc - -

- - + + - + + - - + - -

* NCFAD completed RT-PCR

Current Work

• Adapt assays to new format– Fully automated, integrated system

• magnetic sample preparation• amplification• electronic microarray

– Portable, compact unit for on-site testing

– Fully bio-contained cartridge– Rapid– Low cost (~$10)

CFIA, LethbridgeOliver Lung Dirk Deregt Tara Furukawa-Stoffer Mathew FisherSamuel Ohene-AdjeiKristen Hahn*Anne Beeston*Jennifer Geddes*Billy Mauro*

Nexogen, Inc., San Diego, USA Dalibor Hodko

CFIA, NCFAD, Winnipeg John Pasick Yohannes BerhaneAlfonso Clavijo* Animal and Technical Staff

IAH, Pirbright, UKDonald KingScott Reid

Funding:

CRTI (The Chemical, Biological, Radiological or Nuclear Research and Technology Initiative)

* Former membersCFIA Lethbridge Laboratory

Bovine HC Multiplex PCR DevelopmentVirus Genome Gene

TargetAmplicon Size

Reference

Vesicular Stomatitis

-ssRNA L-gene (polymerase)

270 bp Lung et al. 2010(Unpublished)

Rinderpest -ssRNA F-gene(fusion protein)

235 bp Forsyth and Barrett 1995

MCFV dsDNA AlHV-1 ORF50(tegument protein)

405 bp Traul et al. 2005Li et al. 2000

Blue Tongue dsRNA Segment 1 (polymerase)

342 bp Toussaint et al. 2007 (modified)

BVD +ssRNA 5’ UTR 277 bp Deregt et al, 2006

IBR dsDNA gB 432 bp Adapted from Hindson et al. 2008

PPV dsDNA BL2 gene (envelope protein)

84 bp Nitsche et al. 2006

Enterovirus(Internal Control)

+ssRNA 5’UTR 195 bp Schwab et al. 1995

Specificity

across serotypes

PN Values for heat mapUNQID NAME New York 1Singer Hastings 24515 890 Ames-125cColorado #34V654 V135 #5 Edmonton#39 EdmontonV76 NJ 0185 PNBNJ 0804COE1NJ 1184 HDBNJ 89 GASNJ 92 CLB NJ New Mexico 95IN 85 CLB IN 94 GUB IN 97 CRB IN 98 COE IN ATCC IN 01V1008Kabete O Eldoret Buffalo PakchongWC11 NZ2 Serotype 2 Serotype 8 Serotype 10Serotype 11Serotype 12Serotype 13Serotype 14Serotype 17Entero NTCBVDcom BVDcom 11.76577 7.754208 7.804652 9.714948 3.256222 10.80851 1.35928 1.504666 1.239458 1.622801 1.272246 1.331403 0.812308 1.0339 1.118109 0.970975 1.080153 0.863726 0.851554 0.99503 0.985961 1.195056 0.830047 0.836354 1.187438 1.110822 1.079357 1.169616 7.526438 0.890285 1.117507 0.977176 1.197821 1.189438 1.157167 1.09731 1.171655 1.442352 1BVDV 1 BVDV 1 8.091065 5.345374 6.269013 1.604339 1.028921 1.043356 1.236615 1.314306 1.14814 1.271472 1.20234 1.359998 0.834306 0.943152 0.850309 0.84251 0.978906 0.815397 0.948131 0.986479 1.020446 1.036417 0.849969 0.785233 1.062698 0.992673 0.999221 1.035766 1.123631 0.775337 0.865609 0.918671 1.008658 0.847118 1.020732 1.015398 0.973768 0.990738 1BVDV 2 BVDV 2 1.192644 1.480582 1.0721 4.105763 2.739955 2.324881 1.582017 1.274558 1.205 1.368776 1.372848 1.520939 0.788122 1.147097 0.967384 0.89655 0.918711 0.816881 0.978715 0.894517 1.039074 1.04704 0.820648 0.818492 1.103149 1.034101 0.994347 1.00778 4.092187 0.734854 0.98943 0.976453 1.078531 0.980549 1.01028 0.949754 0.954165 1.002302 1IBR IBR 1.17609 1.252557 1.312911 1.349983 1.359995 1.36139 3.748538 3.589743 3.430233 3.080304 2.624018 2.885781 0.858442 1.030387 1.036894 0.924783 1.015739 0.838732 0.97224 1.034032 1.044263 1.108854 0.870049 0.835017 1.244105 1.061456 0.976465 0.757239 1.438995 0.935944 1.031932 1.112509 1.201936 1.19101 1.233181 1.196953 1.181384 1.25162 1VSV VSV 0.990311 1.340782 1.515463 1.540198 1.380635 1.478721 1.291367 1.813942 1.691816 1.596399 1.256628 1.051135 12.93494 13.58493 5.493361 12.47535 14.00174 13.1101 14.41345 14.32443 12.42949 12.53508 11.66703 12.35545 1.21965 1.259974 1.291757 0.951098 1.169733 0.576439 0.937514 0.852491 0.998242 0.916786 0.942873 0.926176 0.906602 0.90579 1RPV RPV 0.813762 0.850913 0.863405 0.82887 0.879974 0.857929 1.360662 0.7923 0.892522 0.847839 0.935839 0.946198 0.63565 0.803239 0.799678 0.765563 0.931487 0.793653 1.093582 1.026186 0.929206 1.031193 0.672809 0.816834 7.878836 5.648224 6.249232 0.977972 1.061381 0.734397 0.907609 0.929925 0.989575 0.972016 0.952307 0.907815 0.963 0.953902 1MCFV MCFV 1.083654 0.975525 0.961446 1.028907 1.053073 1.185808 1.179411 1.306567 1.144135 1.340048 1.309899 1.281784 0.784625 1.025706 1.009291 0.936185 0.940468 0.817093 0.992365 1.03337 0.994761 1.136021 0.807855 0.830696 1.145384 1.051836 0.936567 3.188325 0.737744 0.722477 0.833866 0.853611 0.899973 0.872598 0.89973 0.904135 0.968358 0.790019 1PPV PPV 1.289462 1.015418 1.235608 1.142404 1.00408 1.10624 1.192285 1.151036 1.198627 1.54175 1.333828 1.400519 0.594427 0.965978 0.944656 0.864962 1.023453 0.882464 1.062199 1.037955 1.088784 1.153146 0.906359 0.868475 1.181598 1.074598 0.954886 0.991382 9.351835 0.505999 0.851102 0.856463 0.889961 0.880337 0.917765 0.884074 0.919851 1.981599 1BTVa BTVa 1.255229 1.220215 1.260638 1.258259 1.18539 1.181823 1.901321 1.527834 1.286354 1.50709 1.462994 1.646308 0.973744 1.069918 0.958822 0.887444 1.052105 0.832121 1.113666 1.083519 1.03506 1.194128 0.96097 0.887396 0.690606 1.077383 1.083188 0.939982 1.170573 13.44441 12.1667 12.78084 12.54584 12.98281 13.00731 10.98303 13.35975 1.434924 1BTVb BTVb 1.104666 1.076057 1.091841 1.098475 1.059821 1.008122 1.126359 0.946015 1.019326 1.26406 1.257478 1.410889 0.799105 1.005829 0.956727 0.903884 0.967964 0.757045 0.958591 0.941938 1.006405 1.101974 0.793738 0.78154 1.049887 0.954058 0.95672 0.951752 1.175902 0.874435 1.218597 1.137489 1.14816 0.954699 0.872045 1.013138 0.938476 0.989955 1Entero Entero 0.717104 1.126007 1.190686 1.215852 1.223931 1.262037 1.191166 1.261282 1.188318 0.995047 1.186688 1.131555 0.8368 1.045319 0.959019 0.90408 1.036192 0.840817 0.902673 0.852565 0.992191 1.085978 0.836666 0.823407 1.052034 1.006911 1.018597 1.129746 1.199476 0.851214 1.020737 1.075995 0.984253 1.046272 1.108857 1.029543 1.023077 2.531845 1Neg ControlNeg Control 1.16786 1.19656 1.251484 1.189663 1.229557 1.2729 1.249676 1.477487 1.320412 1.240144 1.422093 1.485707 1.030243 1.257607 1.184943 1.090684 1.24808 0.986837 1.309383 1.174262 1.103398 1.319519 0.941855 0.857224 1.23534 0.941093 0.908428 0.853934 0.995909 0.742915 0.948758 0.818555 0.985439 0.956848 0.935025 0.911065 0.82454 0.89869 1

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A-T-23 A-T-27 C24 O148 mod1 O-T-16 O-T-8deg ASIA-208mod2

SAT1-215mod3

SAT2-T-23deg

SAT2-T-31 SAT2-T-9deg

SAT3-9 Neg. Cont.Probes

FI

A Iran 1/96 A ARG 2/2001A ARG /87 A COL/85A 22 Iran/99A 22 Iraq 24/64A 24 CruzeioO 1 Manisa O TAW 10/97 O UKG 11/2001 O 1 BFS /860C 1 Noville C 3 Resende ASIA 1 Pak 1/54ASIA 1 ShamirSAT1 Ken 4/98SAT1 Bot 1/68SAT2 Zim 5/81SAT2 Swa 1/69SAT2 Sau 1/2000SAT2 Zim 10/91SAT3 Bec 1/65SAT3 Zim 4/81

Probes

FMD Strain Identification (with strain specific probes)

- Vaccine selection - Epidemiology

FMDPositive+

Bovine HC Pathogen Assays: Overview

Sample from suspect FMD case

Simultaneous detection and serotyping of FMD

FMDNegative

Bovine HC assay Testing for 7 other HC agents

and differentials

-

Foot and Mouth Disease Virus

– Detection (polymerase gene)– Serotyping (VP3 gene)– Strain Identification

Bovine High Consequence Agents

Previously presented

Foot-and-Mouth Disease

• 7 serotypes: A, O, C, Asia 1, SAT 1, 2, 3

• Confirmation requires laboratory diagnosis

• Highly infectious, expensive to control, and economically devastating

• Last cases in Canada:1953 (US 1929)

Clinical Signs:FeverBlisters in mouthExcessive salivationBlisters on feetWeightloss

Current Work

New “Sample-to-Answer” Technology

• Fully Automated Molecular Detection

• Integrated Sample Preparation and DNA Electronic MicroarrayDetection

•Rapid

•Low cost

Portable I nstrument Disposable cartridge

Compact Bio-Contained

New “Sample-to-Answer” Technology

• Fully Automated Molecular Detection

• Integrated Sample Preparation and DNA Electronic MicroarrayDetection

•Rapid

•Low cost

Portable I nstrument Disposable cartridge

Compact Bio-Contained

Rinderpest Oral Swabs

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RPV MCF VSV BTVa BTVb BVDV Com BVDV1 BVDV2 IBR PPV Entero Neg Control

Flu

ore

sc

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t In

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C430 dpi 0

C430 dpi 6

C1035 dpi 0

C1035 dpi 6

NTC

Positive Control Pool

Negative Control

DPI

Rinderpest Oral Swabs

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1

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RPV MCF VSV BTVa BTVb BVDV Com BVDV1 BVDV2 IBR PPV Entero Neg Control

Po

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C430 dpi 0

C430 dpi 6

C1035 dpi 0

C1035 dpi 6

NTC

1. RNA isolation

2. RT-PCR amplification of target

++

Positive Charge

++

Positive Charge

Printing

Biotinylated Capture Probe

Amplicon

Hybridization

Wash

Reporting

NC400

1. Print biotinylated capture probes

2. Add amplicon

Biotinylated capture probe

Amplicon

reporter probe

3. Add reporter probe

4. Measure fluorescence at test site

Electronic Microarray• Electrophoretically driven printing and hybridization

Time (seconds) Time (hours) Electronic Passive

(Movie here)

CMOS Chip PlatinumMicroelectrode

Hydrogel Permeation LayerContaining Streptavidin

50 m diameter test site

Array

• 400 independently activated test sites

1. RNA isolation

2. RT-PCR amplification of target

++

Positive Charge

++

Positive Charge

Printing

Biotinylated Capture Probe

Amplicon

Hybridization

Wash

Reporting

Biotinylated capture probe

NC400

1. Print biotinylated capture probesAmplicon

2. Add amplicon

reporter probe

3. Add reporter probe

4. Measure fluorescence at test site