1. Detection of Bovine High Consequence Agents on an Electronic Microarray Platform Kimberley Burton Hughes Canadian Food Inspection Agency Lethbridge Laboratory

2. 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: Fever Blisters in mouth Excessive salivation Blisters on feet Weight loss

3. FMD Strain Identification (with strain specific probes) - Vaccine selection - Epidemiology FMD Positive + Bovine HC Pathogen Assays: Overview Suspect caseDetect and Serotype FMD 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 FMD Negative

4. 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) 123 VP 1 Example Reference Strain A B C D EF Site 1 Site 2 Site 3 Unknown strain Site 1 Site 2 Site 3 Reference strains Probes

5. FMD O Strain Identification *WRLFMD recommended vaccine strains **Samuel and Knowles, J. Gen Virol. 2001 Reference StrainTopotype** *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 Probes

6. 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

7. Bovine HC Multiplex PCR BVD BHV VSV (NJ) VSV (IND) RPV BTV MCFV ORF BTV VirusAmplicon (bp) BVD277 BHV~432 VSV270 RPV235 MCFV405 PPV84 BTV342 Entero195 Entero Clinical Neg Oral NTC

8. Bovine HC Electronic Microarray Results (PN) Virus Strains BVDV BHV VSV RPV MCFV BTV ORFVEntero NTC 1 2 NJ IND Probes

9. Sensitivity Bovine HC Assay VirusPCRNC400 RP0.1 VSV2-3x10 3 BTV100 BVD31 BHV686.8x10 2 Limit of Detection (TCID 50 /ml) 10 3 10 2 10 1 10 4 10 0 10 -1 10 -2 10 -3 RPV 235 bp TCID 50 /ml: NC400 Results

10. Clinical Samples Tested VirusNo. of Samples No. of Animals Sample Type VSV242 NJ, 2 INDoral/nasal swabs RPV164oral/nasal swabs MCFV242Buffy coat BTV71whole blood BVD6 (spiked)3oral/nasal swabs BHV4 (spiked)2oral/nasal swabs Negative3631oral/nasal swabs

11. Strong, specific reaction starting at 2 dpi. No cross reaction to other probes in assay. Clinical Sample Testing Results

12. Advantages of electronic microarray: - highly multiplexed, type and subtype - robust platform - faster - higher throughput - simplifed data analysis PCR NC400 RT-PCR* DPI-5 1234567891011 Slide Array - -+++++--+-- - -+++++--+-- - --+- inc --- -- - -++-++--+-- * NCFAD completed RT-PCR

13. 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)

14. CFIA, Lethbridge Oliver Lung Dirk Deregt Tara Furukawa-Stoffer Mathew Fisher Samuel Ohene-Adjei Kristen Hahn* Anne Beeston* Jennifer Geddes* Billy Mauro* Nexogen, Inc., San Diego, USA Dalibor Hodko CFIA, NCFAD, Winnipeg John Pasick Yohannes Berhane Alfonso Clavijo* Animal and Technical Staff IAH, Pirbright, UK Donald King Scott Reid Funding: CRTI (The Chemical, Biological, Radiological or Nuclear Research and Technology Initiative) * Former members CFIA Lethbridge Laboratory

16. Bovine HC Multiplex PCR Development VirusGenomeGene Target Amplicon 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. 2005 Li 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

17. Specificity across serotypes

18. PN Values for heat map

19. FMD Strain Identification (with strain specific probes) - Vaccine selection - Epidemiology FMD Positive + Bovine HC Pathogen Assays: Overview Sample from suspect FMD case Simultaneous detection and serotyping of FMD FMD Negative Bovine HC assay Testing for 7 other HC agents and differentials -

20. Foot and Mouth Disease Virus –Detection (polymerase gene) –Serotyping (VP3 gene) –Strain Identification Bovine High Consequence Agents Previously presented

21. 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: Fever Blisters in mouth Excessive salivation Blisters on feet Weightloss

22. Current Work

23. DPI

25. 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

26. Electronic Microarray Electrophoretically driven printing and hybridization Time (seconds) Time (hours) Electronic Passive (Movie here) CMOS Chip Platinum Microelectrode Hydrogel Permeation Layer Containing Streptavidin 50  m diameter test site Array 400 independently activated test sites

27. 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 probes Amplicon 2. Add amplicon reporter probe 3. Add reporter probe 4. Measure fluorescence at test site