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Samuel Ohene-Adjei Canadian Food Inspection Agency. Lethbridge AB CAHLN 2010

Development of Electronic Microarray for Simultaneous Detection and Typing of Avian Influenza and Newcastle Disease Viruses. Samuel Ohene-Adjei Canadian Food Inspection Agency. Lethbridge AB CAHLN 2010. Avian Influenza. Outbreaks of HPAIV

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Samuel Ohene-Adjei Canadian Food Inspection Agency. Lethbridge AB CAHLN 2010

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  1. Development of Electronic Microarray for Simultaneous Detection and Typing of Avian Influenza and Newcastle Disease Viruses Samuel Ohene-Adjei Canadian Food Inspection Agency. Lethbridge AB CAHLN 2010

  2. Avian Influenza Outbreaks of HPAIV 1983 Pennsylvania H5N2 17 million birds mostly chicken and turkeys 2003 Netherlands H7N7 Virus isolated in 241 farms 30 million birds were culled 2004 Canada H7N3 40 Commercial farms 19 million culled (Fraser Valley, BC) Highly infectious High mortality and morbidity rates Transmission - close contact Incubation period – within a few days Economically devastating 16 HA and 9 NA subtypes

  3. Newcastle Disease virus 2000- Mexico - Chicken 2002-2003 USA (S. California, Nevada, Arizona, Texas)- Game fowl 2002 California (USA) – Transmission to commercial farms 3.5 million birds depopulated 9 Serotypes but 1 serotype associated with defined disease Remains infectious in bone marrow and muscle 6 months at -20ºC in infected egg for over 1 year at 4ºC Highly contagious – incubation period 4-6 days Economically devastating (HP)

  4. Diagnosis Confirmatory lab diagnosis needed Complications in veterinary diagnosis: AIV and NDV may present same clinical symptoms Possibility of mixed infection of AIV and NDV • Objectives • To simultaneously detect AIV and NDV • To discriminate between high and low pathogenic NDV

  5. Overview of assay development SAMPLE PCR #2 AIV-HA PCR #3 AIV-NA • PCR #1 • - AIV-Matrix (M) • NDV-Matrix (M) • NDV-Fusion (F) • NC #1 • - AIV and NDV detection • - NDV pathotyping NC #2 AIV-HA subtyping NC #3 AIV-NA subtyping • Specificity Experiments • Multiplex RT-PCR • Microarray • AIV lab strains • 22 NDV strains

  6. NEG AIV 2 AIV 3 AIV 4 AIV 5 AIV 6 AIV 7 AIV 8 AIV 9 AIV 10 AIV 11 AIV 12 AIV 13 AIV 14 AIV 15 AIV 16 AIV 17 AIV 18 Gel images of RT-PCR amplified cDNA from lab strains NEG NDV-1 NDV-2 NDV-3 NDV-4 NDV-6 D10 D11 D12 E1 E2 E3 AIV NDV AIV 19 AIV 20 AIV 21 AIV 22 AIV 23 AIV 24 AIV 25 AIV 26 AIV 27 AIV 28 AIV 29 AIV 30 AIV 31 AIV 32 AIV 33 AIV 34 AIV 35 AIV 36 E4 E5 E6 E7 E8 E9 E10 E11 E12 F1 F2 AIV 37 AIV 38 AIV 39 AIV 40 AIV 41 AIV 42 AIV 43 NDV 1 NDV 4 NDV 6

  7. Electronic Microarray • Electrophoretically driven printing of probes and minimal amplicon hybridization time • 400 independently activated test sites

  8. + + Positive Charge Positive Charge Printing Hybridization Reporting Biotinylated Capture Probe Amplicon Wash

  9. Heat map – P:N data. Probe specificity to AI and NDV Detection probes Pathotyping probes VC22 VC22 NC22 NC22 007 deg 008 deg 23 23 23 23 - - 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 - - 4 4 8 8 01 01 10 10 - - - - - - - - - - - - - - - - - - - - - - - - HP HP HP8 HP8 LP8 LP8 - - - - AT AT HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP LP LP LP LP AT AT - - - - - - M M M M - - - - - - - - - - - - - - - - - - - - - - - - - - M M M M - - - - - - - NDV1 NDV1 FMD FMD NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV FMD FMD NDV NDV NDV NDV AIV AIV AIV 41 AIV strains 41 AIV strains 8 HP NDV strains 22 NDV strains 14 LP NDV strains

  10. Sensitivity Detection probe Pathotyping probe DYH Amazon (HP) B2 (LP) Limit of detection 103 TCID50 /ml Limit of detection 103 TCID50 / ml Limit of detection for NDV B2 using detection probe 101 TCID50/ml pathotyping probe 102 TCID50 / ml

  11. NDV Clinical Testing Clinical samples 10 birds infected with APMV-1 Amazon Sampling: Swabs: oropharynx, cloaca Dpi: 1, 2, 3 Total = 60 samples Field negative samples = 40 • Samples not detected • RT-PCR Ct values close to cutoff (36) • Faint bands in PCR for array • Results highly comparable with • RT-PCR Comparison of Microarray to RT-PCR

  12. AIV Heat map of P:N ratio data Probes . 2(qiu) 014long1 NA Specificity 021 026 008 002 022 017 005 015 022 - Neg.Cont - - - - - - - - - - NA6 N1 N1 N2 N2 N3 N4 N5 N7 N8 N9 N1 N2 Strains N3 N4 N5 N6 N7 N8 N9 NTC Avian HA and NA subtyping 40/41 strains correctly HA subtyped All 41 strains correctly NA subtyped Sensitivity: comparable to real time PCR data Limit of detection of NA assay 43 - 2320 EID50 Limit of detection of HA assay 4 - 23 EID50 • Summary • Simultaneous detection of AIV and NDV and further pathotype NDV • No cross reactions observed • Assay useful for single and mixed infection • Results highly comparable to RT-PCR

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

  14. Outline • Outbreaks • Electronic Microarray • Overview of Avian HC Assay: Design and Development • Results

  15. NDV/AIV Multiplex RT-PCR Design and Development 280 bp AIV Matrix (M) gene 1 1027 400 bp NDV Matrix (M) gene 1 1097 344 bp NDV Fusion (F) gene 1 1662 Cleavage Region

  16. 50 m diameter test site Array Platinum Microelectrode Hydrogel Permeation Layer Containing Streptavidin CMOS Chip Electronic Microarray • Electrophoretically driven printing and hybridization • 400 independently activated test sites (Movie here) Time (seconds) Time (hours) Electronic Passive

  17. H2N3 A/Mallard/AB/77/1977 H2N3 A/Mallard/AB/77/1977 H3N2 A/TY/Eng/1969 H3N2 A/TY/Eng/1969 H4N8 A/Parrot/England/1970 H4N8 A/Parrot/England/1970 H5N1 A/CK/Scot/1959 H5N1 A/CK/Scot/1959 H5N2 A/CK/Hild/26654 H5N2 A/CK/Hild/26654 1368/1994 1368/1994 - - - - H5N2 A/CK/Penn/1370 H5N2 A/CK/Penn/1370 H5N2 A/TK/Minn/3689 H5N2 A/TK/Minn/3689 1551/1981 1551/1981 - - - - 6/2000 6/2000 H5N2 A/DK/BC/26 H5N2 A/DK/BC/26 - - - - H5N3 A/TY/CA/35621/1984 H5N3 A/TY/CA/35621/1984 H5N8 A/TY/Ireland/1963 H5N8 A/TY/Ireland/1963 H5N9 A/TY/ON/7732/1966 H5N9 A/TY/ON/7732/1966 H5N9 A/TY/Wisconsin/1968 H5N9 A/TY/Wisconsin/1968 H6N2 A/TY/Mass/1965 H6N2 A/TY/Mass/1965 H6N8 A/TY/ON/1963 H6N8 A/TY/ON/1963 H7N1 A/TY/ON/18 H7N1 A/TY/ON/18 - - - - 2/2000 2/2000 H7N3 A/CK/Aust/3634/1992 H7N3 A/CK/Aust/3634/1992 H7N3 A/CK/BC/514/2004 H7N3 A/CK/BC/514/2004 H7N7 A/CK/Victoria/32972/1985 H7N7 A/CK/Victoria/32972/1985 H7N8 A/MR/China/28710/1995 H7N8 A/MR/China/28710/1995 H8N4 A/TY/ON/6118/1967 H8N4 A/TY/ON/6118/1967 H9N2 A/TY/Minn/12../1981 H9N2 A/TY/Minn/12../1981 H10N7 A/CK/Germany/1949 H10N7 A/CK/Germany/1949 H11N1 A/DK/Eng/1956 H11N1 A/DK/Eng/1956 H12N5 A/DK/AB/60/1976 H12N5 A/DK/AB/60/1976 H13N1 A/Gull/MD/704/1977 H13N1 A/Gull/MD/704/1977 H14N5 A/Mallard/Gurjev/263/1982 H14N5 A/Mallard/Gurjev/263/1982 H15N8 A/DK/Aust/341/1983 H15N8 A/DK/Aust/341/1983 H16N3 A/BHG/Sweden/5/1999 H16N3 A/BHG/Sweden/5/1999 H2N9 A/Pintail/AB/293/7 H2N9 A/Pintail/AB/293/7 H4N6 A/DK/Czech/1956 H4N6 A/DK/Czech/1956 H9N9 A/Pheasant/WA/373 H9N9 A/Pheasant/WA/373 H3N8 A/Mallard/AB/431/1983 H3N8 A/Mallard/AB/431/1983 H5N2 A/Mallard/QC/2323 H5N2 A/Mallard/QC/2323 25/2006 25/2006 - - - - H1N1 A/Mallard/AB/306/06 H1N1 A/Mallard/AB/306/06 H5N2 A/Goose/MB/428/2006 H5N2 A/Goose/MB/428/2006 H5N2 A/Mallard/BC/8038/2006 H5N2 A/Mallard/BC/8038/2006 H10N7 A/CK/ON/01/1999 H10N7 A/CK/ON/01/1999 H11N1 A/Goose/MB/321/2006 H11N1 A/Goose/MB/321/2006 H13N6 A/Gull/SK/283/1999 H13N6 A/Gull/SK/283/1999 H1N1 A/Mallard/ON/269/2005 H1N1 A/Mallard/ON/269/2005 H1N2 A/Goose/MB/325/2006 H1N2 A/Goose/MB/325/2006 WIN WIN - - - - AH AH AH AH - - - - 2008 2008 - - - - OTH OTH OTH OTH - - - - 0027 276 KI ICE 0027 276 KI ICE NDV CK/US ROAKIN/48 2 CE NDV CK/US ROAKIN/48 2 CE (NDV) DYHAMAZON ICE 99 (NDV) DYHAMAZON ICE 99 - - - - 10 10 10 10 - - - - 25 25 25 25 PMV PMV - - - - 1 Australia 1 Australia Velogenic Velogenic d10 d10 d10 d10 PMV PMV - - - - 1 California 1 California Kimber Kimber Velogenic Velogenic PMV PMV - - - - 1 Fontana 1083 1 Fontana 1083 Velogenic Velogenic PMV PMV - - - - 1 G.B. Texas 1 G.B. Texas Velogenic Velogenic NDV B1 NDV B1 NDV B1 NDV B1 PMV PMV - - - - 1 Ireland/Ulster E3/76 1 Ireland/Ulster E3/76 PMV PMV - - - - 1 1 1 1 LaSota LaSota Lentogenic Lentogenic PMV PMV - - - - 1 1 1 1 Conneticut Conneticut 1960 1960 WIN WIN WIN WIN - - - - AH AH - - - - 2008 2008 - - - - OTH OTH OTH OTH - - - - 0069 0069 WIN WIN WIN WIN - - - - AH AH AH AH - - - - 2003 2003 2003 2003 - - - - CN CN CN CN - - - - 00013 00013 00013 00013 WIN WIN WIN WIN - - - - AH AH AH AH - - - - 2003 2003 2003 2003 - - - - CN CN CN CN - - - - 0009 0009 0009 0009 WIN WIN WIN WIN - - - - AH AH AH AH - - - - 2001 2001 2001 2001 - - - - NS NS NS NS - - - - 01416 01416 01416 01416 WIN WIN WIN WIN - - - - AH AH AH AH - - - - 2008 2008 2008 2008 - - - - FAV FAV FAV FAV - - - - 0001 0001 0001 0001 WIN WIN WIN WIN - - - - AH AH AH AH - - - - 2005 2005 2005 2005 - - - - NS NS NS NS - - - - 03042 03042 03042 03042 WIN WIN WIN WIN - - - - AH AH AH AH - - - - 2006 2006 2006 2006 - - - - IM IM IM IM - - - - 00028 00028 00028 00028 WIN WIN WIN WIN - - - - AH AH AH AH - - - - 2006 2006 2006 2006 - - - - NS NS NS NS - - - - 000924 000924 000924 000924 WIN WIN WIN WIN - - - - AH AH AH AH - - - - 2001 2001 2001 2001 - - - - CN CN CN CN - - - - 00017 00017 00017 00017 96CNN0057 96CNN0057 96CNN0057 96CNN0057 NTC NTC NTC NTC Heat map – P:N data. Reactivity of probes to AI and NDV Detection probes Pathotyping probes VC22 VC22 NC22 NC22 007 deg 008 deg 23 23 01 01 10 10 - - 23 23 - - 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 - - - - - - 4 4 8 8 AT AT - - - - - - - - - - - - - - - - - - - - - - - - - - M M M M HP HP HP8 HP8 LP8 LP8 M M M M - - AT AT HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP - - - - LP LP LP LP - - - - - - - - - - - - - - - - - - - - - - - - - - - - - FMD FMD NDV NDV NDV NDV AIV AIV AIV NDV1 NDV1 FMD FMD NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV NDV 41 AIV strains 41 AIV strains 8 HP NDV strains 00W1015 00.09.28 98 IMP 134 2CE 00W1015 00.09.28 98 IMP 134 2CE 22 NDV strains 14 LP NDV strains

  18. Sensitivity of detection probes Limit of detection 103 TCID50 /ml Limit of detection 101 TCID50/ml

  19. Sensitivity of LP probe 14 12 10 8 P:N 6 4 2 0 LP8 NC22 FMD-AT-23 LP8 NC22 FMD-AT-23 Probe 10^5 10^4 10^3 10^2 10^1 10^0 10^-1 10^-2 NTC Sensitivity of Pathotyping Probes B2 (LP) DYH Amazon (HP) DYH Amazon (HP) B2 (LP) Limit of detection 103 TCID50 / ml Limit of detection 102 TCID50 / ml

  20. NDV Clinical Testing Clinical samples 10 birds infected with APMV-1 Amazon Sampling: Swabs: oropharynx, cloaca Dpi: 1, 2, 3 Total = 60 samples Field negative samples = 40 Comparison of Microarray to RT-PCR

  21. BSE facts 1997 Rum to rum feed ban – pet food. Livestock feedd, fetrilizers, skull, brain, eyes tonsils, CFIA control collection 2001 National TSE network 2001 Canadian cattle ID May 2003 National BSE case TSE Lab Network scrapie associated fibrils

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