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

Highly infectious

High mortality and morbidity rates

Transmission - close contact

Incubation period – within a few days

Economically devastating

16 HA and 9 NA subtypes

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)

Newcastle Disease virus

2000- Mexico - Chicken

2002-2003USA (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)

Objectives•To simultaneously detect AIV and NDV

•To discriminate between high and low pathogenic NDV

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

SAMPLE

PCR #1

- AIV-Matrix (M)

- NDV-Matrix (M)

- NDV-Fusion (F)

NC #1

- AIV and NDV detection

- NDV pathotyping

PCR #2AIV-HA

PCR #3AIV-NA

NC #2AIV-HA

subtyping

NC #3AIV-NA

subtyping

Overview of assay development

Specificity ExperimentsMultiplex RT-PCRMicroarray 42 AIV lab strains22 NDV strains

NE

G

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

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

AIV

37

AIV

38

AIV

39

AIV

40

AIV

41

AIV

42

AIV

43

ND

V 1

ND

V 4

ND

V 6

NE

G

ND

V-1

ND

V-2

ND

V-3

ND

V-4

ND

V-6

D10

D11

D12

E1

E2

E3

E4

E5

E6

E7

E8

E9

E10

E11

E12

F1

F2

Gel images of RT-PCR amplified cDNA from lab strains

AIV NDV

Electronic Microarray

• Electrophoretically driven printing of probes

and minimal amplicon hybridization time

• 400 independently activated test sites

++

Positive Charge

++

Positive Charge

Printing

Biotinylated Capture Probe

Amplicon

Hybridization

Wash

Reporting

Heat map – P:N data. Probe specificity to AI and NDV

AIV

M-0

07 d

eg

AIV

- M-0

08 d

eg

ND

V- M

-01

ND

V- M

-10

FM

D-A

T-2

3

M-

AIV

M-

ND

V- M

-01

ND

V- M

-10

FM

D-A

T-2

3

41 A

IV s

tra

ins

22 N

DV

str

ains

Detectionprobes

41 A

IV s

tra

ins

ND

V1-

HP

ND

V- H

P-1

ND

V- H

P-2

ND

V- H

P-3

ND

V- H

P-4

ND

V- H

P-5

ND

V- H

P-6

ND

V- H

P-7

ND

V- H

P-8

ND

V-H

P8-

VC

22

ND

V-L

P8-

NC

22

ND

V- L

P-4

ND

V- L

P-8

FM

D-A

T-2

3

8 H

P N

DV

str

ain

s1

4 L

P N

DV

str

ain

s

ND

V1-

HP

ND

V- H

P-1

ND

V- H

P-2

ND

V- H

P-3

ND

V- H

P-4

ND

V- H

P-5

ND

V- H

P-6

ND

V- H

P-7

ND

V- H

P-8

ND

V-H

P8-

VC

22

ND

V-L

P8-

NC

22

ND

V- L

P-4

ND

V- L

P-8

FM

D-A

T-2

3Pathotypingprobes

Sensitivity

Sensitivity of detection probe to DYH Amazon (HP)

0

5

10

15

20

25

NDV-M-10 FMD-AT-23

Probe

P:N

10̂ 5 10̂ 4 10̂ 3 10̂ 2 10̂ 1 10̂ 0 10̂ -1 NTC

Limit of detection 103 TCID50 /ml

Limit of detection for NDV B2 using detection probe 101 TCID50/ml

pathotyping probe 102 TCID50 / ml

Limit of detection 103 TCID50 / ml

Detection probe Pathotyping probe

Sensitivity of HP probe

0

5

10

15

20

25

HP 3 FMD-AT-23 HP3 FMD-AT-23

Probe

P:N

10̂ 5 10̂ 4 10̂ 3 10̂ 2 10̂ 1 10̂ 0 10̂ -1 10̂ -2 NTC

DYH Amazon (HP) B2 (LP)

NDV Clinical Testing

Comparison of Microarray to RT-PCR

Bird 642 Cloacal swab

02468

101214161820

NDV-M-01 NDV-HP-3 FMD-AT-23

Probe

P:N

1 dpi

2 dpi

3 dpi

NTC

Cloaca

DPI Array RT-PCR

1 7 9

2 10 10

3 7 10

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

Avian HA and NA subtyping40/41 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

N1 -

021

N1 -

026

N2 -

008

N2 -

014l

ong1

N3 -

002

N4 -

022

N5 -

017

NA

6-2(

qiu)

N7 -

005

N8 -

015

N9 -

022

Neg

.Con

t.

NA Specificity

Probes

Str

ains

AIV Heat map of P:N ratio data

N1

N2

N3

N4

N5N6

N7

N8

N9

NTC

CFIA, LethbridgeOliver Lung Dirk Deregt *Tara Furukawa-Stoffer Kimberley Burton HughesMathew FisherAnne Beeston*Kristen Hahn*Jennifer Geddes*Billy Mauro*

Nexogen, Inc. Dalibor Hodko

CFIA, NCFAD, Winnipeg John Pasick Yohannes BerhaneAnimal and Technical Staff Alfonso Clavijo*

IAH, Pirbright, UKDonald KingScott Reid

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

* Former members

CFIA Lethbridge Laboratory

Outline

Outbreaks Outbreaks

Electronic MicroarrayElectronic Microarray

Overview of Avian HC Assay: Design and DevelopmentOverview of Avian HC Assay: Design and Development

ResultsResults

NDV/AIV Multiplex RT-PCR Design and Development

NDV Matrix (M) gene1 1097

NDV Fusion (F) gene16621

Cleavage Region

400 bp

344 bp

AIV Matrix (M) gene1 1027

280 bp

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

Heat map – P:N data. Reactivity of probes to AI and NDV

AIV

M-0

07 d

eg

AIV

- M-0

08 d

eg

ND

V- M

-01

ND

V- M

-10

FM

D-A

T-2

3

00W1015 00.09.28 98 IMP 134 2CE

M-

AIV

M-

ND

V- M

-01

ND

V- M

-10

FM

D-A

T-2

3

H2N3 A/Mallard/AB/77/1977H3N2 A/TY/Eng/1969H4N8 A/Parrot/England/1970H5N1 A/CK/Scot/1959H5N2 A/CK/Hild/26654-1368/1994H5N2 A/CK/Penn/1370H5N2 A/TK/Minn/3689-1551/1981

H5N2 A/DK/BC/26-6/2000

H5N3 A/TY/CA/35621/1984H5N8 A/TY/Ireland/1963H5N9 A/TY/ON/7732/1966H5N9 A/TY/Wisconsin/1968H6N2 A/TY/Mass/1965H6N8 A/TY/ON/1963H7N1 A/TY/ON/18-2/2000H7N3 A/CK/Aust/3634/1992H7N3 A/CK/BC/514/2004H7N7 A/CK/Victoria/32972/1985H7N8 A/MR/China/28710/1995

H8N4 A/TY/ON/6118/1967H9N2 A/TY/Minn/12../1981H10N7 A/CK/Germany/1949H11N1 A/DK/Eng/1956H12N5 A/DK/AB/60/1976H13N1 A/Gull/MD/704/1977H14N5 A/Mallard/Gurjev/263/1982H15N8 A/DK/Aust/341/1983H16N3 A/BHG/Sweden/5/1999H2N9 A/Pintail/AB/293/7H4N6 A/DK/Czech/1956H9N9 A/Pheasant/WA/373H3N8 A/Mallard/AB/431/1983H5N2 A/Mallard/QC/2323-25/2006H1N1 A/Mallard/AB/306/06H5N2 A/Goose/MB/428/2006H5N2 A/Mallard/BC/8038/2006H10N7 A/CK/ON/01/1999H11N1 A/Goose/MB/321/2006H13N6 A/Gull/SK/283/1999H1N1 A/Mallard/ON/269/2005H1N2 A/Goose/MB/325/2006

WIN-AH-2008-OTH-0027 276 KI ICENDV CK/US ROAKIN/48 2 CE

(NDV) DYHAMAZON ICE 99-10-25PMV-1 Australia Velogenic d10PMV-1 California Kimber VelogenicPMV-1 Fontana 1083 VelogenicPMV-1 G.B. Texas VelogenicNDV B1PMV-1 Ireland/Ulster E3/76PMV-1 LaSota LentogenicPMV-1 Conneticut 1960 WIN-AH-2008-OTH-0069WIN-AH-2003-CN-00013WIN-AH-2003-CN-0009WIN-AH-2001-NS-01416WIN-AH-2008-FAV-0001WIN-AH-2005-NS-03042WIN-AH-2006-IM-00028WIN-AH-2006-NS-000924WIN-AH-2001-CN-0001796CNN0057NTC

-

--

-

-

-AH- -OTH-

-10-25- d10---

NDV B1--1 -1

WIN- - -OTH-WIN-AH-2003-CN-00013WIN-AH-2003-CN-0009WIN-AH-2001-NS-01416WIN-AH-2008-FAV-0001WIN-AH-2005-NS-03042WIN-AH-2006-IM-00028WIN-AH-2006-NS-000924WIN-AH-2001-CN-0001796CNN0057NTC

41 A

IV s

tra

ins

22 N

DV

str

ains

Detectionprobes

41 A

IV s

tra

ins

ND

V1-

HP

ND

V- H

P-1

ND

V- H

P-2

ND

V- H

P-3

ND

V- H

P-4

ND

V- H

P-5

ND

V- H

P-6

ND

V- H

P-7

ND

V- H

P-8

ND

V-H

P8-

VC

22

ND

V-L

P8-

NC

22

ND

V- L

P-4

ND

V- L

P-8

FM

D-A

T-2

3

8 H

P N

DV

str

ain

s1

4 L

P N

DV

str

ain

s

ND

V1-

HP

ND

V- H

P-1

ND

V- H

P-2

ND

V- H

P-3

ND

V- H

P-4

ND

V- H

P-5

ND

V- H

P-6

ND

V- H

P-7

ND

V- H

P-8

ND

V-H

P8-

VC

22

ND

V-L

P8-

NC

22

ND

V- L

P-4

ND

V- L

P-8

FM

D-A

T-2

3Pathotypingprobes

00W1015 00.09.28 98 IMP 134 2CE

H2N3 A/Mallard/AB/77/1977H3N2 A/TY/Eng/1969H4N8 A/Parrot/England/1970H5N1 A/CK/Scot/1959H5N2 A/CK/Hild/26654-1368/1994H5N2 A/CK/Penn/1370H5N2 A/TK/Minn/3689-1551/1981

H5N2 A/DK/BC/26-6/2000

H5N3 A/TY/CA/35621/1984H5N8 A/TY/Ireland/1963H5N9 A/TY/ON/7732/1966H5N9 A/TY/Wisconsin/1968H6N2 A/TY/Mass/1965H6N8 A/TY/ON/1963H7N1 A/TY/ON/18-2/2000H7N3 A/CK/Aust/3634/1992H7N3 A/CK/BC/514/2004H7N7 A/CK/Victoria/32972/1985H7N8 A/MR/China/28710/1995

H8N4 A/TY/ON/6118/1967H9N2 A/TY/Minn/12../1981H10N7 A/CK/Germany/1949H11N1 A/DK/Eng/1956H12N5 A/DK/AB/60/1976H13N1 A/Gull/MD/704/1977H14N5 A/Mallard/Gurjev/263/1982H15N8 A/DK/Aust/341/1983H16N3 A/BHG/Sweden/5/1999H2N9 A/Pintail/AB/293/7H4N6 A/DK/Czech/1956H9N9 A/Pheasant/WA/373H3N8 A/Mallard/AB/431/1983H5N2 A/Mallard/QC/2323-25/2006H1N1 A/Mallard/AB/306/06H5N2 A/Goose/MB/428/2006H5N2 A/Mallard/BC/8038/2006H10N7 A/CK/ON/01/1999H11N1 A/Goose/MB/321/2006H13N6 A/Gull/SK/283/1999H1N1 A/Mallard/ON/269/2005H1N2 A/Goose/MB/325/2006

WIN-AH-2008-OTH-0027 276 KI ICENDV CK/US ROAKIN/48 2 CE

(NDV) DYHAMAZON ICE 99-10-25PMV-1 Australia Velogenic d10PMV-1 California Kimber VelogenicPMV-1 Fontana 1083 VelogenicPMV-1 G.B. Texas VelogenicNDV B1PMV-1 Ireland/Ulster E3/76PMV-1 LaSota LentogenicPMV-1 Conneticut 1960 WIN-AH-2008-OTH-0069WIN-AH-2003-CN-00013WIN-AH-2003-CN-0009WIN-AH-2001-NS-01416WIN-AH-2008-FAV-0001WIN-AH-2005-NS-03042WIN-AH-2006-IM-00028WIN-AH-2006-NS-000924WIN-AH-2001-CN-0001796CNN0057NTC

-

--

-

-

-AH- -OTH-

-10-25- d10---

NDV B1--1 -1

WIN- - -OTH-WIN-AH-2003-CN-00013WIN-AH-2003-CN-0009WIN-AH-2001-NS-01416WIN-AH-2008-FAV-0001WIN-AH-2005-NS-03042WIN-AH-2006-IM-00028WIN-AH-2006-NS-000924WIN-AH-2001-CN-0001796CNN0057NTC

Sensitivity of detection probes

Sensitivity of detection probe to APMV-1 Amazon (HP)

0

5

10

15

20

25

NDV-M-10 FMD-AT-23

Probe

P:N

10^5

10^4

10^3

10^2

10^1

10^0

10^-1

NTC

Sensitivity of detection probe to NDV B2 (LP)

0

5

10

15

20

25

NDV-M-10 FMD-AT-23

Probe

P:N

10^5

10^4

10^3

10^2

10^1

10^0

10^-1

10^-2

NTC

Limit of detection 103 TCID50 /ml

Limit of detection 101 TCID50/ml

Sensitivity of Pathotyping Probes

Sensitivity of HP probe

0

2

4

6

8

10

12

14

HP 3 FMD-AT-23 HP3 FMD-AT-23

Probe

P:N

10^5 10^4 10^3 10^2 10^1 10^0 10^-1 10^-2 NTC

DYH Amazon (HP) B2 (LP) DYH Amazon (HP)B2 (LP)

Limit of detection 103 TCID50 / ml

Limit of detection 102 TCID50 / ml

Sensitivity of LP probe

0

2

4

6

8

10

12

14

LP8 NC22 FMD-AT-23 LP8 NC22 FMD-AT-23

Probe

P:N

10^5 10^4 10^3 10^2 10^1 10^0 10^-1 10^-2 NTC

NDV Clinical Testing

Comparison of Microarray to RT-PCR

Bird 642 Cloacal swab

02468

101214161820

NDV-M-01 NDV-HP-3 FMD-AT-23

Probe

P:N

1 dpi

2 dpi

3 dpi

NTC

Bird 642 Oropharyngeal swab

02468

101214161820

NDV-M-01 NDV-HP-3 FMD-AT-23

Probe

P:N

1 dpi

2 dpi

3 dpi

NTC

Cloaca Oropharynx

DPI Array RT-PCR Array RT-PCR

1 7 9 0 1

2 10 10 5 8

3 7 10 8 10

Clinical samples

10 birds infected with APMV-1 Amazon

Sampling: Swabs: oropharynx, cloaca

Dpi: 1, 2, 3

Total = 60 samples

Field negative samples = 40

BSE facts1997 Rum to rum feed ban – pet food. Livestock feedd, fetrilizers, skull, brain, eyes tonsils, CFIA control collection2001 National TSE network2001 Canadian cattle IDMay 2003 National BSE case

TSE Lab Network scrapie associated fibrils