© 2014 Her Majesty the Queen in Right of Canada

(Canadian Food Inspection Agency), all rights reserved. Use without permission is prohibited.

Data Interpretation Challenges: the Example of

Genetic and Pathobiological Characterization of

HPAI H5N2 and H5N1 Viruses in Canada

July 15-18, 2015

Y. Berhane

National Center For Foreign Animal Diseases

Winnipeg, MB

Event Summary

• In December, 2014, HPAI caused by novel reassortant H5N2

virus was detected in commercial turkey and chicken farms in

Fraser Valley, British Columbia (BC)

• Dec 1-19, 2014: 11 commercial premises and 1 non-commercial

premise were identified with HPAI H5N2 in BC

• Feb 2, 2015: HPAI caused by novel reassortant H5N1 virus was

identified in backyard chicken layer flock

• March 31, 2015 HPAI H5N8 virus was isolated from frozen

hunter killed duck tissues (Date of kill 2014 hunting season)

• Apr 5-18, 2015: three commercial farms in Oxford county,

Ontario (ON) were identified with HPAI H5N2

2

3

Location of H5N2 infected premises in Fraser valley, BC.

The Map shows 11 Index Premises (IPs) and a BY flock from

where novel reassortant HPAI H5N2 viruses were isolated.

Canada

USA

4 km

4

Location of H5N1 and H5N8 infected birds in relation to

the 12 IPs

5

Location of H5N2 infected Premises in Oxford county,

Ontario

4 km

6

Information of Virus Isolates

• The H5N8 – 8 gene segments of EA origin

• Not involved in domestic poultry outbreaks in Canada

• Involved in 2 outbreaks in commercial poultry in California

• Detected in backyard flocks, captive wild birds and wild ducks in a

number of American states

• The H5N2 viruses found in BC and Ontario are novel reassortant viruses

• 5 gene segments (PB2, PA, HA, M & NS) from EA H5N8 virus

• 3 gene segments (PB1, NP & NS) from NA wild birds origin AI viruses

• The BC and ON H5N2 viruses are closely related to viruses

responsible for similar outbreaks in USA

• Similar H5N2 viruses were isolated from wild captive birds, wild

ducks and backyard flocks in different parts US

Information of Virus Isolates

7

• The H5N1 from BC is also a novel reassortant virus

• Different from the ‘EA HPAI H5N1’

• Mixture of EA H5N8 and low pathogenic wild bird origin N.

American viruses

• 4 gene segments (PB2, HA, NP, M) from H5N8

• 4 gene segments (PB1, PA, NA, NS) from North American wild

bird origin AI viruses

• Identical H5N1 viruses were isolated from wild ducks in WA

state

• H5N1 from BC has 19 AA deletion at the stalk of NA gene

compared to the H5N1 from WA

• Deletions in the NA gene are genotypic markers of AI

adaptation to gallinaceous poultry

• Using NGS, we tested all original samples (n=12) and weren’t

able to identify if the deletion occurred in the BY flock

8

A/Ck/BC/FAV01/2015 (H5N1) NA Gene

9

PB2

PB1

PA

HA

NP

NA

M

NS

PB2

PB1

PA

HA

NP

NA

M

NS

Eurasian Gene Segments

North American Gene Segments

H5N8 H5N2 H5N1

Turkeys, Chickens, domestic

Geese Backyard Chickens

PB2

PB1

PA

HA

NP

NA

M

NS

Unknown NA

A/Eurasian wigeon/Netherlands/emc-1/2014 (H5N8)

A/turkey/Germany-MV/R2472/2014 (H5N8)

A/duck/Chiba/26-372-61/2014 (H5N8)

A/duck/Chiba/26-372-48/2014 (H5N8)

A/turkey/Italy/14VIR7898-10/2014 (H5N8)

A/Eurasian wigeon/Netherlands/emc-2/2014 (H5N8)

A/duck/England/36254/2014 (H5N8)

A/chicken/Netherlands/14015531/2014 (H5N8)

A/chicken/Netherlands/14015526/2014 (H5N8)

A/Baikal teal/Korea/Donglim3/2014(H5N8)

A/broiler duck/Korea/Buan2/2014(H5N8)

A/chicken/Miyazaki/7/2014 (H5N8)

A/chicken/Kumamoto/1-7/2014 (H5N8)

A/Baikal teal/Korea/H96/2014(H5N8))

A/crane/Kagoshima/KU1/2014 (H5N8)

A/gyrfalcon/Washington/41088-6/2014(H5N8))

A/broiler/BC/FAV-008/2014 (H5N2)

A/broiler/BC/FAV-009/2014 (H5N2)

A/turkey/BC/FAV-0010/2014 (H5N2)

A/Northern pintail/Washington/40964/2014(H5N2)

A/duck/Beijing/CT01/2014 (H5N8)

A/duck/Beijing/FS01/2014 (H5N8)

A/duck/Guangdong/GD01/2014 (H5N6)

A/duck/Jiangxi/95/2014 (H5N6)

A/duck/Jiangsu/k1203/2010(H5N8)

A/breeder duck/Korea/Gochang1/2014 (H5N8)

A/mallard/Shanghai/SH-9/2013(H5N8)

A/goose/Shandong/k1204/2009(H5N5)

A/duck/Eastern China/108/2008(H5N1)

A/Hunan/1/2009(H5N1)

A/Hunan/2/2009(H5N1)

A/avian/Hong Kong/0828/2007(H5N1)

A/chicken/Bac Giang/07-74/2007(H5N1)

A/environment/Guizhou/4/2009(H5N1)

A/chicken/Viet Nam/TMU013/2008(H5N1)

A/chicken/China/k0602/2010(H5N1)

A/chicken/Shandong/k0603/2010(H5N1)

A/chicken/Korea/Gimje/2008(H5N1)

A/water/Hunan/7/2009(H5N1)

A/duck/Vietnam/QB1207/2012(H5N1)

A/Alberta/01/2014 (H5N1)100

90

100

100

100

100

94

100

100

100

100

96

95

99100

94

88

97

97

0.01

2.3

.4.4

2.3.4.1

2.3.4.3

2.3.4.2

2.3.4.5

2.3.2.1

Phylogenetic characterization of the HA genes of

novel re-assortanat HPAI H5N2 viruses isolated from

Canadian poultry with other EA viruses of H5 subtype

10

11

IP-1

IP-3

IP-4

Movem

ent of bre

edin

g

sto

ck

IP-5

IP-6

IP-10 IP-11

IP-2

IP-7

IP- 8

Ca

tch

ing

cre

w

En

viro

nm

en

tal/lo

ca

lize

d

sp

rea

d

IP-9

Hypothesized spread of HPAI among commercial

premises in the 2014 H5N2 outbreak in BC.

En

viro

nm

en

tal/lo

ca

liz

ed

sp

rea

d

12

Clu

ste

r 1

C

lus

ter

2

Clu

ste

r 3

13

Location of clusters of H5N2 infected premises based

field and molecular epidemiology studies

Canada

USA

3 km

I

II

III

14

Conclusions

• H5N8 originating from Eurasia introduced into the Pacific

migratory flyway during 2014

• H5N8 reassorted with North American origin viruses and

generated novel reassortant viruses with genes from both

EA and NA origin AIVs

• The EA H5N8 and its North American reassorted variant -

H5N2 and H5N1 are highly pathogenic to gallinaceous

poultry

• Still we don’t know why the H5N2 virus only is causing

almost all the outbreaks in USA and Canada

• Why ‘novel H5N1’ and EA H5N8 are not involved in any

outbreaks in states/provinces located on central or

Mississippi flyways

15

Experimental Studies with H5N2/H5N1

• Wild ducks are natural reservoir AIVs and can also be

potentially long-distance vectors

• Implicated in the long-range spread of H5N1 from

Asia to Europe and Africa

• EA H5N1 has been associated with mortality in

domestic and wild waterfowl

• EA/NA H5N2 was isolated from wild ducks in Pacific,

Central and Mississippi flyways

• H5N8 and EA/NA H5N1 only in the Pacific flyway

• H5N8 lethal to chickens, but attenuated, albeit

efficiently transmitted in ducks

• Information gaps exists as how the new novel

reassortant H5N2/H5N1 viruses behave waterfowl

Determining chicken lethal dose 50

(CLD50)

16

• 3 groups of SPF chickens were infected with

different dilutions of H5N1 or H5N2 viruses

• Chickens were observed for mortality for 14 days

• CLD50 calculation was determined based on

mortality

Survival curves for SPF chickens infected with

different doses Of H5N2

CLD50 ~30 PFU

CLD50~ 23 PFU

Survival curves for SPF chickens infected with

different doses Of H5N1

19

Survival curves of juvenile turkeys infected with

H5N1

20

Turkeys - H5N1

Turkey H5N1 DPI 3

Pancreas Liver Spleen

Turkey H5N1 DPI 3

Heart

Brain Duodenum

23

Experimental Design H5N2/H5N1

in domestic waterfowl

Juvenile Muscovy ducks Adult Chinese Geese

10,000 PFU

H5N1 or H5N2

5 Contact ducks

5 SPF chickens in cages

24 hrs after infection

Transmission Study

5 Contact Geese

5 SPF chickens in cages

Birds were monitored for clinical signs, mortality, shedding

pattern and sero-conversion

24

Muscovy Ducks – H5N2

Oral shedding Cloacal shedding

25

Chinese Geese - H5N2

Oral shedding Cloacal shedding

Muscovy Ducks Chinese Geese

Survival curves of Muscovy ducks and Chinese geese

infected with H5N2

27

0

20

40

60

80

100

120

Pre-Bleed 7dpi 10dpi 14dpi Final Bleed

Perc

en

t In

hib

itio

n

NP cELISA H5N2

Inoculated Geese

Contact Geese

Inoculated Duck

0

20

40

60

80

100

120

140

160

180

200

Pre-Bleed 7dpi 10dpi 14dpi Final Bleed

Tit

re

Titre de l'axe

Hemagglutination Inhibition H5N2

Inoculated Geese

Contact Geese

Inoculated Ducks

Days Post-Inoculation

0 2 4 6 8 10 12P

erc

en

t S

urv

iva

l

0

20

40

60

80

100HPAI H5N1 inoculated geese

Contact geese

Contact chickens

Days Post-Inoculation

0 2 4 6 8 10 12

Pe

rce

nt

Su

rviv

al

0

20

40

60

80

100

HPAI H5N1 inoculated ducks

Contact ducks

Contact chickens

Survival curve of Canada geese and Muscovy

ducks infected with H5N1

28

Days post HPAI H5N1 Inoculation

2dpi 3dpi 5dpi 7dpi 10dpi 13dpi

Ct In

f A

ma

trix

re

al-

tim

e R

T-P

CR

ora

l sw

ab

0

10

20

30

40

Days post HPAI H5N1 Inoculation

2dpi 3dpi 5dpi 7dpi 10dpi 13dpiC

t In

f A

ma

trix

re

al-

tim

e R

T-P

CR

clo

aca

l sw

ab

0

10

20

30

40

Chinese Geese H5N1

Oral shedding Cloacal shedding

Days post H5N1 Inoculation

2dpi 3dpi 5dpi 7dpi 10dpi 13dpi

Ct In

f A

ma

trix

re

al-

tim

e R

T-P

CR

ora

l sw

ab

0

10

20

30

40

Days post HPAI H5N1 Inoculation

2dpi 3dpi 5dpi 7dpi 10dpi 13dpi

Ct In

f A

ma

trix

re

al-

tim

e R

T-P

CR

clo

aca

l sw

ab

0

10

20

30

40

Muscovy Ducks H5N1

Oral shedding Cloacal shedding

0

20

40

60

80

100

120

Pre-Bleed 7dpi 13dpi Final Bleed

Perc

en

t In

hib

itio

n

NP cELISA H5N1

Inoculated Geese

Contact Geese

Inoculated Duck

Contact Duck

0

20

40

60

80

100

120

140

160

180

200

Pre-Bleed 7dpi 13dpi Final Bleed

Tit

re

Hemagglutination Inhibition H5N1

Inoculated Geese

Contact Geese

Inoculated Duck

Contact Duck

33

34

H5N2

H5N1

Goose DPI 4, H5N2 versus H5N1 antigen distribution

Liver Brain Pancreas 35

H5N2 DPI 5

H5N1 DPI 5

Duck H5N2 versus H5N1 antigen distribution

Liver Brain Pancreas 36

H5N2

H5N1

Goose DPI 4, H5N2 versus H5N1 antigen distribution

Spleen Heart

37

Conclusions from animal experiments

38

• Novel reassortant H5N1 is very lethal to juvenile

turkeys

• H5N1 and H5N2 replicated systemically in both ducks

and geese

• H5N2 infection was more lethal to juvenile Muscovy

ducks compared to adult Chinese geese

• H5N1 infection was more lethal to adult Chinese

geese compared to juvenile Muscovy ducks

• H5N1 was able to transmit efficiently to contact ducks,

geese as well as chickens

• H5N2 was able to transmit efficiently to contact ducks

and geese, but not from geese to contact chickens

39

Acknowledgments

NCFAD

Dr John Pasick &

Staff

PHAC SPU Unit

• Dr Darwyn Kobasa

• Anders Leung

Animal Care Unit

Dr Carissa Embury-

Hyatt & Pathology

Unit

CFIA Epidemiologist

Dr Caroline Dube

Prof. Soren Alexandersen