1

FAO/OIE International Scientific Conference

on Avian Influenza and Wild Birds

May 30-31, 2006, Rome

The importance of surveillanceThe importance of surveillanceThe importance of surveillanceThe importance of surveillance

ofofofof avian and swine influenzaavian and swine influenzaavian and swine influenzaavian and swine influenza

HIROSHI KIDA and YOSHIHIRO SAKODA

Hokkaido University

Graduate School of Veterinary Medicine

Research Center for Zoonosis Control

Sapporo, Japan

1. Ecology and evolution of influenza viruses

-- Mechanism of antigenic variation of IV --

2. Mechanism of the emergence of pandemic

strains in humans and HPAIV in chickens

3. Do the H5N1 HPAIV strains perpetuate in

the lakes where migratory birds nest ?

4. Is the H5N1 HPAIV alone as a candidate of

pandemic strain?

5. What is the best measure for the control of

bird flu?

Points of Discussion

Host animals and HA and NA subtypes Host animals and HA and NA subtypes Host animals and HA and NA subtypes Host animals and HA and NA subtypes of influenza A virusof influenza A virusof influenza A virusof influenza A virus

2

Duck influenza◆ Each of the known subtypes (H1-15, N1-9) of

influenza A virus has been isolated from ducks.◆ In ducks, viruses replicate in the colon, being shed

with feces.◆ Water-borne fecal-oral transmission◆ Inapparent infection, apathogenic strains◆ Carry and provide viruses during migration and

overwintering.→→→→ Migratory duck is the natural host of

influenza A viruses.

H. KIDA, Hokkaido University Graduate School of Veterinary Medicine

Amino acid sub-stitutions on the HAs of 7 viruses isolated from ducks are scat-tered throughout the HA molecule and not confined to the antigenic sites,

indicating that

viruses are

circulating in the

absence of anti-

body selection

pressure.

Evolutionally

stasis

Amino acid sub-

stitutions on the

HAs of viruses

isolated from

humans are

confind to 5

antigenic sites,

indicating that

viruses are

circulating in

the presence of

antibody select-

ion pressure.

Extensive

antigenic

variation

H3 HAs

Why are viral genomes highly

conserved in ducks ?◆ Viruses replicating in intestine are not under the antibody selection pressure. ◆ 40 to 50 % of population of migratory ducks are juvenile birds that hatched in summer. ◆ Preserved in frozen lake water in winter.

H. KIDA, Hokkaido University Graduate School of Veterinary Medicine

HAs of H3N2 viruses isolated

from pigs in southern China◆ Closely related with those of A/Hong Kong/68 (H3N2) and H3 viruses isolated from migratory ducks◆ Amino acid sequences at the receptor-binding site on the HA indicate either specificity to human or avian viruses

H. KIDA, Hokkaido University Graduate School of Veterinary Medicine

3

Amino acid sequences at the receptor-binding site

of the H3 HA of swine viruses compared to

those of human and avian strains

Amino acid sequences at the receptor-binding site

of the H3 HA of swine viruses compared to

those of human and avian strains

Amino Acid sequence

226 227 228

Human Viruses Leu - Ser - Ser

Avian Viruses Gln - Ser - Gly

Sw/Taiwan/82 Gln - Ser - Gly

Sw/China/78 Leu - Ser - Ser

Aichi/68 and

VirusVirusVirusVirusAcHNAcHNAcHNAcHN OOOO OOOOOOOONHAcNHAcNHAcNHAcCHCHCHCH2222OHOHOHOHOHOHOHOHOOOOOHOHOHOHOHOHOHOHCHCHCHCH2222OHOHOHOHHOHOHOHO COCHCOCHCOCHCOCHOOOO CHOHCHOHCHOHCHOH OOOOOHOHOHOHHOHOHOHOαααααααα2, 2, 2, 2, 2, 2, 2, 2, 66666666αααα

NeuAc Gal GlcNAc

OOOO OOOOOOOONHAcNHAcNHAcNHAcCHCHCHCH2222OHOHOHOHOHOHOHOHOHOHOHOHOHOHOHOHCHCHCHCH2222OHOHOHOHHOHOHOHO COCHCOCHCOCHCOCHOOOO CHCHCHCH2222OHOHOHOH OOOOOHOHOHOHHOHOHOHOαααααααα2, 2, 2, 2, 2, 2, 2, 2, 33333333AcHNAcHNAcHNAcHN OOOO２２６－２２７－２２２６－２２７－２２２６－２２７－２２２６－２２７－２２８２８２８２８LeuLeuLeuLeu –––– Ser Ser Ser Ser ---- SerSerSerSer２２６－２２７－２２２６－２２７－２２２６－２２７－２２２６－２２７－２２８２８２８２８GlnGlnGlnGln –––– Ser Ser Ser Ser ---- GlyGlyGlyGly

Amino acid residues 226 and 228 of the HA1 subunit of

H3 HA determine the receptor specificity.

Virus HA Receptor

Ito et al, (1998) J Virol

Influenza viruses isolated from

domestic ducks in southern China◆◆◆◆ HAs of influenza viruses isolated from domestic ducks

in southern China antigenically and genetically closely

related with those of viruses isolated from pigs in

southern China, from migratory ducks, and A/Hong

Kong/68.◆◆◆◆ Each of the viruses was isolated on the Pacific flyway

of migratory ducks.◆◆◆◆ Migratory duck Migratory duck Migratory duck Migratory duck →→→→ domestic duck domestic duck domestic duck domestic duck →→→→ pigpigpigpig →→→→ humanshumanshumanshumans

H. KIDA, Hokkaido University Graduate School of Veterinary Medicine

4

Hong Kong

Virus shedding

Migratory

duckSiberia

Genetic Genetic Genetic Genetic reassortmentreassortmentreassortmentreassortment

A/Hong Kong/68A/Hong Kong/68A/Hong Kong/68A/Hong Kong/68(H3N2H3N2H3N2H3N2)

A/Asian/67A/Asian/67A/Asian/67A/Asian/67(H2H2H2H2N2N2N2N2)

A/duck/xxA/duck/xxA/duck/xxA/duck/xx(H3H3H3H3N?N?N?N?)))) Domestic

duckPig

SouthernChina

The role of pigs in the emergence of

pandemic strains◆ Pigs are susceptible to avian influenza viruses

of each of the HA subtypes.◆ Genetic reassortants were generated in the

cells lining upper respiratory tract of pig upon

concurrent infection with mammalian and

avian strains.

H. KIDA, Hokkaido University Graduate School of Veterinary Medicine

Preparing for pandemic influenza◆ H1 to H16 and N1 to N9 subtypes of influenza A viruses

perpetuate in lakes where ducks nest in nature. ◆ 1957 H2N2 and 1968 H3N2 viruses are reassortants

between AIV and the preceding human strains.◆◆◆◆ Pigs are susceptible to AIVs and mammalian IVs,

generating reassortants.→→→→ Avian viruses of any subtype can contribute genes for

reassortants : None of the 16 HA and 9 NA subtypes

can be ruled out as potential candidates for future

pandemics.→→→→Global surveillance of swine flu

as well as avian flu

Preparing for pandemic influenzaPreparing for pandemic influenza◆ H1 to H16 and N1 to N9 subtypes of influenza A viruses

perpetuate in lakes where ducks nest in nature. ◆ 1957 H2N2 and 1968 H3N2 viruses are reassortants

between AIV and the preceding human strains.◆◆◆◆ Pigs are susceptible to AIVs and mammalian IVs,

generating reassortants.→→→→ Avian viruses of any subtype can contribute genes for

reassortants : None of the 16 HA and 9 NA subtypes

can be ruled out as potential candidates for future

pandemics.→→→→ Global surveillance of swine fluGlobal surveillance of swine flu

as well as avian fluas well as avian flu

5

1992 1993 1994 1994sum m er sum m er sum m er autum n1/41/41/41/4 0/20/3 0/30/40/10/40/5 0/10 7/137/137/137/13 3/213/213/213/211/21/21/21/2 0/5 0/170/50/30/12/232/232/232/23 0/28 7/307/307/307/30 3/213/213/213/21TotalPotter M arshW estchester LagoonLake HangerC anvasback LakeC orville DeltaM allard Lake M allard Lake M allard Lake M allard Lake Heart Lake

Isolation of influenza viruses from w ater sam ples of lakes in Alaska in 1992-1994FairbanksBig M into LakeBig M into LakeBig M into LakeBig M into Lake

No. of sam ples with virus/total no. of sam ples testedLake Hood/Spenard LocationLake C heney Ptropavlovsk-Kamchatsky(58)Lake Kanicheva(95)Kobyaysky(820)H4N6H4N9H11N1H11N6H11N9 Magadan(295)× × × ×××× × ××Yakutsk(232)H3N8H13N6 Wakkanai (958)H1N1H3N8H5N3H5N4H6N2H6N7H8N1H8N3H9N2H11N9

Elavga(66)Khabarovsk(23) Malyshevo(90) H3N840 Ilands(1321) White Lake(1136)Buotama(51)Kharyyalah(146)Kenkeme(32)Irkutsk(290) 1996199619961996199719971997199719981998199819981999199919991999H1N1H4N5H4N6H7N7Taiwan

HPAIV(H5 or H7)(H5 or H7)(H5 or H7)(H5 or H7)

Acquisition of pathogenicity

of avian influenza virus in chicken

APAIV

LPAIV

6～～～～9 Months

6

Amino acid sequences

at the cleavage sites of influenza A virus HAs

IQSR GLF IESR GLF

KQTR GLF KASR GLF RKKR GLF

RETR GLF IETR GLF

KKRKKR GLF

KKTR GLF VEPR GLF RSSR GLF VQGR GLF IASR GLF

VQDR GLF ISNR GLF

KQAK GLF IRTR GLF

Senne et al, 1996a, Kovacova et al, 2002b

Dk/Alberta/35/76(H1N1)b

Mal/MT/Y61(H2N2)b

Dk/Menphis/928/74(H3N8)b

Dk/Czechoslovakia/56(H4N6)b

Ck/Scotland/59(H5N1)b

Ty/MN/3/92(H5N2)a

Shw/Australia/1/72(H6N5)b

FPV/Rostock/34(H7N1)b

Mal/Alberta/195/89(H7N3)a

Ty/Ontario/6118/68(H8N4)b

Ty/Wisconsin/66(H9N2)b

Ck/Germany/N/49(H10N7)b

Dk/England/56(H11N6)b

Dk/Alberta/60/76(H12N5)b

Gl/Maryland/704/77(H13N6)b

Mal/Gurjev/263/82(H14N5)b

Shw/Australia/2576/79(H15N9)b

H1H2H3H4H5H5H6H7H7H8H9H10H11H12H13H14H15

Subtype StrainsAmino acid sequences

Pathogenicity of influenza virus

for chicken

Cleavability of the HA protein into HA1 and HA2 is crucial and consecutive alignment of basic amino acids (R and K) at the cleavage site is related tothe pathogenicity for chicken.

Cleavage activation of the HA by ubiquitous protease →→→→ penetration by fusion into host cell→→→→ extensive replication →→→→ systemic infection

Ohita

Yamaguchi Kyoto

HPAI outbreaks in Japan, 2004

HPAIH5N115,000 broilers3/ 5/04Kyoto Pref.4

HPAIH5N1225,325 layers2/28/04Kyoto Pref.3

HPAIH5N113 pet chickens2/17/04Oita Pref.2

HPAIH5N134,000 layers1/12/04Yamaguchi Pref.1

1. Ck/Yamaguchi/7/04 (H5N1) is highly pathogenic for birds.

2. Ck/Yamaguchi/7/04 (H5N1) is not highly pathogenicfor mice with low mortality and does not infect pigs.

PathogenicityPathogenicityPathogenicityPathogenicity of A/ck/Yamaguchi/7/04 (H5N1) of A/ck/Yamaguchi/7/04 (H5N1) of A/ck/Yamaguchi/7/04 (H5N1) of A/ck/Yamaguchi/7/04 (H5N1)

for birds and mammals for birds and mammals for birds and mammals for birds and mammals

7

HPAI viruses in feral water birds, HPAI viruses in feral water birds, HPAI viruses in feral water birds, HPAI viruses in feral water birds, 2005200520052005ChinaChinaChinaChina：：：： Whooper swans, BarWhooper swans, BarWhooper swans, BarWhooper swans, Bar----headed geese, headed geese, headed geese, headed geese, etcetcetcetcHong KongHong KongHong KongHong Kong：：：： Peregrine falcon, Grey heron, Peregrine falcon, Grey heron, Peregrine falcon, Grey heron, Peregrine falcon, Grey heron, etcetcetcetcCroatiaCroatiaCroatiaCroatia：：：： Mute swans Mute swans Mute swans Mute swans MongoliaMongoliaMongoliaMongolia：：：： Whooper swans, BarWhooper swans, BarWhooper swans, BarWhooper swans, Bar----headed geeseheaded geeseheaded geeseheaded geeseRomania: Swan, HeronRomania: Swan, HeronRomania: Swan, HeronRomania: Swan, HeronKazakhustanKazakhustanKazakhustanKazakhustan: Geese, ducks: Geese, ducks: Geese, ducks: Geese, ducks

Dk/China/E319.2/03Ph/ST/44/04Dk/HN/5806/03Dk/HN/114/05Ck/ST/810/05Ck/Indonesia/BL/03Dk/Indonesia/MS/04Ck/HK/YU324/03Dk/YN/6255/03Ck/YN/493/05Egret/HK/757.3/03HK/212/03BHG/HK/12.1/03GH/HK/861.1/03Dk/HK/821/02Ck/HK/FY157/03Ck/Thailand/1/04Thailand/1(Kan-1)/04Dk/Viet Nam/11/04Viet Nam/1194/04

Gs/ST/1621/05Pf/HK/D0028L/04

Ck/ST/4231/03Ck/Yamaguchi/7/04Whooper Swan/Mongolia/3/05Whooper Swan/Mongolia/4/05Whooper Swan/Mongolia/6/05BH gull/QH/3/05Bar-headed Gs/QH/12/05Bar-headed Gs/QH/5/05GBH gull/QH/2/05Bar-headed Gs/Mongolia/1/05

Env/HK/437.10/99Gs/GD/1/96HK/156/97Ck/HK/YU562/01

PhylogeneticPhylogeneticPhylogeneticPhylogenetic tree of HA tree of HA tree of HA tree of HA genesgenesgenesgenesof HPAI viruses isolatedof HPAI viruses isolatedof HPAI viruses isolatedof HPAI viruses isolatedfrom water birdsfrom water birdsfrom water birdsfrom water birds Chicken ・Fatality 100% (8weeks) ・IVPI index = 2.95Duck ・Fatality30% （4weeks） ・Neurological signsDuck ・Fatality 100% （3days） ・Neurological signsMouse ・Fatality50% （4weeks）Pig ・Infected without showing（4weeks） clinical signs

8

Nucleotide sequence identity （％）（％）（％）（％） between the HA genes of H5 influenza

viruses isolated from birds and humans in different areas in the world98.998.998.998.9----Ck/Nigeria/641/06 (H5N1)Ck/Nigeria/641/06 (H5N1)Ck/Nigeria/641/06 (H5N1)Ck/Nigeria/641/06 (H5N1) 94.094.094.094.096.296.296.296.2Ck/Guangdong/174/04 (H5N1)Ck/Guangdong/174/04 (H5N1)Ck/Guangdong/174/04 (H5N1)Ck/Guangdong/174/04 (H5N1) 93.693.693.693.696.396.396.396.3Ck/Yamaguchi/7/04 (H5N1)Ck/Yamaguchi/7/04 (H5N1)Ck/Yamaguchi/7/04 (H5N1)Ck/Yamaguchi/7/04 (H5N1) 93.793.793.793.794.994.994.994.9Thailand/2(SPThailand/2(SPThailand/2(SPThailand/2(SP----33)/04 (H5N1)33)/04 (H5N1)33)/04 (H5N1)33)/04 (H5N1) 93.793.793.793.794.994.994.994.9Viet Nam/1194/04 (H5N1)Viet Nam/1194/04 (H5N1)Viet Nam/1194/04 (H5N1)Viet Nam/1194/04 (H5N1) 93.093.093.093.094.594.594.594.5Dk/Yokohama/aqDk/Yokohama/aqDk/Yokohama/aqDk/Yokohama/aq----10/03 (H5N1)10/03 (H5N1)10/03 (H5N1)10/03 (H5N1) 92.492.492.492.493.893.893.893.8HK/156/97 (H5N1)HK/156/97 (H5N1)HK/156/97 (H5N1)HK/156/97 (H5N1) 92.192.192.192.193.493.493.493.4HK/483/97 (H5N1)HK/483/97 (H5N1)HK/483/97 (H5N1)HK/483/97 (H5N1) 87.387.387.387.389.989.989.989.9Dk/Hok/69/00 (H5N3)Dk/Hok/69/00 (H5N3)Dk/Hok/69/00 (H5N3)Dk/Hok/69/00 (H5N3) 86.386.386.386.389.289.289.289.2Dk/Hok/101/04 (H5N3)Dk/Hok/101/04 (H5N3)Dk/Hok/101/04 (H5N3)Dk/Hok/101/04 (H5N3) 88.788.788.788.791.191.191.191.1Swan/Hok/67/96 (H5N3)Swan/Hok/67/96 (H5N3)Swan/Hok/67/96 (H5N3)Swan/Hok/67/96 (H5N3) 86.786.786.786.788.788.788.788.7Dk/HK/698/79 (H5N3)Dk/HK/698/79 (H5N3)Dk/HK/698/79 (H5N3)Dk/HK/698/79 (H5N3) 86.786.786.786.788.288.288.288.2Dk/Miyagi/54/76 (H5N3)Dk/Miyagi/54/76 (H5N3)Dk/Miyagi/54/76 (H5N3)Dk/Miyagi/54/76 (H5N3) 83.783.783.783.786.086.086.086.0Tern/S.A/61 (H5N3)Tern/S.A/61 (H5N3)Tern/S.A/61 (H5N3)Tern/S.A/61 (H5N3) 77.477.477.477.477.877.877.877.8Ck/Ibaraki/1/05 (H5N2)Ck/Ibaraki/1/05 (H5N2)Ck/Ibaraki/1/05 (H5N2)Ck/Ibaraki/1/05 (H5N2) 77.177.177.177.179.479.479.479.4Dk/Hok/84/02 (H5N3)Dk/Hok/84/02 (H5N3)Dk/Hok/84/02 (H5N3)Dk/Hok/84/02 (H5N3) 75.475.475.475.476.376.376.376.3Ck/Gtml/45511Ck/Gtml/45511Ck/Gtml/45511Ck/Gtml/45511----3/00 (H5N2)3/00 (H5N2)3/00 (H5N2)3/00 (H5N2) 76.176.176.176.178.478.478.478.4Ck/Mexico/232/94 (H5N2)Ck/Mexico/232/94 (H5N2)Ck/Mexico/232/94 (H5N2)Ck/Mexico/232/94 (H5N2) 75.875.875.875.880.080.080.080.0Dk/Penn/10218/84 (H5N2)Dk/Penn/10218/84 (H5N2)Dk/Penn/10218/84 (H5N2)Dk/Penn/10218/84 (H5N2) 75.375.375.375.379.179.179.179.1Ty/Ontario/7732/66 (H5N9)Ty/Ontario/7732/66 (H5N9)Ty/Ontario/7732/66 (H5N9)Ty/Ontario/7732/66 (H5N9)HVAIV Isolated in our lab

99.499.499.499.499.699.699.699.6BarBarBarBar----headed Gs/QH/5/05 (H5N1)headed Gs/QH/5/05 (H5N1)headed Gs/QH/5/05 (H5N1)headed Gs/QH/5/05 (H5N1) 98.998.998.998.999.399.399.399.3Whooper swan/Mongolia/3/05 (H5N1)Whooper swan/Mongolia/3/05 (H5N1)Whooper swan/Mongolia/3/05 (H5N1)Whooper swan/Mongolia/3/05 (H5N1) ----98.998.998.998.9Mute swan/Croatia/1/05 (H5N1)Mute swan/Croatia/1/05 (H5N1)Mute swan/Croatia/1/05 (H5N1)Mute swan/Croatia/1/05 (H5N1) Mute swan/Croatia/1/05 (H5N1)Mute swan/Croatia/1/05 (H5N1)Mute swan/Croatia/1/05 (H5N1)Mute swan/Croatia/1/05 (H5N1)Ck/Nigeria/641/06 (H5N1)Ck/Nigeria/641/06 (H5N1)Ck/Nigeria/641/06 (H5N1)Ck/Nigeria/641/06 (H5N1)6666----9Months9Months9Months9Months

goosegoosegoosegoose turkeyturkeyturkeyturkey

quailquailquailquail

ApathogenicApathogenicApathogenicApathogenic

AIVAIVAIVAIV

Low PathogenicLow PathogenicLow PathogenicLow Pathogenic

AIVAIVAIVAIV HPAIVHPAIVHPAIVHPAIVHPAIVHPAIVHPAIVHPAIVHPAIVHPAIVHPAIVHPAIVchickenschickenschickenschickens

X Pandemic Pandemic Pandemic Pandemic

virusvirusvirusvirus

Pandemic Pandemic Pandemic Pandemic Pandemic Pandemic Pandemic Pandemic

virusvirusvirusvirusvirusvirusvirusvirus

Human virusHuman virusHuman virusHuman virus

Genetic Genetic Genetic Genetic

ReassortantReassortantReassortantReassortant

virusvirusvirusvirus

pigpigpigpig

duckduckduckduck

HPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strainsHPAI virus and human pandemic virus strains

Present status of the library of vaccine strain candidatesPresent status of the library of vaccine strain candidatesPresent status of the library of vaccine strain candidatesPresent status of the library of vaccine strain candidatesPresent status of the library of vaccine strain candidatesPresent status of the library of vaccine strain candidatesPresent status of the library of vaccine strain candidatesPresent status of the library of vaccine strain candidates

Influenza viruses of 49

combinations of the HA

and NA subtypes have been isolated from fecal

samples of ducks inAlaska, Siberia, Mongolia,

Taiwan, China, and Japan (blackblackblackblack).

So far, 76 other combinat-

ions have been generated by genetic reassortment

procedure in the laboratory (redredredred)..

Thus, avian influenza viruses of 123 combinations of HA and NA subtypes have been stocked as vaccine strain candidates. Their pathogenicity, antigenicity, genetic

information and yield in chicken embryo have been analyzed, and being databased.

1. Ecology and evolution of influenza viruses

-- Mechanism of antigenic variation of IV --

2. Mechanism of the emergence of pandemic

strains in humans and HPAIV in chickens

3. Do the H5N1 HPAIV strains perpetuate in

the lakes where migratory birds nest ?

4. Is the H5N1 HPAIV alone as a candidate of

pandemic strain?

5. What is the best measure for the control of

bird flu?

Points of Discussion