Molecular Epidemiology of Avian Influenza in Thailand 2004-2008 PRESENTED BY Sunchai Payungporn, Ph.D. Center of Excellence in Clinical Virology Chulalongkorn.

Molecular Epidemiology of Avian Influenza in Thailand 2004-2008

PRESENTED BY

Sunchai Payungporn, Ph.D.

Center of Excellence in Clinical VirologyChulalongkorn University, Bangkok, Thailand

Family Family OrthomyxoviridaeOrthomyxoviridae

Genus Genus Influenzavirus AInfluenzavirus A

Type speciesType species Influenza A virusInfluenza A virus

SubtypeSubtype HA 16 subtypesHA 16 subtypes

NA 9 subtypesNA 9 subtypes

Negative Negative ssRNA viruses ssRNA viruses

8 segmented RNA genome8 segmented RNA genome

envelopedenveloped virusvirus

PB2, PB1, PA, HA, NP, NA, M1, M2, NS1 and NS2

H5N1 avian influenza virus spreading worldwideH5N1 avian influenza virus spreading worldwide

Majors problems:• Public health• Economics• Society

Epidemics in:• Asian• Europe• Africa

H5N1 avian influenza virus spreading worldwideH5N1 avian influenza virus spreading worldwide

In human, until now 348 cases were reported with 216 deaths (62%)

The Nation, Jan 22, 2004The Nation, Jan 22, 2004

In Thailand, the outbreak of H5N1 virus was formally reported since January 2004

• Before 2004, Thailand has been the world’s major poultry exporters (~ 1 billion chickens / year)

• After the spread of H5N1 avian influenza virus in Thailand, more than 100 million birds were killed

First strain in chicken of Thailand:

AA//ChickenChicken//NakornNakorn--PathomPathom//ThailandThailand//CUCU--K2/04K2/04

The genome sequence analysis of H5N1 avian influenza A virus The genome sequence analysis of H5N1 avian influenza A virus isolated from the outbreak among poultry populations in Thailandisolated from the outbreak among poultry populations in Thailand

Virology 2004, 328:169-76Virology 2004, 328:169-76

Molecular characterizationMolecular characterization

HA geneHA gene - a common characteristic of a highly pathogenic AI (HPAI)

NA geneNA gene - a 20-codon deletion

NS geneNS gene - a 5-codon deletion

M2 and PB2 genesM2 and PB2 genes - polymorphisms

Kalij Pheasant

Crow White peafowl

Openbill Ostrich

The example of avian species that infected by H5N1 virusThe example of avian species that infected by H5N1 virus

Characterization of the Hemagglutinin and Characterization of the Hemagglutinin and Neuraminidase genes of recent influenza virus isolates Neuraminidase genes of recent influenza virus isolates

from different avian species in Thailandfrom different avian species in Thailand

Acta VirolActa Virol 2005, 492005, 49::277-80277-80

Avian Influenza H5N1 virus in FelineAvian Influenza H5N1 virus in Feline

Zoo in Suphanburi provinceZoo in Suphanburi province

• 2 Tigers 2 Tigers (Panthera tigris)(Panthera tigris)

• 2 Leopards2 Leopards ((PantheraPanthera parduspardus))

EID 2004, 10:2189-91EID 2004, 10:2189-91

Clinical signs:• high fever• respiratory distress

• Animals had been fed on fresh chicken carcasses from a local slaughterhouse

• Many chickens in Suphanburi were dying from H5N1 influenza infection

• Sriracha tiger Zoo in Chonburi provinceSriracha tiger Zoo in Chonburi province

(the biggest tiger zoo in Thailand)(the biggest tiger zoo in Thailand)

• Probable tiger-to-tiger transmission ofProbable tiger-to-tiger transmission of

H5N1 avian influenza virus H5N1 avian influenza virus

• 147 Tigers 147 Tigers (Panthera tigris) (Panthera tigris) dieddied

EID 2005, 11:699-701

Symptoms of Sick TigerSymptoms of Sick Tiger

Hair standing on the head

High fever

Respiratory distress

Severe leukopenia

Thrombocytopenia

Increased level of Liver enzyme

Nasal discharge

Neurological sign

Tigers and Bird fluTigers and Bird flu

Avian Influenza H5N1 in Naturally Infected Domestic CatAvian Influenza H5N1 in Naturally Infected Domestic Cat

2-year-old male cat (Felis catus) had eaten a pigeon (Columba levia) carcass 5 days before illness onset.

EID 2006, 12:681-3Strain of virus in cat:

AA//catcat//ThailandThailand//KU-02/04KU-02/04

Fatal Avian Influenza A H5N1 in a DogFatal Avian Influenza A H5N1 in a Dog

5 days later 5 days later

- high fever - high fever

- panting- panting

- lack of energy - lack of energy

- bloody nasal discharge- bloody nasal discharge

Strain of virus in dog:

AA//dogdog//ThailandThailand--SuphanburiSuphanburi//KU-08/04KU-08/04

One-year old dog (Native) from Suphanburi One-year old dog (Native) from Suphanburi

province had eaten HPAI H5N1 infected duck province had eaten HPAI H5N1 infected duck

carcasses in the outbreak areacarcasses in the outbreak area

EID 2006, 12:1744-47

Phylogenetic analysis of the HA and NA gene of the Phylogenetic analysis of the HA and NA gene of the H5N1 influenza virus isolated from dog and catH5N1 influenza virus isolated from dog and cat

DogDog DogDog

CatCat

Virus research 2006

TigerTiger

humanhuman

ck/Nongkhai/NIAH400802/07ck/TH/NP172/06

Guangzhou/1/06JapaneseWhiteEye/HK/1038/06

Anhui/1/05dk/Laos/3295/06ck/Malaysia/935/06

commonmagpie/HK/645/06Zhejiang/16/06

JapaneseWhiteEye/HongKong/73720/07WhiteBackedMunia/HongKong/82820/07

Guangxi/1/05dk/Guiyang/3009/05dk/Guiyang/3242/05

gs/Guiyang/3422/05ck/Guiyang3/055/05

gs/Yunnan/4494/05gs/Guangxi/3017/05

gs/Guangxi/345/05gs/Guangxi/3316/05

dk/Hunan/127/05dk/Hunan/149/05dk/Hunan/152/05

dk/Hunan/139/05Egypt/0636NAMRU320/07

Egret/Egypt/1162NAMRU3/06dk/Egypt/22533/06

Egypt/14724NAMRU320/06turkey/Turkey1//05

WhooperSwan/Mongolia/244/05Nigeria/6e20/07

ck/Nigeria/641/06Turkey/15/06

Iraq/207NAMRU3/06Azerbaijan/001161/06

ck/Krasnodar/01/06swan/Iran/754/06

ck/Liaoning/23/05Barhdgs/Qinghai12/05

Barhdgs/Qinghai1A/05ck/Kyoto/3/04

crow/Kyoto/53/04ck/Yamaguchi/7/04

ck/Korea/ES/03dk/Guangxi/13/04

ck/YN/115/04ck/YN/374/04

Indonesia/CDC1046/07Indonesia/CDC103220/07

Indonesia/CDC1047/07Indonesia/283H/06Indonesia/326N/06

Indonesia/CDC742/06Indonesia/370E/06

Indonesia/5/05Indonesia/CDC940/06

Indonesia/546bH/06Indonesia/596/06Indonesia/599/06

Indonesia/625/06dk/Indonesia/MS/04ck/Indonesia/4/04

ck/Indonesia/11/03ck/Indonesia/7/03VN/JP14/05ck/Cambodia/013LC1b/05

VN/1194/04VN/1203/04

VN/HN3/0408/05TH/16/04

TH/676/05VN/JPHN30321/05

HK/213/03ck/Henan/16/04

ck/Henan/01/04ck/Henan/13/04ck/Henan/12/04

dk/Guangxi/50/01ck/HK/YU777/02

ck/HK/YU22/02migdk/Jiangxi1653/05

dk/Guangxi/2775/05ck/Hunan/41/04

blbird/Hunan1/04treesparrow/Henan/4/04

dk/Hubei/wg/02sw/Anhui/ca/04

dk/Guangxi/1378/04dk/Guangxi/1681/04

ck/Hunan/2292/06ck/Shanxi/2/06

ck/Myanmar/06010011B/06dk/Guiyang/504/06

ck/Guiyang237/06gs/Guiyang/337/06

gs/Guiyang/1325/06ck/Guiyang441/06ck/Guiyang1218/06ck/Guiyang846/06

ck/HK/8911/01ck/HK/SF219/01ck/HK/8791/01

gs/VNGZ3/05HK/156/97

gs/Guangdong/1/96

ck/YN/115/04ck/YN/374/04

ck/Indonesia/11/03ck/Indonesia/7/03

ck/YN/115/04ck/YN/374/04

ck/Indonesia/11/03ck/Indonesia/7/03VN/JP14/05ck/Cambodia/013LC1b/05

VN/1194/04VN/1203/04

VN/HN3/0408/05TH/16/04

TH/676/05VN/JPHN30321/05

HK/213/03ck/Henan/16/04

ck/Henan/01/04ck/Henan/13/04ck/Henan/12/04

dk/Guangxi/50/01ck/HK/YU777/02

ck/HK/YU22/02migdk/Jiangxi1653/05

dk/Guangxi/2775/05ck/Hunan/41/04

blbird/Hunan1/04treesparrow/Henan/4/04

dk/Hubei/wg/02sw/Anhui/ca/04

ck/Hunan/2292/06ck/Shanxi/2/06

ck/Myanmar/06010011B/06dk/Guiyang/504/06

ck/Guiyang237/06gs/Guiyang/337/06

gs/Guiyang/1325/06ck/Guiyang441/06ck/Guiyang1218/06ck/Guiyang846/06

ck/HK/8911/01ck/HK/SF219/01ck/HK/8791/01

gs/VNGZ3/05HK/156/97

gs/Guangdong/1/96

Unified Nomenclature System Unified Nomenclature System for the HPAI H5N1 for the HPAI H5N1 Avian Influenza VirusesAvian Influenza Viruses

WHO/OIE/FAO H5N1 Evolution Working Group

Clade descriptions:0 = early progenitors; 1996-2002 from Hong Kong (HK) and China

3 = 2000-2001 from HK, China, Vietnam

4 = 2002/2003 lineage from HK and China; 2005/2006 from Guiyang Prov.

5 = 2000-2003 from China and Vietnam; 2004 lineage from Guangxi Province

6 = 2002/2004 from China

7 = 2002/2004 from China; 2005/2006 from Yunnan, Hebei, Shanxi Provinces

8 = 2001-2004 from HK and China

9 = 2003-2005 from China

1 = 2002/2003 from HK; 2003-2006 from Vietnam, Cambodia, Thai, Laos, Malaysia

2.1 = 2003-2007 from Indonesia

2.2 = 2005 from Qinghai Lake outbreak and Mongolia;

2005-2007 from Eastern and Western Europe, the Middle East, and Africa

2.3 = 2003-2006 from China, HK, Vietnam, Thailand, Laos, and Malaysia

2.4 = 2002-2005 from China (predominately Yunnan and Guangxi Provinces)

2.5 = 2003/2004 from Korea, Japan, China; 2006 lineage from Shantou Prov.

http://www.who.int/csr/disease/ avian_infl uenza/guidelines/nomenclature/en

A/Anhui/1/05 A/duck/Laos/3295/06

A/chicken/Thailand/NP-172/06 A/Guangxi/1/05 A/duck/Hunan/5152/05 A/duck/Hunan/5106/05

A/duck/Guiyang/3242/05 A/goose/Guiyang/3422/05 A/goose/Guangxi/3017/05

A/goose/Yunnan/4494/05 A/duck/Guangxi/351/04

A/Bar-headed Goose/Qinghai/12/05 A/turkey/Turkey/1/05

A/chicken/Kyoto/3/04 A/chicken/Korea/ES/03

A/Indonesia/283H/06 A/Indonesia/5/05

A/Indonesia/CDC1046/07 A/Indonesia/546bH/06

A/duck/Indonesia/MS/04 A/chicken/Indonesia/7/03

A/Hong Kong/213/03 A/chicken/Thailand/Kanchanaburi/CK-160/05 A/chicken/Thailand/Nontaburi/CK-162/05

A/quail/Thailand/Nakhon Pathom/QA-161/05 A/Thailand/NK165/05

A/chicken/Thailand/PC-170/06 A/pigeon/Thailand/KU-03/04

A/tiger/Suphanburi/Thailand/Ti-1/04 A/leopard/Suphanburi/Thailand/Leo-1/04 A/white peafowl/Bangkok/Thailand/CU-16/04 A/crow/Bangkok/Thailand/CU-15/04

A/Viet Nam/1203/04 A/chicken/Ayutthaya/Thailand/CU-23/04

A/dog/Thailand-Suphanburi/KU-08/04 A/tiger/Thailand/CU-T7/2004 A/duck/Chonburi/Thailand/CU-5/04

A/chicken/Nakorn-Patom/Thailand/CU-K2/04 A/chicken/Thailand/PC-168/06 A/chicken/Thailand/PC-340/08

A/chicken/Thailand/NS-339/08 A/Chicken/Henan/16/04

A/migratory duck/Jiangxi/1653/05 A/tree sparrow/Henan/4/04

A/blackbird/Hunan/1/04 A/duck/Guangxi/1378/04

A/chicken/Guiyang/846/06 A/Chicken/Hong Kong/8911/01

A/chicken/Shanxi/2/06 A/Goose/Guangdong/1/96

A/HongKong/156/97

Clade 2.3.4

Clade 2.3.1

Clade 2.3.3

Clade 2.3.2

Clade 2.4

Clade 2.2

Clade 2.5

Clade 2.1.3

Clade 2.1.2Clade 2.1.1

Clade 1

Clade 8Clade 9Clade 6Clade 5Clade 4Clade 3Clade 7Clade 0

Thailand 2005

Thailand 2004

Thailand 2008

Genetics CharaterizationGenetics Charaterization

1. Hemagglutinin (HA) gene

- Cleavage site - HPAI

2. Neuraminidase (NA) gene

- Stalk region - 20 amino acids deletion

- Oseltamivir resistance - H274Y

4. Polymerase Basic protein 2 (PB2) gene

- amino acid position 627 - E627K

5. Nonstructural protein (NS) gene

- 5 amino acids deletion

- amino acid position 92 – D92E

- amino acids at C-terminal – ESEV/EPEV

- Amantadine resistance - L26I, V27A, A30S, S31N

3. M2 ion channel gene

The Cleavage site between HPAI and LPAIThe Cleavage site between HPAI and LPAI

LPAILPAI HPAIHPAI(Low Pathogenic Avian Influenza) (High Pathogenic Avian Influenza)

Picture from Nature reviews microbiology 3:591-600

LPAI do not contain a series of basic amino acid at the cleavage site

HPAI virus have multiple basic amino acids at the cleavage site

The difference of cleavage site of Hemagglutinin geneThe difference of cleavage site of Hemagglutinin gene

LPAILPAI

HPAIHPAI

USA 1975 - 83

China 1996 - 97 Thailand

2004 and 2006

Thailand 2005 - 06

Northwestern of China 2005 - 06

Southeastern of China 2005-06 Thailand 2006

There are several variation patterns of inserted basic amino acid

20 amino acids deletion within the stalk region of 20 amino acids deletion within the stalk region of Neuraminidase proteinNeuraminidase protein

All of the viruses isolated from Thailand have 20 aa deletion within the stalk region of NA (may increase retention of virions at the plasma membrane)

Oseltamivir sensitive / resistance in NA Oseltamivir sensitive / resistance in NA at amino acid position 274at amino acid position 274

• Mutation of H274Y Oseltamivir resistance• Fortunately, all of the isolates in Thailand were sensitive to Oseltamivir

Amantadine sensitive / resistance in M2Amantadine sensitive / resistance in M226 3126 31

Dual amantadine resistance mutations at positions 26 and 31 were found in

most of H5N1 influenza isolated in Thailand

Different amino acid at position 627 of PB2Different amino acid at position 627 of PB2

627627

• Viruses isolated from avian species contain 627E (Glu)• Viruses isolated from mammalian species contain 627K (Lys)

5 amino acids deletion and C-terminal of NS5 amino acids deletion and C-terminal of NS9292

• 5 amino acid deletion (80- 84): may enhance the virulence• D92E resistance to IFN and TNF-alpha treatment• Carboxyl-terminus (ESEV): may correlate with virulence in mammals

Conclusions

• Bioinformatics are crucial for molecular epidemiology of H5N1 avian influenza– Phylogenetic analysis– Genetic characterizations

• LPAI or HPAI• Anti-viral drugs resistance mutant • Virulence of the virus

AcknowledgementAcknowledgement

National Center for Genetic Engineering and Biotechnology, Thailand

Thailand Research Fund (Senior Research Scholar)

Royal Golden Jubilee Ph.D Program

National Research Council Of Thailand

Center of Excellence in Viral Hepatitis Research, Chulalongkorn University

Chulalongkorn University emergency fund

Faculty of Veterinary Science, Chulalongkorn University , Bangkok, Thailand

Chulalongkorn University and Hospital

The department of Livestock Development, National Institute of Animal Health, Bangkok, Thailand

Faculty of Veterinary Medicine, Kasetsart University, Kumphaengsaen Campus, Nakorn Pathom, Thailand

The department of Pediatrics, Faculty of Medicine, Srinakharinwirot University Nakhon Nayok, Thailand

The Department of Medical Sciences (DMSc) Ministry of Public Health, Nonthaburi, Thailand

The Department of Microbiology, Siriraj Hospital, Mahidol University, Bangkok, Thailand

The ASEAN-China Cooperation fund

Molecular Epidemiology of Avian Influenza in Thailand 2004-2008 PRESENTED BY Sunchai Payungporn, Ph.D. Center of Excellence in Clinical Virology Chulalongkorn.

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