lecular Epidemiology of Avian Influenz in Thailand 2004-2008 PRESENTED BY Sunchai Payungporn, Ph.D. Center of Excellence in Clinical Virology Chulalongkorn University, Bangkok, Thailand
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
WHO
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
Quail
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
Avian Influenza H5N1 virus in FelineAvian Influenza H5N1 virus in Feline
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
Avian Influenza H5N1 virus in FelineAvian Influenza H5N1 virus in Feline
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
CatCat
Virus research 2006
TigerTiger
TigerTiger
humanhuman
humanhuman
2.5
2.2
2.4
2.3.1
2.3.2
2.3.3
2.3.4
2.1.3
2.1.2
2.1.1
1
3
5
6
4
7
9
8
99
96
100
99
91
99
93
85
93
100
100
100
82
99
100
100
100
100
99
0
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/CDC938/06Indonesia/CDC887/06
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
dk/Guangxi/1311/04dk/Guangxi/2396/04
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
0.005
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/CDC938/06Indonesia/CDC887/06
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/7/03
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/CDC938/06Indonesia/CDC887/06
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
dk/Guangxi/1311/04dk/Guangxi/2396/04
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
0.005
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
0.005
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)
HAHA
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