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Molecular Epidemiology of Avian Influenza
in Thailand 2004-2008
PRESENTED BY
Sunchai Payungporn, Ph.D.
Center of Excellence in Clinical Virology
Chulalongkorn University, Bangkok, Thailand
Family
Orthomyxoviridae
Genus
Influenzavirus A
Type species
Influenza A virus
Subtype
HA 16 subtypes
NA 9 subtypes
Negative ssRNA viruses
8 segmented RNA genome
enveloped virus
WHO
PB2, PB1, PA, HA, NP, NA, M1, M2, NS1 and NS2
H5N1 avian influenza virus spreading worldwide
Majors problems:
• Public health
• Economics
• Society
Epidemics in:
• Asian
• Europe
• Africa
H5N1 avian influenza virus spreading worldwide
In human, until now 348 cases were reported with 216 deaths (62%)
The 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
The genome sequence analysis of H5N1 avian influenza A virus
isolated from the outbreak among poultry populations in Thailand
First strain in chicken of Thailand:
A/Chicken/Nakorn-Pathom/Thailand/CU-K2/04
Molecular characterization
HA gene - a common characteristic
of a highly pathogenic AI (HPAI)
NA gene - a 20-codon deletion
NS gene - a 5-codon deletion
Virology 2004, 328:169-76
M2 and PB2 genes - polymorphisms
The example of avian species that infected by H5N1 virus
Kalij Pheasant
Crow
White peafowl
Quail
Openbill
Ostrich
Characterization of the Hemagglutinin and
Neuraminidase genes of recent influenza virus isolates
from different avian species in Thailand
Acta Virol 2005, 49:277-80
Avian Influenza H5N1 virus in Feline
Avian Influenza H5N1 virus in Feline
Zoo in Suphanburi province
• 2 Tigers (Panthera tigris)
• 2 Leopards (Panthera pardus)
Clinical signs:
• high fever
• respiratory distress
EID 2004, 10:2189-91
• Animals had been fed on fresh chicken carcasses from a local slaughterhouse
• Many chickens in Suphanburi were dying from H5N1 influenza infection
Avian Influenza H5N1 virus in Feline
• Sriracha tiger Zoo in Chonburi province
(the biggest tiger zoo in Thailand)
• Probable tiger-to-tiger transmission of
H5N1 avian influenza virus
• 147 Tigers (Panthera tigris) died
EID 2005, 11:699-701
Symptoms 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 flu
Avian 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.
Strain of virus in cat:
A/cat/Thailand/KU-02/04
EID 2006, 12:681-3
Fatal Avian Influenza A H5N1 in a Dog
One-year old dog (Native) from Suphanburi
province had eaten HPAI H5N1 infected duck
carcasses in the outbreak area
5 days later
EID 2006, 12:1744-47
Strain of virus in dog:
A/dog/Thailand-Suphanburi/KU-08/04
- high fever
- panting
- lack of energy
- bloody nasal discharge
Phylogenetic analysis of the HA and NA gene of the
H5N1 influenza virus isolated from dog and cat
Dog
Dog
Cat
Tiger
Tiger
human
Cat
human
Virus research 2006
ck/Nongkhai/NIAH400802/07
ck/TH/NP172/06
Guangzhou/1/06
JapaneseWhiteEye/HK/1038/06
Anhui/1/05
dk/Laos/3295/06
ck/Malaysia/935/06
commonmagpie/HK/645/06
Zhejiang/16/06
JapaneseWhiteEye/HongKong/73720/07
WhiteBackedMunia/HongKong/82820/07
Guangxi/1/05
dk/Guiyang/3009/05
100
dk/Guiyang/3242/05
gs/Guiyang/3422/05
ck/Guiyang3/055/05
gs/Yunnan/4494/05
gs/Guangxi/3017/05
100
gs/Guangxi/345/05
gs/Guangxi/3316/05
dk/Hunan/127/05
100 dk/Hunan/149/05
dk/Hunan/152/05
dk/Hunan/139/05
Egypt/0636NAMRU320/07
Egret/Egypt/1162NAMRU3/06
dk/Egypt/22533/06
Egypt/14724NAMRU320/06
turkey/Turkey1//05
WhooperSwan/Mongolia/244/05
Nigeria/6e20/07
ck/Nigeria/641/06
Turkey/15/06
Iraq/207NAMRU3/06
Azerbaijan/001161/06
ck/Krasnodar/01/06
100
swan/Iran/754/06
ck/Liaoning/23/05
Barhdgs/Qinghai12/05
Barhdgs/Qinghai1A/05
ck/Kyoto/3/04
crow/Kyoto/53/04
99
ck/Yamaguchi/7/04
ck/Korea/ES/03
dk/Guangxi/13/04
ck/YN/115/04
ck/YN/374/04
Indonesia/CDC1046/07
Indonesia/CDC103220/07
Indonesia/CDC938/06
Indonesia/CDC887/06
Indonesia/CDC1047/07
Indonesia/283H/06
100
Indonesia/326N/06
Indonesia/CDC742/06
Indonesia/370E/06
Indonesia/5/05
Indonesia/CDC940/06
Indonesia/546bH/06
100 Indonesia/596/06
Indonesia/599/06
Indonesia/625/06
dk/Indonesia/MS/04
ck/Indonesia/4/04
ck/Indonesia/11/03
ck/Indonesia/7/03
VN/JP14/05
ck/Cambodia/013LC1b/05
VN/1194/04
VN/1203/04
VN/HN3/0408/05
TH/16/04
TH/676/05
VN/JPHN30321/05
HK/213/03
99
Unified Nomenclature System
for the HPAI H5N1
Avian Influenza Viruses
2.3.4
2.3.3
2.3.2
2.3.1
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.
2.5
5 = 2000-2003 from China and Vietnam; 2004 lineage from Guangxi Province
2.4
82
6 = 2002/2004 from China
7 = 2002/2004 from China; 2005/2006 from Yunnan, Hebei, Shanxi Provinces
2.1.3
8 = 2001-2004 from HK and China
9 = 2003-2005 from China
2.1.2
1 = 2002/2003 from HK; 2003-2006 from Vietnam, Cambodia, Thai, Laos, Malaysia
100
2.1.1
2.1 = 2003-2007 from Indonesia
1
2.2 = 2005 from Qinghai Lake outbreak and Mongolia;
93
ck/Henan/16/04
2005-2007 from Eastern and Western Europe, the Middle East, and Africa
85
2.3 = 2003-2006 from China, HK, Vietnam, Thailand, Laos, and Malaysia
8
ck/HK/YU22/02
migdk/Jiangxi1653/05
dk/Guangxi/2775/05
ck/Hunan/41/04
blbird/Hunan1/04
treesparrow/Henan/4/04
dk/Hubei/wg/02
sw/Anhui/ca/04
dk/Guangxi/1378/04
91
dk/Guangxi/1681/04
dk/Guangxi/1311/04
dk/Guangxi/2396/04
93
2.4 = 2002-2005 from China (predominately Yunnan and Guangxi Provinces)
ck/Henan/01/04
ck/Henan/13/04
ck/Henan/12/04
dk/Guangxi/50/01
ck/HK/YU777/02
9
99
6
2.5 = 2003/2004 from Korea, Japan, China; 2006 lineage from Shantou Prov.
WHO/OIE/FAO H5N1 Evolution Working Group
http://www.who.int/csr/disease/
avian_infl uenza/guidelines/nomenclature/en
5
ck/Hunan/2292/06
ck/Shanxi/2/06
ck/Myanmar/06010011B/06
dk/Guiyang/504/06
ck/Guiyang237/06
gs/Guiyang/337/06
gs/Guiyang/1325/06
ck/Guiyang441/06
ck/Guiyang1218/06
ck/Guiyang846/06
99
100
4
96
ck/HK/SF219/01
ck/HK/8791/01
99
0.005
ck/HK/8911/01
gs/VNGZ3/05
HK/156/97
gs/Guangdong/1/96
3
0
7
2.2
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 2.1.2
Clade 2.1.1
Thailand 2005
*
Clade 1
Thailand 2004
*
0.005
Thailand 2008
Clade 8
Clade 9
Clade 6
Clade 5
Clade 4
Clade 3
Clade 7
Clade 0
Genetics Charaterization
1. Hemagglutinin (HA) gene
- Cleavage site - HPAI
2. Neuraminidase (NA) gene
- Stalk region - 20 amino acids deletion
- Oseltamivir resistance - H274Y
3. M2 ion channel gene
- Amantadine resistance - L26I, V27A, A30S, S31N
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
The Cleavage site between HPAI and LPAI
HA
LPAI
(Low Pathogenic Avian Influenza)
LPAI do not contain a series of
basic amino acid at the cleavage site
HPAI
(High Pathogenic Avian Influenza)
HPAI virus have multiple
basic amino acids at the cleavage site
Picture from Nature reviews microbiology 3:591-600
The difference of cleavage site of Hemagglutinin gene
LPAI
USA
1975 - 83
China
1996 - 97
Thailand
2004 and 2006
HPAI
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
Neuraminidase 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
at amino acid position 274
• Mutation of H274Y  Oseltamivir resistance
• Fortunately, all of the isolates in Thailand were sensitive to Oseltamivir
Amantadine sensitive / resistance in M2
26
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 PB2
627
• Viruses isolated from avian species contain 627E (Glu)
• Viruses isolated from mammalian species contain 627K (Lys)
5 amino acids deletion and C-terminal of NS
92
• 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
Acknowledgement
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