1. 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

2. Family Orthomyxoviridae Genus Influenzavirus A Type species Influenza A virus SubtypeHA 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

3. H5N1 avian influenza virus spreading worldwide Majors problems: Public health Economics Society Epidemics in: Asian Europe Africa

4. H5N1 avian influenza virus spreading worldwide In human, until now 348 cases were reported with 216 deaths (62%)

5. The Nation, Jan 22, 2004 In Thailand, the outbreak of H5N1 virus was formally reported since January 2004

6. 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

7. A/Chicken/Nakorn-Pathom/Thailand/CU- K2/04 First strain in chicken of Thailand: A/Chicken/Nakorn-Pathom/Thailand/CU- K2/04 The genome sequence analysis of H5N1 avian influenza A virus isolated from the outbreak among poultry populations in Thailand Virology 2004, 328:169-76 Molecular characterization HA gene HA gene - a common characteristic of a highly pathogenic AI (HPAI) NA gene NA gene - a 20-codon deletion NS gene NS gene - a 5-codon deletion M2 and PB2 genes M2 and PB2 genes - polymorphisms

8. Kalij Pheasant Quail CrowWhite peafowl OpenbillOstrich The example of avian species that infected by H5N1 virus

9. Characterization of the Hemagglutinin and Neuraminidase genes of recent influenza virus isolates from different avian species in Thailand Acta Virol 2005, 49:277-80

10. Avian Influenza H5N1 virus in Feline

11. Zoo in Suphanburi province 2 Tigers (Panthera tigris) 2 Tigers (Panthera tigris) 2 Leopards (Panthera pardus) 2 Leopards (Panthera pardus) EID 2004, 10:2189-91 Avian 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

12. Sriracha tiger Zoo in Chonburi province Sriracha tiger Zoo in Chonburi province (the biggest tiger zoo in Thailand) (the biggest tiger zoo in Thailand) Probable tiger-to-tiger transmission of Probable tiger-to-tiger transmission of H5N1 avian influenza virus H5N1 avian influenza virus 147 Tigers (Panthera tigris) died 147 Tigers (Panthera tigris) died EID 2005, 11:699-701 Avian Influenza H5N1 virus in Feline

13. 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

14. Tigers and Bird flu

15. 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. EID 2006, 12:681-3 Strain of virus in cat: A/cat/Thailand/KU-02/04

16. Fatal Avian Influenza A H5N1 in a Dog 5 days later - high fever - panting - lack of energy - bloody nasal discharge Strain of virus in dog: A/dog/Thailand-Suphanburi/KU- 08/04 One-year old dog (Native) from Suphanburi province had eaten HPAI H5N1 infected duck carcasses in the outbreak area EID 2006, 12:1744-47

17. Phylogenetic analysis of the HA and NA gene of the H5N1 influenza virus isolated from dog and cat Dog Dog Cat Cat Virus research 2006 Tiger Tiger human human

18. Unified Nomenclature System for the HPAI H5N1 Avian 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

19. 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 Clade 1 Clade 8 Clade 9 Clade 6 Clade 5 Clade 4 Clade 3 Clade 7 Clade 0 Thailand 2005 Thailand 2004 Thailand 2008 * * *

20. Genetics 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

21. The Cleavage site between HPAI and LPAI LPAIHPAI (Low Pathogenic Avian Influenza)(High Pathogenic Avian Influenza) HA 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

22. The difference of cleavage site of Hemagglutinin gene LPAI HPAI 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

23. 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)

24. 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

25. 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

26. 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)

27. 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

28. 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

29. 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