Molecular epidemiological analysis of highly pathogenic avian influenza H5N1 subtype isolated from poultry and wild bird in Thailand

Virus Research 138 (2008) 70–80

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Molecular epidemiological analysis of highly pathogenic avian influenza H5N1 subtype isolated from poultry and wild bird in Thailand Yuko Uchida a,b , Kridsada Chaichoune c , Witthawat Wiriyarat c , Chiaki Watanabe b , Tsuyoshi Hayashi a,b , Tuangthong Patchimasiri d , Bandit Nuansrichay d , Sujira Parchariyanon d , Masatoshi Okamatsu b , Kenji Tsukamoto b , Nobuhiro Takemae a,b , Parntep Ratanakorn c , Shigeo Yamaguchi b , Takehiko Saito a,b,∗ a

Zoonotic Diseases Collaboration Center (ZDCC), Kasetklang, Chatuchak, Bangkok 10900, Thailand Research Team for Zoonotic Diseases, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Kannondai, Tsukuba, Ibaraki 305-0856, Japan c Faculty of Veterinary Science, Mahidol University Salaya, Phuttamonthon, Nakorn-pathom 73170, Thailand d National Institute of Animal Health, Kasetklang, Chatuchak, Bangkok 10900, Thailand b

a r t i c l e

i n f o

Article history: Received 17 June 2008 Received in revised form 8 August 2008 Accepted 19 August 2008 Available online 8 October 2008 Keywords: H5N1 Influenza Thailand Poultry Wild bird

a b s t r a c t A comprehensive molecular epidemiological analysis was performed on highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype derived from poultry and wild bird during 2004–2007 in Thailand. Sequence analysis followed by phylogenetic analysis was applied to all eight segments of the viruses. Viruses belonging to clades 1 and 2.3.4 in the HA phylogenetic tree have been shown to circulate in Thailand. Our analysis revealed differential evolution of the HPAI viruses among clade 1 strains. Isolates from Phichit province in 2006 resided in two distinct branches, designated 1.p1 and 1.p2. A hemagglutination inhibition test with a panel of monoclonal antibodies demonstrated a possible antigenic drift between the Phichit isolates. Involvement of free-grazing duck practice in the area was discussed as a cause of the differential evolution among the Phichit isolates. A branch, designated 1-TGWB and consisting exclusively of isolates from zoological tigers and wild birds, was evident in all phylogenetic trees constructed in the study. The branch’s existence indicated that the HPAI viruses could have been maintained in the wild bird population for a certain period, although no involvement of wild birds in HPAI transmission to poultry was evident in this study. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Influenza A viruses infect humans as well as a variety of avian and mammalian species. Wild waterfowl are considered the virus’s natural reservoir. Sixteen and nine subtypes are recognized for the hemagglutinin (HA) and neuraminidase (NA) surface antigens, respectively. Only a portion of the H5 and H7 avian influenza subtypes, each with a series of basic amino acids at the cleavage site of the HA molecule, are highly pathogenic for poultry species, such as chicken and quail (Wood et al., 1993).

∗ Corresponding author at: Research Team for Zoonotic Diseases, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Tel.: +81 29 838 7802; fax: +81 29 838 7802. E-mail address: [email protected] (T. Saito). 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.08.007

The first H5N1 outbreak in poultry in Asian countries was recognized in 1996 at a goose farm in Guangdong province, China (Tang et al., 1998; Xu et al., 1999). Serious outbreaks in poultry, accompanied by human casualties, followed in Hong Kong in 1997. Despite the successful eradication of those outbreaks in Hong Kong, outbreaks re-emerged in 2001 in terrestrial poultry (Guan et al., 2002) and in 2002 in wild waterfowl (Sturm-Ramirez et al., 2004). The current wave of HPAI H5N1 in east and southeast Asia has been recognized since December 2003, when an outbreak of the HPAI H5N1 was confirmed for the first time in South Korea. By the beginning of 2004, H5N1 outbreaks had followed in Vietnam, Japan, Thailand, Cambodia, China, Laos, and Indonesia. By 2006, HPAI had spread among poultry species, resulting in high rates of mortality throughout central Asia, Europe, and Africa (OIE, 2008a), bringing a heavy economic burden to those countries. In Thailand, outbreaks of the HPAI H5N1 subtype in poultry were reported on 23 January 2004 for the first time in that country (Tiensin et al., 2005). The first wave of outbreaks continued

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Table 1 H5N1 viruses isolated in Thailand sequenced in this study Period of outbreaks (wave)a

Strain nameb

Date of specimen collection (mm/dd/yy) c

Place of isolation

2004/1/23-5/24 (1)

A/chicken/Kalasin/NIAH316/04 A/chicken/Kalasin/NIAH317/04 A/chicken/Kohn Kaen/NIAH330/04c A/open-bill stork/Thailand/VSMU-2-NSN/04 A/open-bill stork/Thailand/VSMU-9-BKK/04c A/duck/PhangNga/NIAH181/04 A/open-bill stork/Thailand/VSMU-20-AYA/04c

2004/7/3-2005/4/12 (2)

A/quail/Phathumthani/NIAH2711/04c A/chicken/Nonthaburi/NIAH2879/04 A/chicken/NaraThiwat/NIAH1703/04c A/chicken/Loei/NIAH2373/04 A/chicken/Samutprakan/NIAH6604/04 A/chicken/Nakhon Sawan/NIAH01502/04 A/chicken/Nakhon Sawan/NIAH01503/04 A/chicken/Suphanburi/NIAH7540/04c A/chicken/Suphanburi/NIAH7618/04 A/duck/Angthong/NIAH8246/04c A/chicken/Angthong/NIAH8334/04

7/19/2004 7/21/2004 7/22/2004 8/2/2004 8/5/2004 10/22/2004 10/22/2004 10/22/2004 10/22/2004 11/2/2004 11/3/2004

Phathum Thani Nonthaburi Nara Thiwat Loei Samut Prakan Nakhon Sawan Nakhon Sawan Suphanburi Suphanburi Angthong Angthong

2005/7/1-11/9 (3)

A/chicken/Suphanburi/NIAH108192/05c A/common myna/Thailand/VSMU-10-BRM/05 A/pigeon/Thailand/VSMU-11-KRI/05c A/pigeon/Thailand/VSMU-13-KRI/05c A/tree sparrow/Thailand/VSMU-12-KRI/05 A/tree sparrow/Thailand/VSMU-14-KRI/05c A/open-bill stork/Thailand/VSMU-15-ATG/05c A/tree sparrow/Rachaburi/VSMU-16-RBR/05c A/quail/Nakhon Pathom/NIAH7562/05c A/chicken/Kalasin/NIAH3776/05 A/open-bill stork/Thailand/VSMU-29-NSN/05

7/15/2005 8/24/2005 9/15/2005 9/15/2005 9/15/2005 9/15/2005 9/26/2005 10/3/2005 10/6/2005 11/3/2005 12/21/2005

Suphanburi Buri Ram Kanjanaburi Kanjanaburi Kanjanaburi Kanjanaburi Angthong Ratchaburi Nakhon Pathom Kalasin Nakornsawan

2006

A/chicken/Phichit/NIAH606988/06c

a b c

1/30/2004 1/30/2004 1/30/2004 2/5/2004 2/12/2004 2/20/2004 4/30/2004

7/26/2006

Kalasin Kalasin Khon Kaen Nakornsawan Bangkok Phang Nga Ayudthya

Phichit

Outbreak wave number. Year of isolation in the strain name was shortened to two digits. The strains whose internal genes were sequenced.

until May 2004, followed by two more waves continuing into 2005 (Buranathai et al., 2007; Tiensin et al., 2005). Sporadic outbreaks were reported from 2006 to the beginning of 2008 (Amonsin et al., 2006a; Buranathai et al., 2007; Chutinimitkul et al., 2006; OIE, 2008b). Approximately 63 million birds in three outbreaks were culled to prevent the spread of infection (Buranathai et al., 2007). Thailand has experienced significant damage in the international poultry trade due to the HPAI outbreaks. Humans have also suffered from HPAI infection in Thailand. As of April 2008, 25 confirmed cases have been reported, including 17 fatalities (WHO, 2008). The involvement of wild birds as carriers of HPAI viruses has been a focus of debate during the course of the worldwide HPAI spread. A mass die-off of wild birds was recorded at Qinghai Lake during May and June 2005 (Chen et al., 2005). Migratory birds have been blamed for the dissemination of the strains related to the Qinghai Lake die-off to Europe and Africa since May 2005. The spread of the outbreaks coincided with migratory flyways from Qinghai Lake to Europe and Africa (Chen et al., 2005; OIE, 2008a). The first evidence for the transmission of the influenza virus between passerine birds and poultry was provided in a report on H7N7 influenza virus infection (Nestorowicz et al., 1987). In Thailand, the open-bill stork was suspected as a virus distributor, since it migrates long distances and a mass of infected and dead ones with the H5N1 virus was found in 2004 (BirdLife International, 2004; Melville and Shortridge, 2006; Payungporn et al., 2004). Since the second outbreak of poultry in October 2004, the Thai government began to monitor wild birds for the HPAI virus (ProMED-mail, 2004). On the other hand, among the multiple introductions of Qinghai-related strains into Nigeria in 2006, some of the outbreaks were caused not by wild birds

but by imports of poultry and poultry products (Ducatez et al., 2006). The present study included a comprehensive molecular epidemiological analysis of the HPAI viruses derived from poultry and wild birds in Thailand during 2004–2007. Besides phylogenetic sequence analyses, detailed information on viruses, such as date of specimen collection and place of isolation, was collected. Such information helped us in characterizing the epidemiological factors in the spread and recurrence of HPAI outbreaks in Thailand. 2. Materials and methods 2.1. Virus isolation Viruses derived from poultry were isolated from cloacal swabs of live birds or carcasses and inoculated to 10-day-old embryonated eggs. Those from wild birds were isolated from cloacal swabs of live or dead birds collected for the surveillance of the H5N1 viruses in wild birds. Specimens were inoculated to 10-day-old embryonated eggs or Madin–Darby canine kidney (MDCK) cells. Viruses were detected by hemagglutination assay according to WHO recommendation. Subtypes of surface antigens were identified by RT-PCR with the H5 and N1 specific primers (Lee et al., 2001). 2.2. Viral RNAs and data on viruses Viral RNAs accompanied by epidemiological data, such as date, place, and host of specimens collected for isolation, were provided by the National Institute of Animal Health of Thailand (NIAHT), five Regional Veterinary Research and Development Centers (RVRDC), and the faculty of veterinary science of Mahidol Uni-

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Table 2 List of the H5N1 viruses isolated in Thailand whose HA sequences in databases were used for phylogenetic analysis Period of outbreaks (wave)a

Strain nameb

Date of specimen collection (mm/dd/yy) d

Place of isolation

2004/1/23-5/24 (1)

A/tiger/Suphanburi/Thailand/Ti-1/04 A/Thailand/16/04c A/Thailand/Kan353/04c A/Thailand/SP83/04c A/chicken/Thailand/1/04c A/chicken/Thailand/2/04c A/bird/Thailand/3.1/04c A/chicken/Suphanburi/1/04c A/quail/Angthong/71/04c A/duck/Angthong/72/04c A/duck/Thailand/71.1/04c A/Thailand/1(KAN-1)/04 A/Thailand/2(SP-33)/04 A/chicken/Thailand/9.1/04c A/quail/Thailand/57/04c A/chicken/Thailand/73/04c A/Goose/Thailand/79/04c A/Thailand/Chaiyaphum/622/04c A/little grebe/Thailand/Phichit-01/04c A/chicken/Nakorn-Patom/Thailand/CU-K2/04 A/tiger/Thailand/VSMU-11-SPB/04c A/Thailand/5(KK-494)/04 A/Thailand/4(SP-528)/04

12/XX /2003 1/3/2004 1/6/2004 1/19/2004 1/21/2004 1/21/2004 1/21/2004 1/21/2004 1/23/2004 1/23/2004 1/23/2004 1/23/2004 1/23/2004 1/24/2004 1/24/2004 1/26/2004 1/26/2004 1/29/2004 1/30/2004 1/XX/2004 2/1/2004 2/18/2004 n.s.e

Suphanburi Suphanburi Kanchanaburi Suphanburi Suphanburi Suphanburi Suphanburi Suphanburi Angthong Angthong Angthong Kanchanaburi Suphanburi Kamphaeng Phet Uttaradit Bangkok Kalasin Chaiya Phum Phichit Nakhon Pathom Suphanburi Khon Kaen Suphanburi

2004/7/3-2005/4/12 (2)

A/chicken/Ayutthaya/Thailand/CU-23/04 A/duck/Kamphaengphet/NIAH-6-2-0041/04c A/tiger/Thailand/VSMU-23-CBI/04c A/crested eagle/Belgium/01/04 A/tiger/Thailand/CU-T3/04 A/tiger/Thailand/CU-T4/04 A/tiger/Thailand/CU-T5/04 A/tiger/Thailand/CU-T6/04 A/tiger/Thailand/CU-T7/04 A/tiger/Thailand/CU-T8/04 A/duck/Kamphaengphet/NIAH6-2-0043/04c

7/XX/2004 10/15/2004 10/26/2004 10/XX/2004 10/XX/2004 10/XX/2004 10/XX/2004 10/XX/2004 10/XX/2004 10/XX/2004 11/2/2004

Ayutthaya Kamphaeng Phet Chonburi Bangkok Chonburi Chonburi Chonburi Chonburi Chonburi Chonburi Kamphaeng Phet

2005/7/1-11/9 (3)

A/great barbet/Thailand/VSMU-2-CBI/05c A/chicken/Thailand/Kanchanaburi/CK-160/05 A/quail/Thailand/Nakhon Pathom/QA-161/05 A/chicken/Thailand/Nontaburi/CK-162/05 A/pigeon/Thailand/VSMU-25-BKK/05c A/chicken/Kamphaengphet/NIAH6-3-0013/05c A/chicken/Kamphaengphet/NIAH6-3-0014/05c A/Thailand/NK165/05 A/brown-headed gull/Thailand/VSMU-28-SPK/05c

6/22/2005 10/XX/2005 10/XX/2005 10/XX/2005 11/24/2005 12/7/2005 12/7/2005 12/9/2005 12/14/2005

Chonburi Kanchanaburi Nakhon Pathom Nonthaburi Bangkok Kamphaengphet Kamphaengphet Nakhon Nayok Samut-Prakarn

2006

A/chicken/Thailand/PC-168/06 A/chicken/Thailand/PC-170/06 A/chicken/Nakhon phanom/NIAH113718/06c A/chicken/Thailand/NP-172/06

7/XX/2006 7/XX/2006 7/30/2006 7/XX/2006

Phichit Phichit Nakhon Phanom Nakhon Phanom

2007

A/chicken/Nong khai/NIAH400802/07c

1/24/2007

Nong Khai

a b c d e

Outbreak wave number. Year of isolation in the name was shortened to two digits. Epidemiological data of the strains analyzed as described in Section 2. XX means the exact date could not be specified. Not specified other than during wave 1.

versity (MU) (Table 1). Epidemiological data on the Thai isolates whose sequences were obtained from the Influenza Database at the National Center for Biotechnology Information (NCBI) were found in the literature or, when no data in the literature were available, were provided by the National Institute of Animal Health, Regional Veterinary Research and Development Centers, the Department of Livestock Development (DLD) of Thailand, and Mahidol University (Table 2). Epidemiological data on human isolates that could not been found in the literature were obtained from Dr. Rebecca Garten of the Centers for Disease Control and Prevention, Atlanta, Georgia, USA. Sequence data with epidemiological data were used to make the HA phylogenetic tree only; the phylogenetic trees of the other segments were constructed with as many sequences as possible,

which included those without epidemiological data to avoid bias of sampling in constructing trees and to enhance accuracy of the trees. 2.3. Genomic sequencing and phylogenetic analysis Extracted viral RNAs were reverse-transcribed to cDNA by Superscript III (Invitrogen). Those DNAs were amplified by polymerase chain reaction (PCR) with Ex Taq polymerase (Takara Bio) and specific primers against the eight segments of the H5N1 influenza virus and cDNA as a template. The information of primers is offered for demand. The surface antigen genes of 30 strains, 19 from poultry and 11 from wild birds, were sequenced com-

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Table 3 Amino acid substitutions in the putative antigenic, receptor binding, and cleavage sites among viruses used for antigenic analysis Strain

Regions corresponding to the antigenic sites defined in the H3 HA molecule Site A

Ck/Suphanburi/1/04 Ck/Loei/NIAH2373/04 Ck/Kalasin/NIAH3776/05 Ck/Phichit/NIAH606988/06 Ck/Thailand/PC-170/06 Ck/Nong khai/NIAH400802/07b a b

Site B

Receptor binding site a

Site C

Site D

138a

140a

141a

124

129

40

210

212

83a

Site E 86a

Q · · L · ·

E K K K K T

S · · · · P

S · · · · D

L · · · · V

K · · · R ·

V · · · E ·

R · · · · K

A · · · P ·

V · · · A A

Cleavage site

129

325

L · · · · S

R · K · K ·

Positively selected amino acids predicted previously (G.J. Smith et al., 2006). CkNK11371806 and CkNP17206 possess the same amino acid substitutions in the list to CkNK40080207.

pletely. The accession numbers of the sequences obtained are AB450502–AB450657. Phylogenetic trees were constructed based on alignments of 1514 and 1110 bp for HA and NA, respectively. Representative strains for the sequencing of internal genes were chosen based on the topologies in the HA and NA phylogenetic trees and the host species of origin. Nine and seven viruses derived from poultry and wild birds, respectively, were selected (Table 1). The nucleotide lengths used for phylogenetic analysis were 1994, 2029, 1244, 780, 1472, and 901 bp in the PB2, PB1, PA, NS, NP, and M genes, respectively. BioEdit software (Hall, 1999) was used for sequence alignment, and phylogenetic trees based on nucleotide information were constructed by MEGA 3.1 with the neighbor-joining method. The bootstrap value was calculated from 1000 replicates. Maximum parsimony method was also used to analyze phylogeny of the HA gene by MEGA 4 with 500 replication of bootstrap analysis. 2.4. Antigenic analysis The antigenic characteristics of the viruses isolated in Thailand were analyzed by the hemagglutinin inhibition test (HI) using monoclonal antibodies (MAb) described by the WHO manual on animal influenza diagnosis and surveillance (WHO, 2002). Formalin-inactivated antigens prepared for HPAI diagnosis by the National Institute of Animal Health of Thailand were used for the test, except for the Ck/Thailand/PC-170/06 (CkPC17006) which was isolated at Chulalongkorn University, therefore, not available for diagnosis at NIAH. The virus with the modified HA gene of CkPC17006 to be used as an antigen for the HI test was prepared by the reverse genetics method (Hoffmann et al., 2000). The HA gene with modification at the cleavage site was synthesized artificially based on the published sequence (Accession No. DQ999887) and was inserted into the pHW2000 plasmid provided by Dr. Erich Hoffmann of St. Jude Children’s Research Hospital. The viral sequence at the cleavage site was modified from REKRRKKR to RETR. NA and internal genes were used from Ck/Yamaguchi/7/04 (H5N1) and A/Puerto Rico/8/34 (PR8), respectively, for the reverse-geneticsderived influenza virus. PCR-BluntIITOPO vector (Invitrogen) was used for the cloning of NA and internal genes, which were inserted into the BsmBIor BsaIsite of the pHW2000 plasmid. Eight synthesized plasmids were transfected to 293 T cells cocultured with MDCK cells, and the reverse-genetics-derived virus was propagated in MDCK cells with 5 ␮g/ml acetylated trypsin. pHW2000 plasmids containing each gene segment from A/Puerto Rico/8/34 were kindly provided by St. Jude Children’s Research Hospital. MAbs against H5 antigen, VN04-2, VN04-3, VN04-10, VN04-12, VN04-15, CP24, CP25, and CP58 were kindly provided by St. Jude Children’s Research Hospital, and Y38/3 and Y86/1 were produced against Ck/Yamaguchi/7/2004 by the National Institute of Animal Health of Japan.

3. Results Nucleotide sequence analysis of the HA gene, followed by comparison of the nucleotide and deduced amino acid sequence identities, differentiated the Thai HPAI viruses examined in this study into two groups based on their mutual sequence identities. Both the nucleotide and amino acid sequence identities among the Thai strains examined, except for Ck/Nakhonphanom/NIAH113718/2006 (CkNK11371806), Ck/Thailand/NP-172/2006 (CkNP17206), and Ck/Nongkhai/NIAH400802/2007 (CkNK40080207), were above 98%, indicating that they were genetically homogeneous. It was clear that the three exceptional viruses, isolated in late 2006 and 2007, were distinct from the viruses that circulated during the earlier stage of the Thai HPAI outbreaks. Phylogenetic analysis by neighbor-joining method (Fig. 2A) confirmed the two distinct lineages among Thai isolates described previously (G.J.D. Smith et al., 2006). The strains isolated in 2004–2006, whose identities were 98% or higher among each other, resided in clade 1 (Thai–Vietnam strains) by the WHO/OIE/FAO H5N1 Evolution Working Group (WHO, 2007). As indicated above, CkNK11371806, CkNP17206, and CkNK40080207 belonged to a distinct lineage that is currently designated clade 2.3.4 (Fujian-like strains). Compared to the clade 1 strains, each of the three Thai strains involved in clade 2.3.4 had five amino acid substitutions, at S124D, L129S, K140T, S141P, and R212K, within the regions homologous to the putative antigenic sites of the H3 HA (Wiley et al., 1981; Wilson et al., 1981), raising the possibility of an antigenic difference from those belonging to clade 1. This notion was confirmed by antigenic analysis of a panel of MAbs, as shown in Table 4. Two MAbs – VN04-2 and VN04-3 – reacted significantly less to CkNK40080207 than did the clade 1 strains used in the analysis. The three Thai strains involved in clade 2.3.4 also had L129S within the receptor binding site of the HA molecule. Putative N-glycosylation sites at 11, 23, 154, 165, 193, 286, and 483 were found among all of the Thai strains examined except for CkNP17206, which lacked a site at 193. Although isolated in the same province (Phichit) in July 2006, three isolates – Ck/Thailand/PC-168/2006 (CkPC16806), Ck/Phichit/NIAH606988/2006 (CkPC60698806), and Ck/Thailand/PC-170/2006 (CkPC17006) – were differentiated into two branches, which we designated 1.p1 (CkPC16806 and CkPC60698806) and 1.p2 (CkPC17006) (Fig. 2A). The bootstrap values for the branches were 63 and 97%, respectively. Eight amino acid differences were found between the 1.p1 and 1.p2 strains (Table 3). Among them, five substitutions – 40, 83, 86, 138, and 210 – were located within regions homologous to the antigenic epitopes A, D, and E of the H3 HA molecule (G.J. Smith et al., 2006; Wiley et al., 1981; Wilson et al., 1981), suggesting that antigenicity may differ among those strains (Table 3 and Fig. 2B). Two of them, at 138 in site A and 83 in site E, were also recognized previously as positively selected amino acids (G.J.

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Fig. 1. Geographical location of the provinces where the viruses mentioned in this text were isolated.

Smith et al., 2006). Again, antigenic analysis with MAbs demonstrated antigenic differences among the Phichit isolates (Table 4). CkPC17006, along with Ck/Loei/NIAH2373/2004 (CkLoei237304) and Ck/Kalasin/NIAH3776/2005 (CkKalasin377605), reacted with MAbs Y38/3 and Y86/1. On the other hand, CkPC60698806 did not react with those MAbs. A similar reaction was observed with VN04-15: it reacted to CkPC60698806 with a titer of 100 and to CkPC17006 and CkKalasin377605 with titers that were more than 16-fold higher. No single amino acid difference among the strains examined appeared to correlate with the reaction patterns observed for Y38/3 and Y86/1. Glutamine at 138 in combination with lysine at 140 might be involved in an antigenic epitope recognized by Y38/3 and Y86/1. Ck/Yamaguchi/7/2004, which was used as an antigen for producing those two MAbs, has arginine 140. Lysine and arginine are similar in their chemical properties, such as molecular charge and mass. For epitope mapping, H5 HA antigenicity needs to be analyzed in detail. Substitution at R325K was found at the cleavage site between the HA1 and HA2 molecule. Th/NK165/2005 (NK16505), which is closely related to CkPC17006, was isolated from a 5-year-old male patient who died on 7 December 2005, in Nakhon Nayok province (Chutinimitkul et al., 2006). It had four amino acid differences from CkPC17006, at 40, 129, 210, and 505 (Fig. 2B). Only NK16505 had a valine at amino acid 129 which was thought to be a positively selected amino acid residing in the receptor binding site, when compared to the other Thai strains. This substitution, when combined with A134V, was previously thought to alter the receptor recognition from 2–3 to both 2–3 and 2–6 sialic acid linkages (Auewarakul et al., 2007). The places where the viruses associated with branches 1.p1 and 1.p2 in the phylogenetic trees were isolated coincided with two regions affected in wave 3. Outbreaks in wave 3 were reported mainly from Kamphaengphet province, in the lower north region of Thailand, and in nine provinces in the central region, such as Kanchanaburi and Nontaburi (Buranathai et al., 2007; OIE, 2005). Viruses related to 1.p1 were isolated in Kamphaengphet province, which neighbors Phichit province, and those related to 1.p2 were from Kanchanaburi and Nontaburi provinces (Fig. 1). A wild bird isolate, Tree sparrow/Thailand/VSMU-14-KRI/2005 (Ts14KRI05), resided in a sister branch of 1.p1. A cluster with a relatively long branch length was observed in the HA tree (Fig. 2A). This cluster, designated 1-TGWB

in this study, was composed exclusively of isolates from wild birds and tigers in the HA tree. Wild bird isolates in this cluster were Tree sparrow/Thailand/VSMU-12-KRI/2005 (Ts12KRI05), Tree sparrow/Rachaburi/VSMU-16-RBR/2005 (Ts16RBR05), PigeonThailand/vsmu-13-KRI/2005 (P13KRI05), Open-bill stork/Th/VSMU-15-ATG/2005 (OS15ATG05), Openbill stork/Th/VSMU-29-NSN/2005 (OS29NSN05) and Great barbet/Th/VSMU-2-CBI/2005. Those from tigers were Tiger/Thailand/CU-T3/2004-tiger/Thailand/CU-T8/2004 (TGCUT3804) and Tiger/Thailand/VSMU-23-CBI/2004 (TG23CBI04). The tiger strains within the cluster were isolated in October 2004 in Chonburi province (Amonsin et al., 2006b), and the wild bird strains were isolated from June to December 2005 in several provinces within a 200 km radius from where the tiger strains were isolated. N402H was a specific amino acid substitution in 1-TGWB strains compared to the other viruses residing in clade 1. Although this substitution is a characteristic of the 1-TGWB strains, the significance of this substitution remains to be elucidated. Multiple introductions of the HPAI viruses into the openbill stork population in Thailand during 2004 and 2005 were demonstrated in the HA tree as well as in other trees described below in this study. Among the five strains isolated from openbill storks, two of them, OSvsmu1505 and OSsmu2905, were located in the 1-TGWB cluster in the HA tree, whereas Openbill stork/Thailand/VSMU-2-NSN/2004 (OS2NSN04) and Open-bill stork/Thailand/VSMU-20-AYA/2004 (OS20AYA04) both resided in a different branch that was closely related to isolates in Vietnam (Fig. 2A). Open-bill stork/Th/VSMU-9-BKK/2004 (OS9BKK04) also resided in a different position within clade 1, being distinguishable from other open-bill stork isolates. Similar topology for 1.p1, 1.p2 and 1-TGWB clades was observed in the HA trees constructed by neighbor-joining and maximum parsimony methods (data not shown). Phylogenetic analysis of the NA gene among the Thai strains also demonstrated that the differentiation of 1.p1 and 1.p2 as well as of the 1-TGWB cluster, shown in the HA tree, could be applied to the NA gene (Fig. 3). A majority of the Thai isolates in the NA tree resided in an equivalent of clade 1 in the HA tree. The clade 1 Thai strains shared sequence identities to each other above 99% in the NA gene. The differentiation of three Phichit isolates into 1.p1 (CkPC60698806 and CkPC16806) and 1.p2 (CkPC17006) appeared to be applicable to the NA tree, although the bootstrap values for those branches were lower. CkNP17206 belonged to a distinct lineage, an equivalent to clade 2.3.4 in the HA tree. The sequence identities of CkNP17206 to clade 1 Thai isolates were as low as 94.5–96%. A cluster of wild bird isolates, which was described as 1-TGWB in the HA tree, was also observed in the NA tree (Fig. 3). The same strains observed in 1-TGWB in the HA tree, along with Ts14KRI05, Ck/Kalasin/NIAH316/2004 (Ck31604), and Ck/Samutprakan/NIAH6604/2004 (Ck660404), formed the cluster in the NA tree. Ts14KRI05 was involved in 1-TGWB in the NA tree, though not in 1-TGWB in the HA tree. All internal gene segments of Ts14KRI05 also belonged to clade 1-TGWB in each tree as described below, suggesting a genetic reassortment among the wild bird population. A80T in the NA molecule distinguished 1-TGWB strains from the other clade 1 strains. In addition, Ck31604 in the 1-TGWB cluster had three unique substitutions at S25N, M27I, and V321I among 1-TGWB strains. The NA molecule of all the 2004–2007 Thai isolates possessed a 20-amino acid deletion in the NA stalk region (N1 numbering: 49–68). Among the NAs of the Thai strains, there was no sign of the amino acid substitutions related to sialic acid binding (Claas et al., 1998; Garcia et al., 1996), neuraminidase inhibitor resistance (Kiso et al., 2004; Le et al., 2005), or the stability of sialidase activity under low pH demonstrated in N2 (Suzuki et al., 2005; Takahashi et al., 2003).

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Fig. 2. (A) Phylogenetic tree of the HA gene of H5N1 subtype influenza A viruses, including representative viruses and isolation in Thailand. The nucleotides were analyzed by the neighbor-joining method. The tree was rooted to A/goose/Guangdong/96. Direction of the root is shown as arrow. Bootstrap values more than 50% are shown at each branch. The isolated host, country, and year in each strain name were abbreviated as chicken: Ck; duck: Dk; quail: Qa; goose: Gs; tiger: Tg; Thailand: Th; Vietnam: VN; 1997–2007: 97–07. (B) Enlarged sub-trees in the HA phylogenetic tree where 1. p1 and 1. p2 belong. Amino acid substitutions at each branch are shown. *Positively selected amino acid.

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Fig. 2. (Continued ).

Phylogenetic analysis of the internal gene segments, PB2 (Fig. 4), PB1, PA, NP, M, and NS (data provided as the supplemental), among the Thai isolates revealed similar evolutional patterns throughout those segments. The most intriguing similarity among those trees was the existence of a cluster consisting of isolates from tigers and wild bird strains in each internal segment, as in the 1-TGWB cluster in the HA and NA trees. In particular, the 1-TGWB cluster in the trees of PB2, PB1, NP, and NS genes consisted exclusively of tiger and wild bird strains, whereas other strains from poultry were involved within the 1-TGWB cluster in the PA and M trees. Bootstrap values for the 1-TGWB clade in the PB2, PB1, and NP trees were higher – 62, 68, and 62 – though the values in the PA, NS, and M trees were 33, 26, and 7%. However, nucleotide and amino acid identities were as high as 99.7% among the wild bird strains within the 1-TGWB strains, suggesting that they are genetically closely related each other in all segments examined. Substitution at E627K in PB2 was exclusively found in the strains belonging to 1-TGWB among the Thai isolates. These observations suggested that wild bird isolates belonging to the 1-TGWB cluster were derived from those of infected tigers. E96D in PB1 and P33L in PB1-F2, described previously (G.J. Smith et al., 2006) as positively selected amino acids, were also found in 1-TGWB viruses. Bootstrap values for the cluster in those trees were much less than that obtained in the HA tree. Three Phichit isolates were similarly separated into two branches in the internal gene trees, as seen in the HA and NA trees, indicating the co-evolution of all the segments of those viruses. There was no evidence of a reassortment between clade 1 viruses and clade 2.3.4 viruses. This is probably because the outbreaks caused by clade 2.3.4 viruses were isolated events and because the prevalence of HPAI viruses was dramatically decreased in the later outbreaks in Thailand. Substitution at 66 in PB1-F2 amino acid (N66S), which is a factor increasing virulence against the infected host (G.J. Smith et al., 2006), was found in Ck/Kohn Kaen/NIAH330/04 (CkKK33004), although the significance of this substitution was not evident in this study. All of the Thai strains sequenced in this study contained a deletion at 80–84 and the PDZ binding sequence motif at the C-terminal in the NS1 protein (Krug, 2006; Obenauer et al., 2006), and either the L26I or S31N substitution in the M2 protein, which confers amantadine resistance (Cheung et al., 2006). 4. Discussion Extensive phylogenetic and antigenic analysis in this study demonstrated the genetic evolution within the Thai HPAI strains

belonging to clade 1, which used to be called the Thai–Vietnam lineage. The inclusion of 19 newly sequenced poultry isolates and 11 isolates from wild birds in Thailand, accompanied by the date and place of each isolation, allowed us to perform a comprehensive molecular evolutional study of the HPAI viruses in Thailand. Genetic comparison of three strains from Phichit province isolated in July 2006 revealed the divergence even among clade 1 isolates in that year. The isolation of genetically divergent strains within the same province may suggest that viruses that evolved in separate environments may have been brought together in Phichit province by unidentified movements. Such movements might be those of poultry, poultry-related materials, or others. Free-grazing ducks could contribute to the genetic evolution shown in this study. Free-grazing ducks were considered a reservoir and distributor of HPAI viruses affecting domestic chickens, because flocks of ducks were moved long distances in one season and showed few or no clinical signs when they were infected with the HPAI viruses (Gilbert et al., 2006; Songserm et al., 2006a). The H5N1 viruses that emerged after 2002 have been shown to cause lethal infection in ducks (Sturm-Ramirez et al., 2004), although infection did not always result in high mortality. The places where the progenitors of the three Phichit strains were isolated corresponded to a free-grazing duck range in Thailand. After harvest, farmers allow free-grazing ducks to move around the paddy fields to eat leftover rice grains, insects, and snails. The regions of such movement extend from the central to the lower north regions, including Phichit province (Gilbert et al., 2006; Songserm et al., 2006a). The evolution could also be attributed to host immune pressure in the ducks. Among the amino acid differences between 1.p1 and 1.p2 viruses, three of them, residing in putative antigenic epitopes at 83, 86, and 138, were thought to be in positions under positive selective pressure (G.J. Smith et al., 2006). A specific antibody against the H5 virus was detected in ducks that tolerated experimental infection by an H5N1 virus (Isoda et al., 2006). Free-grazing duck populations could possess antibodies induced by primary infection, and these antibodies could either protect themselves from re-infection or serve as a selective pressure of antigenic variants in vivo. Possible antigenic drift was demonstrated in this study by the HI analysis with a panel of MAbs (Table 4). It was previously suggested that replication in ducks, even at first exposure, propagated antigenic variants during prolonged infection (Hulse-Post et al., 2005). Although we cannot rule out the possibility that selective pressures could be posed on those sites in chickens, this is not likely, since HPAI viruses would kill most, if not all, infected chickens. Thus, the viruses may have been maintained and evolved after

Y. Uchida et al. / Virus Research 138 (2008) 70–80

Fig. 3. Phylogenetic tree of NA gene. Clade names and abbreviations are the same as described in Fig. 2A.

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Fig. 4. Phylogenetic tree of PB2 gene. Clade names and abbreviation are the same as described in Fig. 2A.

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Table 4 Antigenic analysis of viruses isolated in Thailand by HI test using monoclonal antibodies Strain

Monoclonal antibodies VN04-2

VN04-3

VN04-10

VN04-12

VN04-15

CP24

CP25

CP58

Y38/3

Y86/1

Ck/Suphanburi/1/04 Ck/Loei/NIAH2373/04 Ck/Kalasin/NIAH3776/05 Ck/Phichit/NIAH606988/06 Ck/Thailand/PC-170/06 Ck/Nong khai/NIAH400802/07

25,600 12,800 25,600 25,600 51,200 400

25,600 6,400 12,800 12,800 25,600 200

12,800 3,200 3,200 3,200 6,400 1,600

800 1600 1600 3200 3200 800

<100 400 1600 100 3200 <100

1600 400 100 400 400 6400

100 200 100 200 800 100

3200 800 200 1600 800 800

<100 200 1600 <100 800 <100

<100 1,600 3,200 <100 12,800 <100

transmission from other poultry into free-grazing duck populations and then were transferred back to chickens. It is likely that the wild bird isolates in the 1-TGWB cluster were introduced from infected zoological tigers and maintained stably among various kinds of small terrestrial birds for a certain period. Small residential wild birds such as crows, pigeons, and tree sparrows may have had contact with tiger carcasses or excretion in the zoo, and subsequently disseminated the viruses widely to other small terrestrial birds. Several cases of infection have been reported in pigeons and crows in Thailand (Pantin-Jackwood and Swayne, 2007; Songserm et al., 2006b). Dead crows infected with H5N1 were found as far as 30 km away from an affected farm in Japan (Tanimura et al., 2006). Because small terrestrial birds either cannot fly such a long distance or be infected persistently (Boon et al., 2007; Perkins and Swayne, 2002), several links in chains of infection and transmission of the viruses should have occurred. It was intriguing to note that those viruses derived from wild birds and tigers exclusively possessed three amino acid substitutions – E627K in PB2, P33L in PB1-F2, and E96D in PB1 – compared to other Thai HPAI isolates. E627K amino acid substitution in PB2 was considered to be related to host range adaptation from birds to mammals (Subbarao et al., 1993). K627 of PB2 was shown to play a role in efficient viral replication in mice infected with Hong Kong H5N1 viruses (Shinya et al., 2004). This substitution was detected in H5N1 viruses isolated from humans in Hong Kong in 1997 (Naffakh et al., 2000). Furthermore, it was found in wild bird strains belonging to clade 2.2, the so-called Qinghai Lake strains that have been spreading toward the Western countries since 2005 (Chen et al., 2006; Zhou et al., 2006). Having lysine at 627 of the PB2 protein is not likely to be disadvantageous in replication in a wild bird population. The functions of the substitution at 33 in PB1-F2, proposed as a positively selected amino acid, and that at 96 in PB1 in wild birds or mammals remain to be studied. The involvement of wild birds in the transmission of the HPAI viruses to poultry remains controversial. Multiple introductions of the HPAI viruses into the open-bill stork population in Thailand were made evident in this study. This analysis also revealed the maintenance of the HPAI viruses in open-bill storks as well as in residential birds. This evidence indicated that the HPAI viruses could have circulated for several months with a few gene mutations among wild bird populations and could have spread across wide areas as the birds flew long distances. However, it could be argued that the open-bill stork might not be a direct transmitter of the viruses to poultry, because they are too large to enter enclosed poultry farms and tend to stay away from human habitation. In contrast, small terrestrial birds – tree sparrows, pigeons, starlings, and others – are more likely to be exposed to farm poultry, free-grazing ducks, and backyard poultry. An experiment with small terrestrial birds inoculated with an H5N1 virus isolated in Hong Kong suggested that they could act as potential transmitters between wild birds and poultry or as a reservoir of the H5N1 virus (Boon et al., 2007). In sparrows and starlings, the virus was detected from oropharyngeal and cloacal swabs after infection; starlings, especially, shed the virus in the long term with no fatalities (Boon et al., 2007). It could

be considered that small terrestrial birds transmitted the viruses from poultry to free-grazing ducks and open-bill storks, which could maintain the viruses for certain periods of time. A reverse flow of the pathway is likely to occur, although our study did not establish evidence of virus re-introduction from wild birds to poultry. Implementation of bio-security measures, by which poultry can be completely separated from wildlife, is a key to intercepting the transmission chain of the HPAI viruses. In this study, molecular analysis of the Thai HPAI isolates, accompanied by epidemiological information such as the place, date, and host of each isolation, revealed several evolutional features of the Thai HPAI viruses. Such epidemiological information is indispensable to obtaining molecular information on causative agents in order to fully understand the epidemiological characteristics of outbreaks. The government of Thailand has implemented strict control and prevention measures against HPAI since that country’s first experience of an outbreak in January 2004. For the early detection of HPAI, the Thai government has begun active surveillance of wild birds and free-grazing ducks in high-risk regions, along with surveillance of poultry. Bio-security has been intensified by improving poultry housing environments and free-grazing duck practices in order to prevent HPAI from spreading (Gilbert et al., 2006; Songserm et al., 2006a). The results of our analysis revealed the importance of Thailand’s preventive measures against HPAI. Acknowledgments This work was supported by a program of the Founding Research Center for Emerging and Reemerging Infectious Diseases launched by a project commissioned by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2008.08.007. References Amonsin, A., Chutinimitkul, S., Pariyothorn, N., Songserm, T., Damrongwantanapokin, S., Puranaveja, S., Jam-on, R., Sae-Heng, N., Payungporn, S., Theamboonlers, A., Chaisingh, A., Tantilertcharoen, R., Suradhat, S., Thanawongnuwech, R., Poovorawan, Y., 2006a. Genetic characterization of influenza A viruses (H5N1) isolated from 3rd wave of Thailand AI outbreaks. Virus Res. 122 (1–2), 194–199. Amonsin, A., Payungporn, S., Theamboonlers, A., Thanawongnuwech, R., Suradhat, S., Pariyothorn, N., Tantilertcharoen, R., Damrongwantanapokin, S., Buranathai, C., Chaisingh, A., Songserm, T., Poovorawan, Y., 2006b. Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand. Virology 344 (2), 480–491. Auewarakul, P., Suptawiwat, O., Kongchanagul, A., Sangma, C., Suzuki, Y., Ungchusak, K., Louisirirotchanakul, S., Lerdsamran, H., Pooruk, P., Thitithanyanont, A., Pittayawonganon, C., Guo, C.T., Hiramatsu, H., Jampangern, W., Chunsutthiwat, S., Puthavathana, P., 2007. An avian influenza H5N1 virus that binds to a humantype receptor. J. Virol. 81 (18), 9950–9955. BirdLife International, 2004. BirdLife Concerned over Thai Stork Cull., http://www.birdlife.org/news/news/2004/07/thai flu.html.

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