1 Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
2 National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi 11000, Thailand
Correspondence
Pilaipan Puthavathana
siput{at}mahidol.ac.th
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ABSTRACT |
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The GenBank accession numbers of the sequences determined in this work are AY626143AY626149, AY627885AY627898, AY555150AY555153, AY577314AY577316, AY649382 and AY649383.
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INTRODUCTION |
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The H2N2 and H3N2 influenza A viruses that caused the human pandemics of 1957 and 1968, respectively, were suggested to be new reassortants that had acquired genetic material from avian viruses. However, it was proposed that influenza virus could not transmit directly from avian species to humans, but required the pig as an intermediate host or a mixing vessel to recombine the avian virus with the pre-existing human influenza virus and generate a new reassortant, which was subsequently transmitted to the human population (Zambon, 2001). Direct transmission of avian influenza virus from domestic poultry to humans was demonstrated for the first time by the outbreak of the highly pathogenic avian H5N1 virus in Hong Kong in 1997 (Claas et al., 1998
; Yuen et al., 1998
) and subsequently confirmed by human infection caused by avian H9N2 influenza viruses in Hong Kong in 1999 (Peiris et al., 1999
), by avian H7N7 viruses in the UK in 1996 (Kurtz et al., 1996
) and in The Netherlands in 2003 (Koopmans et al., 2004
), and again by avian H5N1 viruses in Hong Kong in February 2003 (Peiris et al., 2004
).
The Hong Kong influenza H5N1 virus that infected humans in 1997 was believed to be a reassortant virus that had acquired the HA gene from A/goose/Guangdong/1/96 (H5N1)-like viruses, the NA gene from A/teal/Hong Kong/W312/97(H6N1)-like viruses and the internal genes from A/quail/Hong Kong/G1/97 (H9N2)-like or A/teal/Hong Kong/W312/97 (H6N1)-like viruses (Hatta & Kawaoka, 2002; Subbarao & Shaw, 2000
). Even though the Hong Kong/1997 (H5N1) viruses were eliminated by the culling of millions of poultry, its ancestors remained and have gone through several reassortment events in avian species and generated new genotypes, with reemergence of the disease from time to time thereafter (Guan et al., 2002
; Hatta & Kawaoka, 2002
). In 2001, avian H5N1 isolates were classified into five genotypes designated AE (Guan et al., 2002
). After 2002, these genotypes were not detected and eight new genotypes V, W, X1, X2, X3, Y, Z and Z+ emerged (Li et al., 2004
).
The reemergence of H5N1 influenza A viruses affected a large area of East and Southeast Asia for the first time from late 2003 to May 2004. At least eight countries were involved. According to official reports, the outbreak began in South Korea and then spread to Vietnam, Japan, Thailand, Cambodia, China, Laos and Indonesia (CDC/WHO Avian Influenza Response Team, 2004). However, only Vietnam and Thailand reported the disease in humans (Chotpitayasunondh et al., 2004
; Hien et al., 2004
).
It was presumed that the H5N1 viruses responsible for the outbreaks in these countries in 20032004 had the same origin, and they were antigenically distinguishable from the 1997 and early 2003 viruses (CDC/WHO Avian Influenza Response Team, 2004). Nevertheless, no solid information has yet been published. The present study performed nucleotide and amino acid sequence analyses of all eight genomic segments of three human H5N1 viruses, as well as the HA and NA of two additional human viruses and a chicken virus from Thailand. These data provide an insight into the origin of the reemergence and evolution of the H5N1 viruses.
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METHODS |
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Geographical distribution of the H5N1 strains.
This study included five human and one chicken H5N1 isolates designated A/Thailand/1(KAN-1)/2004 (H5N1), A/Thailand/2(SP-33)/2004 (H5N1), A/Thailand/3(SP-83)/2004 (H5N1), A/Thailand/4(SP-528)/2004 (H5N1), A/Thailand/5(KK-494)/2004 (H5N1) and A/Chicken/Thailand/CH-2/2004 (H5N1). A/Thailand/1(KAN-1) virus was isolated from a 6-year-old boy from Kanchanaburi, a province located in the west of Thailand, approximately 128 km west of Bangkok. A/Thailand/2(SP-33) virus was isolated from a 7-year-old boy, A/Thailand/3(SP-83) was from a 58-year-old woman and A/Thailand/4(SP-528) was from a 1-year-old boy. These three patients lived in different areas of the Supanburi Province located in central Thailand at a distance of about 100 km northwest of Bangkok. A/Thailand/5(KK-494) virus was isolated from a 4-year-old boy in Khon Kaen, a province in the northeast of the country, about 449 km northeast of Bangkok. A/Chicken/Thailand/CH-2 virus was isolated from a chicken in a poultry farm in Chachoengsao, a province in the central part of the country, approximately 82 km east of Bangkok. The locations of these provinces are shown in Fig. 1.
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After infection for 18 h, the amount of influenza virus present in the infected-cell monolayer was determined by measurement of the amount of viral nucleoprotein (NP) by ELISA. Plates of infected-cell monolayers were fixed with 80 % acetone overnight and endogenous peroxidase activity was blocked by incubation with 3 % H2O2. The infected-cell monolayers were then incubated with monoclonal antibody to influenza A virus NP (BV European Veterinary Laboratory) for 1 h, before washing and incubating with goat anti-mouse Ig conjugated to horseradish peroxidase (Southern Biotechnology Associates) for 1 h at 37 °C. 3,3',5,5'-Tetramethylbenzidine peroxidase substrate (KPL) was added for 15 min at room temperature before stopping the reaction with 1 M H2SO4. The OD450 was measured. The mean OD of the infected cultures without drug was equal to the total amount of NP protein (100 %). Cultures infected with amantadine-sensitive virus should produce less than 50 % of the total NP protein.
RNA extraction.
Virus propagated in LLC-MK2 or MDCK cells for one or two passages was used as the source of viral RNA for nucleotide sequencing. RNA was extracted from the culture supernatants using the QIAamp Viral RNA mini kit (Qiagen).
Gene amplification.
RT-PCR was performed using the One-step RNA PCR kit (AMV) (Takara Bio) to amplify the PB2, PB1, PA and HA genes, while the Qiagen One-step RT-PCR kit was used to amplify the NP, NA, M and NS genes. PCR primers in this study were as described by Hoffmann et al. (2001) as follows: Ba-PB2-1 and Ba-PB2-2341R for the PB2 gene; Bm-PB1-1 and Bm-PB1-2341R for the PB1 gene; Bm-PA-1 and Bm-PA-2233R for the PA gene; Bm-HA-1 and Bm-NS-890R for the HA gene (the HA reverse primer was identical to the NS reverse primer); Bm-NP-1 and Bm-NP-1565R for the NP gene; Ba-NA-1 and Ba-NA-1413R for the NA gene; Bm-M-1 and Bm-M-1027R for the M gene; and Bm-NS-1 and Bm-NS-890R for the NS gene. The sizes of the amplified products for PB2, PB1, PA, HA, NP, NA, M and NS were 2341, 2341, 2233, 1778, 1565, 1413, 1027 and 890 bp, respectively, plus 29 mer oligonucleotide primer for all products.
Nucleotide sequencing.
The amplified DNA products were electrophoresed in 1 % agarose gel and pieces of gel containing DNA bands of the expected sizes were purified using the Qiagen Gel Extraction kit. The purified DNA was used as the target for direct nucleotide sequencing using a Cycle Sequencing kit (BigDye Terminator version 3.1; Applied Biosystems), followed by analysis in an ABI PRISM version 310 or 377 DNA sequencer (PE Applied Biosystems).
Sequence analysis.
Nucleotide sequences were edited using the Chromas version 2.23 program (Technelysium Pty Ltd; available at http://www.technelysium.com.au/chromas.html). Alignment and homology analysis were performed using BioEdit v. 5.0.6 (T. Hall, Ibis Therapeutics, Isis Pharmaceuticals; available at http://www.mbio.ncsu.edu/BioEdit/bioedit.html), CLUSTAL X v. 1.8 (Thompson et al., 1997; available at ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/) and GeneDoc v. 2.6.02 (Nicholas et al., 1997
; available at http://www.psc.edu/biomed/genedoc). The phylogram was drawn using PHYLIP v. 3.5c (Felsenstein, 1993
; available at http://evolution.genetics.washington.edu/phylip.html) and TreeView v. 1.6.6 (Page, 1996
; available at http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). All sequences have been deposited in GenBank. The accession numbers are provided in Table 1
.
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RESULTS AND DISCUSSION |
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The Hong Kong/1997 H5N1 viruses isolated from humans were found to be highly lethal in birds and ferrets, but were either of high or low pathogenicity in mice (Gao et al., 1999). This information suggested that factors other than the presence of multiple basic amino acids in the cleavage sequence contributed to the severity of H5N1 viruses in mice. A reverse genetics study has shown that a single amino acid substitution, from glutamic acid to lysine at aa 627 on the PB2 molecule (RNA polymerase subunit), was associated with virulence of H5N1 viruses in mice (Hatta et al., 2001
; Shinya et al., 2004
). H5N1 viruses with lysine at this position (HK483RG and HK6PB2-627K) showed systemic spreading and neurovirulence in mice, while viruses with glutamic acid at this position (HK3PB2-627E and HK486RG) showed non-lethal infection that was limited to the lung (Shinya et al., 2004
). The same mutation was found in an H7N7 virus from a patient who died from severe pneumonia, but not in patients with conjunctivitis during an outbreak in Europe in 2003 (Fouchier et al., 2004
). Among the three Thailand viruses that were fully sequenced, glutamic acid at aa 627 in the PB2 protein was found in one sequence, while lysine was found in the other two. Interestingly, all of these viruses were from fatal cases. In Thailand, there was a total of 12 laboratory-confirmed cases with eight deaths occurring during this outbreak. The fatality rate in the Thai patients was 66·7 %, while it was 80 % (8 of 10 cases) for the Vietnamese patients (Hien et al., 2004
). These figures were much higher than those reported from the Hong Kong outbreak in 1997 (33 %, 6 of 18 cases) (Nicholson et al., 2003
). The reason for this higher level of fatality is unclear.
One additional glycosylation site in the HA protein and a deletion of 20 aa in the NA protein are present in the Thailand viruses
The Thailand viruses, as well as other genotype Z viruses, contained a 20 aa deletion in the stalk of the NA molecule. The position of the deletion was close but not identical to the 19 aa deletion found in the Hong Kong/1997 viruses. As a result of this difference, genotype Z viruses have lost an N-linked glycosylation site that was located just upstream of the deletion site in the 1997 viruses (Fig. 3). The NA protein plays an essential role in the release of viral progeny from infected cells, probably through the cleavage of glycosidic linkages to sialic acids on HA molecules of the progeny virus particles. It has been proposed that deletion of amino acid residues may impair the ability of NA to release the progeny virus particles (Matrosovich et al., 1999
). In eggs, the length of the NA stalk correlates with the efficiency of virus replication: the longer the stalk, the more efficient the replication (Castrucci & Kawaoka, 1993
). Despite a predicted inefficiency due to its short NA stalk, the H5N1 viruses studied here replicated and spread efficiently, suggesting that there might be some additional compensatory changes resulting in the short NA stalk being functional. It was previously suggested that an additional glycosylation site at the head of the HA molecule at aa 156 might compensate for the shortening of the NA stalk. Furthermore, the fact that these two changes coincided with the spread of these strains in land-based poultry raises a possibility that these two changes may have resulted in adaptation of the viruses for more efficient spread in terrestrial domestic poultry (Matrosovich et al., 1999
). Both the 1997 viruses and the genotype Z viruses of the present outbreak have these two mutations, while both the parental goose/Guangdong (Gs/Gd)-like viruses and most viruses isolated between the two outbreaks do not.
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Amantadine resistance of the Thailand viruses was confirmed by a drug-sensitivity assay in MDCK cell cultures. All three H5N1 Thailand viruses were resistant to amantadine. The highest concentration of 8·0 µg ml1 could not inhibit the H5N1 viruses at all titres assayed: 25, 50 and 100 TCID50 (Fig. 4 and data not shown). In contrast, A/New Caledonia/20/1999 (H1N1)-like viruses at a titre of 100 TCID50 could be inhibited almost completely by amantadine at a concentration as low as 0·3 µg ml1 (Fig. 4
). With A/Sydney/05/1997 (H3N2)-like viruses at a titre of 100 TCID50, a 50 % reduction in the amount of NP protein was seen with a drug concentration of 8 µg ml1 (Fig. 4
). It was not clear how amantadine resistance was selected and maintained in the Thailand viruses without selection pressure from the antiviral drug.
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Human influenza viruses preferentially bind to sialyl oligosaccharide receptors with 2,6 linkage to galactose, while avian viruses preferentially bind to cellular receptors with
2,3 linkages (Cross et al., 2001
). Despite the ability to infect humans, the Hong Kong/1997 H5N1 viruses still preferred
2,3-linkage receptors, which suggested that transmission from birds to humans did not require a change in receptor-binding preference (Matrosovich et al., 1999
). The preference of H5N1 viruses for avian receptors was probably the reason why human-to-human transmission was limited.
Host-specific amino acids of the Thailand viruses are mostly avian specific
There were 32 amino acid residues in M1, M2, NP, PA and PB2 that have been described as host-specific residues (Shaw et al., 2002). Of these 32 residues, the Hong Kong H5N1 viruses had avian-like residues at 23 sites, human-like residues at five sites and mixture of the two at four other sites (Shaw et al., 2002
). Our H5N1 viruses, on the other hand, had avian-like residues at 31 of 32 sites and a human-like residue at only one site (valine at aa 28 in M2) (Table 2
).
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In order to understand the evolution and origin of the Thailand viruses, we performed a phylogenetic analysis of all genomic segments of the human H5N1 isolates from Thailand (Fig. 5). All segments of the Thailand isolates were closely related to the genotype Z. The viruses had a 5 aa deletion in NS and a 20 aa deletion in NA. The Thailand viruses and other genotype Z viruses had HA and NA proteins that were in the Gs/Gd-like lineage. PB2, PB1, PA and NS of these viruses had other avian origins in the same lineage as most of the other genotypes, while the NP and M genes were probably from another lineage that was shared by genotypes Y, Z+ and Z for NP and by genotypes V, W, Z+ and Z for M. In all trees, the Thailand sequences formed separate subclusters distinguishable from the most closely related genotype Z (Guan et al., 2004
; Li et al., 2004
).
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ACKNOWLEDGEMENTS |
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Received 16 June 2004;
accepted 25 October 2004.