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Nature - HA mutations responsible for the binding of H5N1 A viruses

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  • Nature - HA mutations responsible for the binding of H5N1 A viruses

    Nature 444, 378-382 (16 November 2006) | <ABBR title="Digital Object Identifier" minmax_bound="true">doi</ABBR minmax_bound="true">:10.1038/nature05264; Received 20 August 2006; Accepted 21 September 2006
    Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors

    Shinya Yamada<SUP minmax_bound="true">1,</SUP><SUP minmax_bound="true">3</SUP>, Yasuo Suzuki<SUP minmax_bound="true">3,</SUP><SUP minmax_bound="true">4</SUP>, Takashi Suzuki<SUP minmax_bound="true">3,</SUP><SUP minmax_bound="true">5</SUP>, Mai Q. Le<SUP minmax_bound="true">6</SUP>, Chairul A. Nidom<SUP minmax_bound="true">7</SUP>, Yuko Sakai-Tagawa<SUP minmax_bound="true">1,</SUP><SUP minmax_bound="true">3</SUP>, Yukiko Muramoto<SUP minmax_bound="true">1,</SUP><SUP minmax_bound="true">3</SUP>, Mutsumi Ito<SUP minmax_bound="true">1,</SUP><SUP minmax_bound="true">3</SUP>, Maki Kiso<SUP minmax_bound="true">1,</SUP><SUP minmax_bound="true">3</SUP>, Taisuke Horimoto<SUP minmax_bound="true">1,</SUP><SUP minmax_bound="true">3</SUP>, Kyoko Shinya<SUP minmax_bound="true">8</SUP>, Toshihiko Sawada<SUP minmax_bound="true">9</SUP>, Makoto Kiso<SUP minmax_bound="true">9</SUP>, Taiichi Usui<SUP minmax_bound="true">10</SUP>, Takeomi Murata<SUP minmax_bound="true">10</SUP>, Yipu Lin<SUP minmax_bound="true">11</SUP>, Alan Hay<SUP minmax_bound="true">11</SUP>, Lesley F. Haire<SUP minmax_bound="true">11</SUP>, David J. Stevens<SUP minmax_bound="true">11</SUP>, Rupert J. Russell<SUP minmax_bound="true">11,</SUP><SUP minmax_bound="true">13</SUP>, Steven J. Gamblin<SUP minmax_bound="true">11</SUP>, John J. Skehel<SUP minmax_bound="true">11</SUP> and Yoshihiro Kawaoka<SUP minmax_bound="true">1,</SUP><SUP minmax_bound="true">2,</SUP><SUP minmax_bound="true">3,</SUP><SUP minmax_bound="true">12</SUP>
    Top of page Abstract

    H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects<SUP minmax_bound="true">1, </SUP><SUP minmax_bound="true">2</SUP>. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-2,6-galactose (SA2,6Gal), whereas the latter prefer those ending in SA2,3Gal (refs 3–6). A conversion from SA2,3Gal to SA2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.

    We used H5N1 viruses isolated from birds and humans to identify amino acid changes in the HA molecule that could enable the viruses to recognize human-type receptors (Supplementary Table 1 and Fig. 1). A/Vietnam/30262III/04 and A/Vietnam/3028II/04 contained a heterogeneous mixture of HAs on sequence analysis, prompting us to plaque-purify the viruses in Madin–Darby canine kidney (MDCK) cells to obtain viral clones with distinct HA sequences (Supplementary Table 1). We also used plaque-purified clones of A/Vietnam/30408/05 that differed in their HAs<SUP minmax_bound="true">7</SUP>. To expand the repertoire of viruses, we synthesized the HAs of eight H5N1 viruses isolated from humans in Thailand, Vietnam and Cambodia, using sequences in the Influenza Sequence Database (https://www.flu.lanl.gov/). Mutant viruses possessing each of these HA genes, the neuraminidase of A/Vietnam/1194/04 (VN1194) and all remaining genes from A/Puerto Rico/8/34 (PR8; H1N1) were then generated by reverse genetics<SUP minmax_bound="true">8</SUP>.
    Figure 1: Receptor-binding activity of H5N1 viruses.

    Direct binding of viruses to sialylglycopolymers containing either 2,3-linked (blue) or 2,6-linked (red) sialic acids was measured. a, Human isolate, Vietnam 2004. b, Virus isolated from duck. ce, Human isolates with capacity to recognize both SA2,6Gal and SA2,3Gal. Data are the mean s.d. of triplicate experiments.
    High resolution image and legend (135K)<!-- -->

    The receptor specificity of the resulting H5N1 viruses was determined with an assay that measured direct binding to sialylglycopolymers possessing either SA2,3Gal or SA2,6Gal. None of the five avian H5N1 isolates bound appreciably to SA2,6Gal, whereas 3 of the 21 human isolates, A/Vietnam/3028II/04clone3 (VN/3028IIcl3), A/Thailand/1-KAN-1/04RG (Thai/KAN), and A/Vietnam/30408/05clone7 (VN/30408cl7), including subclones, bound to both SA2,3Gal and SA2,6Gal; some of the other human H5N1 viruses also recognized SA2,6Gal but to only a limited extent (Fig. 1 and Supplementary Fig. 2). The specificity of the receptor-binding assay was verified as follows. Sialylglycopolymers possessing either SA2,3Gal or SA2,6Gal were treated with Arthrobacter ureafaciens sialidase or mock-treated and then incubated with SA–Gal linkage-specific lectins: Maackia amurensis lectin II (MALII), specific for SA2,3Gal; Sambucus nigra lectin (SNA), specific for SA2,6Gal; and M. amurensis lectin I (MALI), specific for both Gal1-4GlcNAc and SA2,3Gal1-4GlcNAc. The sialylglycopolymer possessing SA2,3Gal reacted only with MALII, whereas that possessing SA2,6Gal reacted only with SNA (Supplementary Fig. 3). The sialylglycopolymers treated with the sialidase no longer bound to the respective SA–Gal linkage-specific lectins, but gained reactivity with MALI, confirming that the sialidase treatment removed only the terminal sialic acid. These lectins did not react with polymers lacking oligosaccharides, as expected. Similarly, viruses that bound to the sialylglycopolymers did not bind to those treated with the sialidase or the polymer backbone without oligosaccharides.
    To identify mutations capable of conferring SA2,6Gal recognition, we focused on the three viruses that bound to the human receptor analogue relatively well: VN/3028IIcl3, Thai/KAN and VN/30408cl7. Comparison of the HA sequences identified two amino acid differences at positions 192 and 223 between VN/3028IIcl3 and VN1194 (a human clade-1 isolate, whose HA is identical to that of avian viruses such as A/duck/Thailand/71.1/2004 and A/chicken/Thailand/9.1/2004 and whose HA structure we determined by X-ray crystallography; see below). Introduction of the Gln192Arg mutation, but not the Ser223Asn mutation, into the HA of VN1194 appreciably enhanced the capacity of the HA to recognize SA2,6Gal, and introduction of both mutations increased the binding capacity further. This finding implicates Gln192Arg as a possible determinant of the shift to recognition of the human receptor by VN/3028IIcl3 (Fig. 2a). The Thai/KAN virus also showed two amino acid changes in HA1 as compared with VN1194: Gly139Arg and Asn182Lys. Introduction of either mutation into the VN1194 HA enhanced SA2,6Gal binding (Fig. 2b), and an additional increase in binding capacity was observed when both mutations were substituted simultaneously. Thus, both Gly139Arg and Asn182Lys seem to contribute to recognition of the human-type receptor.
    Figure 2: Effect of HA mutations on the host cell receptor preference of the VN1194HA.

    Shown is the effect of HA mutations found in VN/3028IIcl3 (a) and Thai/KAN (b) viruses. The mutations (in parentheses) were introduced first singly and then in combination. The HAs of the viruses possessing double mutations, VN1194(Q192R,S223N) in a and VN1194(G139R,N182K) in b, are identical to those of VN/3028IIcl3 and Thai/KAN, respectively. Data are the mean s.d. of triplicate experiments.
    High resolution image and legend (156K)<!-- -->

    The HA of VN/30408cl7 differed from that of VN1194 by four amino acids at positions 75, 123 and 193 in HA1 and 167 in HA2. When introduced singly into the VN1194 HA, none of these amino acid substitutions enhanced binding to SA2,6Gal, with the exception of Asn193Lys, which increased binding capacity slightly (Supplementary Fig. 4a). Different pairs of these amino acid residues substituted at positions 75, 123 and 193 (in HA1) and 167 (in HA2) produced variable increases in SA2,6Gal recognition (Supplementary Fig. 4b), and the introduction of various combinations of three mutations into the VN1194 HA enhanced SA2,6Gal binding even further (Supplementary Fig. 4c). These results suggest that two or more of these changes acting in concert are necessary for SA2,6Gal recognition by the VN/30408cl7 HA.
    A group of H5N1 viruses (clade 2) that are antigenically and genetically distinct from previously circulating viruses (clade 1) have become prevalent<SUP minmax_bound="true">1</SUP> (Supplementary Fig. 1). As it was unclear whether mutations that conferred SA2,6Gal recognition to the HA of clade-1 VN1194 virus would act comparably in the HAs of viruses of different clades, we inserted the Gly139Arg, Asn182Lys, Gln192Arg and Asn193Lys mutations separately into the HA of a clade-2 chicken isolate, A/chicken/Indonesia/N1/05 (CkInd). Either Gln192Arg or Asn193Lys, but not Gly139Arg, enhanced the SA2,6Gal-binding affinity of CkInd to an extent similar to that observed for the HA of VN1194 (Fig. 3). Introduction of Asn182Lys into the CkInd HA nearly abolished its binding to both SA2,3Gal and SA2,6Gal molecules, indicating the incompatibility of lysine at this position in the CkInd HA.
    Figure 3: Effect of mutations responsible for SA2,6Gal recognition by clade-1 HAs on a clade-2 HA.

    Mutations responsible for SA2,6Gal recognition by clade-1 HAs (in parentheses) were introduced into the HA of CkInd. The direct binding of each mutant virus to sialylglycopolymers containing either 2,3-linked (blue) or 2,6-linked (red) sialic acids was then measured at different concentrations of sialylglycopolymer. Data are the mean s.d. of triplicate experiments.
    High resolution image and legend (68K)<!-- -->

    To address the structural basis for the acquisition of human receptor-binding capacity by the H5 HA, we determined the crystal structure of the VN1194 virus. The structure was solved by molecular replacement using coordinates from the A/duck/Singapore/95 HA (relevant crystallographic statistics are given in Supplementary Table 2). The backbone structure of the VN1194 HA is shown in Fig. 4a, superimposed on the backbone of the HA of A/duck/Singapore/95. The two structures are very similar (root mean-square deviation (r.m.s.d.) on all C positions, 0.46 Å), reflecting their 94% sequence identity. Figure 4b shows the receptor-binding domain, located on the globular head of the HA (Fig. 4a), of VN1194 superimposed on that of A/duck/Singapore/95 (ref. 9) and A/Vietnam/1203/04 (VN1203; ref. 10), which overlap with an r.m.s.d. of 0.46 Å and 0.5 Å, respectively, on the 175 C positions of this domain. The overlap of the structure of the interhelical loop region of the HA2 of VN1194 and A/duck/Singapore/95 is shown in Fig. 4d. Again, the close correspondence of these structures presumably reflects their very high sequence identity. We note that the highly similar overall domain arrangement of the H5 HAs of VN1194 and A/duck/Singapore/95 (Fig. 4a) differs from the arrangement reported for VN1203 (ref. 10). Further studies are needed to understand the basis of these differences.
    Figure 4: Crystal structure of VN1194 H5 HA and the location of mutations conferring SA2,6Gal-binding capacity.

    a, Monomer from the crystal structure of the H5 HA from VN1194 (blue) determined at 2.8 Å, superimposed with a monomer of the H5 HA from A/duck/Singapore/95 (green). The mutations discussed in the text are indicated: residues in red represent positions where single substitutions affect receptor binding; those in yellow increase SA2,6Gal binding when introduced in combination with other changes. Asn 223 (blue) increases SA2,6Gal recognition in the presence of Arg 192. b, Superposition of the closely related receptor-binding domains of VN1194 (blue), A/duck/Singapore/95 (green) and VN1203 (grey) with the avian receptor analogue taken from the A/duck/Singapore/95 complex structure. The main secondary structure elements of the binding site (130-loop, 220-loop and 190-helix) and key binding site residues are indicated. c, More detailed view of the receptor-binding domain of the H5 HA of VN1194 with the human receptor analogue docked into the structure from its complex with the H1 HA. Three of the mutations discussed in the text are modelled in red: Asn182Lys, Ser223Asn and Gln192Arg. Note that the residue numbering differs in the deposited coordinate file; for example, Asn 182 here is numbered 186 in the PDB file. d, Close up of the overlap between the interhelical regions of domain F of VN1194 (blue) and A/duck/Singapore/95 (green) and the orientation of the hydrophobic residue Phe 63 (HA2).
    High resolution image and legend (85K)<!-- -->

    Residues 75 in HA1 and 167 in HA2 are distant from the receptor-binding site, and the orientation of residues 139 and 193 precludes receptor contact (Fig. 4a). Clearly, further studies will be required to investigate the role of these residues. However, residues 182 and 192 are located at positions in the structure where it is feasible for them to make stabilizing interactions with sugars joined to sialic acid by 2,6 linkages (Fig. 4a, c). Mutations at residue 182 (the equivalent residue in the H3 HA is 186) have been linked to changes in receptor specificity<SUP minmax_bound="true">9, </SUP><SUP minmax_bound="true">11, </SUP><SUP minmax_bound="true">12, </SUP><SUP minmax_bound="true">13</SUP>, and mutating Asn 182 in silico to a lysine residue leads to a potential hydrogen-bond interaction with the 2-OH of Gal-2 (Fig. 4c). Mutating Gln 192 (196 in the H3 HA) to an arginine residue with selection of a preferred rotamer (from the 'O' database)<SUP minmax_bound="true">14</SUP> generates a conformation that places one of the guanidinium nitrogen atoms 4.5 Å from the 2-OH of Glc-5 (Fig. 4c); thus, the mutant HA may be capable of forming a hydrogen bond with human receptor moieties.
    Serine 123 (128 in the H3 HA) is located on the turn leading into the 130-loop that forms the front edge of the binding site (Fig. 4b, c). Mutation of this residue could alter the orientation of the 130-loop and thus the attachment angle between the sialic acid and the next residue in the polysaccharide chain, thereby influencing the preference for the 2,3 or 2,6 linkage. Ser 223 is located close to the sialic-acid-binding site and in silico substitution of this residue with an asparagine leads to a conformation that places its side-chain nitrogen about 4 Å from the 3-OH of Gal-2 (Fig. 4c). This observation suggests that the mutant HA, with an asparagine at position 223, may be able to form a hydrogen bond with Gal-2 and thus influence SA2,6Gal recognition, although this amino acid substitution only slightly increases the SA2,6Gal-binding capacity of the VN1194 HA (Fig. 2a).
    The influenza A viruses responsible for the pandemics of 1918, 1957 and 1968 all derived their HAs from avian viruses, which typically recognize SA2,3Gal (ref. 3). Yet, the HAs of early isolates from humans infected in these pandemics seem to have recognized SA2,6Gal in preference to SA2,3Gal (ref. 3), suggesting that conversion of the avian HA to one that can recognize SA2,6Gal-terminated polysaccharides on host cells is an important step in the generation of pandemic strains. This concept is supported by studies on the distribution of influenza virus receptors in the human airway<SUP minmax_bound="true">15, </SUP><SUP minmax_bound="true">16</SUP>. The critical amino acid substitutions involved in this shift of receptor recognition were residues 226 and 228 in the H2 and H3 HAs<SUP minmax_bound="true">17</SUP> (equivalent to residues 222 and 224 in the H5 HA). The introduction of these mutations into the H5 HA permitted its binding to an 2,6 glycan<SUP minmax_bound="true">10</SUP>, although neither change has been found in the HAs of H5N1 viruses isolated from humans.
    In our study, both single and combined amino acid substitutions in the avian H5 HA mediated a shift to SA2,6Gal recognition. Moreover, two of these changes, lysine at position 182 and arginine at position 192, were present in the HAs of clade-2 H5N1 viruses isolated from two individuals in Azerbaijan and one individual in Iraq, but not in any of the more than 600 avian isolates examined. Although amino acid substitutions in viral proteins other than the HA, including PB2 (refs 18–20), may be needed to confer full pandemic status to an avian virus efficiently replicating in humans, the amino acid residues identified here may be selected during an early phase of human infection involving cells of the upper respiratory tract. Thus, such residues might provide useful molecular markers in assessments of H5N1 field isolates for their capacity to replicate in humans—an essential indicator of pandemic potential.
    Top of page Methods

    Virus preparation

    Viruses were grown in MDCK cells, which were maintained in minimal essential medium supplemented with 5% newborn calf serum (Sigma) and antibiotics at 37 °C in 5% CO<SUB minmax_bound="true">2</SUB>. All experiments with live H5N1 virus were done in a biosafety level-3 containment laboratory.

    Generation of viruses by reverse genetics

    Reassortant viruses were generated with a plasmid-based reverse genetics system<SUP minmax_bound="true">8</SUP>. The viral complementary DNAs were cloned into a plasmid under control of the human polymerase I promoter and the mouse RNA polymerase I terminator (referred to as PolI plasmids). All viruses generated by reverse genetics possessed the neuraminidase of VN1194 and the internal genes of PR8. To generate the various HAs of human isolates registered in the Influenza Sequence Database (https://www.flu.lanl.gov/), we introduced mutations into pPolI–VN1194HA. Each construct was sequenced to verify the absence of unwanted mutations.

    Receptor specificity assays

    By using a solid-phase binding assay with the sodium salts of sialylglycopolymers (poly -l-glutamic acid backbones containing N-acetylneuraminic acid linked to galactose through either an -2,3 (Neu5Ac2,3Gal1,4GlcNAc-pAP) or an -2,6 (Neu5Ac2,6Gal1,4GlcNAc-pAP) bond) as described<SUP minmax_bound="true">7, </SUP><SUP minmax_bound="true">21, </SUP><SUP minmax_bound="true">22</SUP>, we determined the direct binding capacity of the viruses for the sialylglycopolymers. In brief, polystyrene Universal-Bind microplates (Corning) were incubated with either of the two glycopolymers in PBS at 4 °C for 3 h, and then irradiated under ultraviolet light at 254 nm for 2 min. After removal of the glycopolymer solution, the plates were blocked with 0.1 ml of PBS containing 2% bovine serum albumin (Invitrogen) at room temperature for 1 h. After five washes with PBS, the plates were incubated in a solution containing influenza virus (128 haemagglutination units in PBS) at 4 °C for 12 h. After three washes with PBS, antibody to the virus was added to the plates. After 2 h of incubation at 4 °C, the plates were washed three times with ice-cold PBS and then incubated with horseradish peroxidase (HRP)-conjugated protein A (2000-fold dilution in PBS; Organon Teknika–Cappel) at 4 °C. After four washes with ice-cold PBS, the plates were incubated with O-phenylenediamine (Sigma) in PBS containing 0.01% H<SUB minmax_bound="true">2</SUB>O<SUB minmax_bound="true">2</SUB> for 10 min at room temperature, and the reaction was stopped with 0.05 ml of 1 M HCl. Absorbance was determined at 490 nm.
    To confirm the specificity of the assay, we carried out control experiments as follows. In brief, the glycopolymers were fixed on the plates as described above, 120 l of A. ureafaciens sialidase (80 mU ml<SUP minmax_bound="true">-1</SUP>; Nacalai Tesque) was added and the plates were incubated at 37 °C for 16 h. After three washes with PBS, the plates were blocked with 0.1 ml of PBS containing 1% bovine serum albumin at 4 °C for 16 h. Removal of sialic acids from sialylglycopolymers was confirmed by incubating them with 50 l of biotinylated SNA specific for SA2,6Gal, biotinylated MALII specific for SA2,3Gal, or biotinylated MALI specific for both Gal1-4GlcNAc and SA2,3Gal1-4GlcNAc (all Vector Laboratories), at room temperature for 1 h. After three washes with PBS, the plates were incubated with HRP-conjugated streptavidin (Vector Laboratories) at room temperature for 1 h. After four washes with PBS, the plates were incubated with O-phenylenediamine in PBS containing 0.01% H<SUB minmax_bound="true">2</SUB>O<SUB minmax_bound="true">2</SUB> for 10 min at room temperature, and the reaction was stopped with 0.05 ml of 1 M HCl. Absorbance was determined at 490 nm. Plates containing sialidase-treated glycopolymers and those containing poly -l-glutamic acid backbones were used to confirm the lack of virus binding to the asialoglycopolymers or poly -l-glutamic acid backbones.

    Crystal structure determination

    The H5N1 virus NIBRG-14, modified from A/Vietnam/1194/04 by removal of the polybasic cleavage site in HA, was obtained from the National Institute for Biological Standards and Control. HA was prepared by bromelain digestion of purified egg-grown virus for 90 min at 34 °C in 10 mM Tris-HCl (pH 8.0) and 5 mM mercaptoethanol (virus protein/bromelain ratio 10/1 w/w). The HA was purified as described<SUP minmax_bound="true">23</SUP>. Crystallization conditions were screened by the sitting-drop vapour diffusion method using Crystal Clear strips (Douglas Instruments). The nanodrops were set up with 0.1 l of H5 protein solution (10 mg ml<SUP minmax_bound="true">-1</SUP>) and 0.1 l of reservoir solution by using an Oryx1-6 robot (Douglas Instruments). Diffracting crystals were obtained from Index solution 57 (Hampton Research): namely, 30% (v/v) pentaerythritol ethoxylate, 50 mM ammonium sulphate and 50 mM Bis-Tris (pH 6.5). This solution also acted as a cryoprotectant. The H5 diffraction data were recorded on a Raxis4 detector (100-m scan) mounted on a Rigaku MicroMax 007 HF generator, integrated with Denzo and scaled with Scalepack<SUP minmax_bound="true">24</SUP>. The structure was solved by molecular replacement using AmoRe<SUP minmax_bound="true">25</SUP> using the PDB file 1JSM (ref. 9) as the initial search model. Standard refinement was carried out with a combination of refmac5 (ref. 25) and CNS<SUP minmax_bound="true">26</SUP>, together with manual model building with O<SUP minmax_bound="true">12</SUP>. Molecular figures were created with Pymol (http://pymol.sourceforge.net/).



    Top of page Acknowledgements

    We thank K. Wells for technical assistance, and J. Gilbert for editing the manuscript. The NIMR contributors were responsible for the structural studies and for HA sequencing. This work was supported by CREST (Japan Science and Technology Agency); by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan; by the Ministry of Health, Labour and Welfare, Japan; and by grants from the NIH, NIAID. Structural studies were supported by the UK MRC and by an International Partnership Research Award in Veterinary Epidemiology of the Wellcome Trust. Author Contributions S.Y., Y.S., T.S., M.Q.L., C.A.N., Y.S.T., Y.M., T.H., T.S., M.K, T.U., T.M., Y.L., A.H. and Y.K. were responsible for the virological studies. L.F.H., D.J.S., R.J.R., S.J.G. and J.J.S. were responsible for the structural studies.
    Competing interests statement:

    The authors declared no competing interests.
    Supplementary information accompanies this paper.

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    1. <LI id=a1 minmax_bound="true">Division of Virology, Department of Microbiology and Immunology, and, <LI id=a2 minmax_bound="true">International Research Centre for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan <LI id=a3 minmax_bound="true">Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan <LI id=a4 minmax_bound="true">College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501, Japan <LI id=a5 minmax_bound="true">Department of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences and COE Program in the 21st Century, Yada, Shizuoka 422-8526, Japan <LI id=a6 minmax_bound="true">National Institute of Hygiene and Epidemiology, Hanoi, Vietnam <LI id=a7 minmax_bound="true">Avian Influenza Laboratory, Tropical Disease Centre, Airlangga University, Surabaya, Indonesia <LI id=a8 minmax_bound="true">The Avian Zoonosis Research Centre, Tottori University, Tottori 680-8553, Japan <LI id=a9 minmax_bound="true">Department of Applied Bioorganic Chemistry, The United Graduate School of Agricultural Science, Gifu University, Yanagido, Gifu 501-1193, Japan <LI id=a10 minmax_bound="true">Department of Applied Biological Chemistry, Shizuoka University, Shizuoka 422-8529, Japan <LI id=a11 minmax_bound="true">MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK <LI id=a12 minmax_bound="true">Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA
    2. Present address: Centre for Biomolecular Sciences, University of St Andrews, St Andrews KY16 9ST, UK.
    Correspondence to: Yoshihiro Kawaoka<SUP minmax_bound="true">1,</SUP><SUP minmax_bound="true">2,</SUP><SUP minmax_bound="true">3,</SUP><SUP minmax_bound="true">12</SUP> Correspondence and requests for materials should be addressed to Y.K. (Email: kawaoka@ims.u-tokyo.ac.jp). Coordinates for the H5 structure have been deposited in the Protein Data Bank under accession code 2IBX.

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  • #2
    Re: Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to h

    Editor's Summary

    16 November 2006
    Pandemic potential

    <HR class=separator minmax_bound="true">The fact that the H5N1 bird flu virus circulating in Asia, Europe and Africa is unable to attach to human-type cell receptors has helped to prevent it from causing a worldwide epidemic of a human variant of the disease. Now a study of H5N1 isolates from some of the few humans that have been infected (from Vietnam and Thailand) has identified two mutations in a viral haemagglutinin that allow it to bind to both human and avian receptors. These mutations might be of use as molecular markers for assessing the pandemic potential of H5N1 field isolates.
    Letter: Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors

    Shinya Yamada, Yasuo Suzuki, Takashi Suzuki, Mai Q. Le, Chairul A. Nidom, Yuko Sakai-Tagawa, Yukiko Muramoto, Mutsumi Ito, Maki Kiso, Taisuke Horimoto, Kyoko Shinya, Toshihiko Sawada, Makoto Kiso, Taiichi Usui, Takeomi Murata, Yipu Lin, Alan Hay, Lesley F. Haire, David J. Stevens, Rupert J. Russell, Steven J. Gamblin, John J. Skehel and Yoshihiro Kawaoka
    <ABBR title="Digital Object Identifier" minmax_bound="true">doi</ABBR minmax_bound="true">:10.1038/nature05264

    Comment


    • #3
      Re: Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to h

      Is this the article related to that ?

      ___________________________

      http://today.reuters.co.uk/news/arti...h-C3-Science-2

      Scientists identify key mutations in bird flu virus

      Wed Nov 15, 2006 7:00 PM GMT
      <INPUT id=CurrentSize type=hidden value=13 name=CurrentSize> Email This Article | Print This Article | RSS

      [-] Text [+]

      HONG KONG (Reuters) - Scientists have discovered two spots on the H5N1 bird flu virus which would need to mutate for the virus to infect people more easily.
      H5N1 is largely a disease in animals because it has surface proteins which bind more easily to "receptors" lining respiratory tracts of birds, rather than receptors in humans.
      Experts fear H5N1 will infect more humans and trigger a pandemic killing millions of people if the virus mutates to attach easily to human receptors.


      In the latest issue of Nature, scientists in Japan, Britain and the United States said they had discovered two specific spots on the genes of H5N1 which appeared to determine if the virus attached more easily to bird or human receptors.


      This discovery will help scientists decide if any strain of the H5N1 has the potential to cause a human pandemic.
      "The bottom line is that the changes (on the two spots) can be used as molecular markers to identify the potential of the viruses that may grow well in humans," said Yoshihiro Kawaoka of the Institute of Medical Science at the University of Tokyo.
      Using 21 samples of the H5N1 virus taken from human victims in Indonesia and Vietnam, the team of scientists found that three of them bound especially easily to human receptors.
      "We found many mutations and we tried to identify which mutations were important ... two appeared to be very important," Kawaoka told Reuters by telephone from the United States.
      Kawaoka warned against any over-emphasis on these two spots.
      "It is very important that we shouldn't only focus on these two. The virus can become human-like by many mutations, these two are important but they are not the only ones," he said.
      "But these two will give indication when a virus has changed receptor specificity."

      &#169; Reuters 2006. All Rights Reserved. | Learn more about Reuters

      Comment


      • #4
        G143R N186K Q196R S227N

        The key players (using H3 numbering) are G143R, N186R, Q196R, S227N.

        Comment


        • #5
          Re: Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses

          I don't think this is at all what Niman's talking about when he refers to Egypt having Influenza B polymorphisms in it's H5N1 structure.

          These guys are talking about something else.

          Comment


          • #6
            Re: Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses

            Supplementary Table 1| H5N1 viruses used in this study
            <TABLE class=MsoNormalTable style="MARGIN: auto 6.75pt; BORDER-COLLAPSE: collapse; mso-table-layout-alt: fixed; mso-table-lspace: 9.0pt; mso-table-rspace: 9.0pt; mso-table-anchor-vertical: margin; mso-table-anchor-horizontal: page; mso-table-left: 170.1pt; mso-table-top: 19.7pt; mso-padding-alt: 0in 4.95pt 0in 4.95pt" cellSpacing=0 cellPadding=0 align=left border=0><TBODY><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 0; mso-yfti-firstrow: yes"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=227>
            Virus strain<SUP></SUP>
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            Clade
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>
            Amino acid residues that differ from those in VN1194
            </TD></TR><TR style="HEIGHT: 12.95pt; mso-yfti-irow: 1"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 12.95pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt" width=67>
            Isolates
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 12.95pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132>Vietnam 2004
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 12.95pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt" noWrap width=227>A/Vietnam/1194/04 (RG)
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 12.95pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 12.95pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=416> 
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 2"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
            from
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/1203/04 (RG)
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>T36K
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 3"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
            humans
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/3046/04 (RG)
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>A134V,I198V
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 4"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> >
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Hatay/2004 (RG)
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>D43A,T188I,I256L,N154S(HA2)
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 5"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/30259/04
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>S123P,D128N(HA2),R167K(HA2)
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 6"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/3030/04
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>P74L,V175M,V210A>
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 7"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/3035/04>
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>I45L(HA2)
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 8"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/3047III/04
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>N182D,D183N,I198V
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 9"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/30262III/04 clone2
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>A127P
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 10"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/30262III/04 clone8
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>N50H(HA2)
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 11"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/3028II/04 clone1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>A134V,N244S
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 12"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/3028II/04 clone2
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>A134T
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 13"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/3028II/04 clone3
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>Q192R,S223N>
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 14"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132>Thailand 2004
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt" noWrap width=227>A/Thailand/2-SP-33/04 (RG)†
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>A127V
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 15"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Thailand/1-KAN-1/04 (RG)‡
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>G139R,N182K
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 16"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-alt: solid windowtext .5pt" noWrap width=132>Cambodia 2005
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=227>A/Cambodia/JP52a/05 (RG)
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=416>P136L,K140Q,L175M,T188I
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 17"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132>Vietnam 2005
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/JPHN30321/05 (RG)
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>R53K,N84D,D94N,K140R,L175M,K189R,V219I,D57N(HA2)
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 18"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/30408/05 clone7
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>E75K,S123P,N193K,R167K(HA2)
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 19"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/Vietnam/30408/05 clone9
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>S123L,R167K(HA2)
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 20"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=227>A/Vietnam/30408/05 clone10
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=416>S123P, Q169R,R167K(HA2)
            </TD></TR><TR style="HEIGHT: 32.8pt; mso-yfti-irow: 21"><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 32.8pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 32.8pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 32.8pt; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=227>A/Vietnam/30850/05
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 32.8pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=48>
            2
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 32.8pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" width=416>V86A,D94N,S124D,L129S,K140T,S141P,S155N,V174I,P181 S,
            R212K,E227D,T263A,L269V,M282I,R310K,Q322L,I183T(HA 2)
            </TD></TR><TR style="HEIGHT: 13.75pt; mso-yfti-irow: 22"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 13.75pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 13.75pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=132>Vietnam 2004
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 13.75pt; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=227>A/chicken/Vietnam/G04/04
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 13.75pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 13.75pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" width=416>K140R
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 23"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132>Vietnam 2005
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent" noWrap width=227>A/duck/Vietnam/5001/05
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt" noWrap width=416>S123P,S124N,R167K(HA2)
            </TD></TR><TR style="HEIGHT: 11.45pt; mso-yfti-irow: 24"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" noWrap width=67>
            Isolates
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=227>A/duck/Vietnam/5003/05
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=48>
            1
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 11.45pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=416>S123P,K140R,V210I,R167K(HA2)
            </TD></TR><TR style="HEIGHT: 31pt; mso-yfti-irow: 25"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: #d4d0c8; HEIGHT: 31pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt" vAlign=top width=67>
            from poultry
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 31pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=132> 
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 31pt; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=227>A/chicken/Vietnam/TY31/05
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 31pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=48>
            2
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 31pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" width=416>K35R,R53K,V86A,V87Y,D94N,S124D,K140N,S155D,T156A,
            A184E,K189R,I198V,V200I,R212K,M226I,E227D,A249T,T2 63A,L269V,R310K,I183T(HA2),A198V(HA2)
            </TD></TR><TR style="HEIGHT: 25.95pt; mso-yfti-irow: 26; mso-yfti-lastrow: yes"><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 50.5pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 25.95pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=67>
             
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 99.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 25.95pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=132>Indonesia 2005
            </TD><TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 170.35pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 25.95pt; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=227>A/chicken/Indonesia/N1/05
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: windowtext 1pt solid; WIDTH: 35.65pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 25.95pt; BACKGROUND-COLOR: transparent; mso-border-left-alt: solid windowtext .5pt; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" noWrap width=48>
            2
            </TD><TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 4.95pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 4.95pt; PADDING-BOTTOM: 0in; BORDER-LEFT: #d4d0c8; WIDTH: 312.2pt; PADDING-TOP: 0in; BORDER-BOTTOM: windowtext 1pt solid; HEIGHT: 25.95pt; BACKGROUND-COLOR: transparent; mso-border-right-alt: solid windowtext .5pt; mso-border-bottom-alt: solid windowtext .5pt" width=416>V86A,D94N,S124D,L129S,Q138L,K140T,S141P,K189R,R212 K,
            T263A,R127K(HA2), N146D(HA2),D158N(HA2),V203M(HA2)>
            </TD></TR></TBODY></TABLE><SUP></SUP>Viruses generated by reverse genetics are labelled “RG”.
            †A/Thailand/2-SP-33/04 HA differs from A/Vietnam/1194/04 HA by three amino acids (A127 in HA1, and D37A and R127K in HA2). Introduction of the two mutations in the HA2 prohibited virus generation, so a virus possessing the A/Vietnam/1194/04 HA containing only the A127A mutation was generated.
            A/Thailand/1-KAN-1/04 HA differs from A/Vietnam/1194/04 HA by four amino acids (G139R and N182K in the HA1 and Y24T and H25T in HA2). Introduction of the two mutations in the HA2 prohibited virus generation, so a virus possessing the A/Vietnam/1194/04 HA with only HA1 mutations (i.e., G139R and N182K) was generated.


            Add 4 to each number to get H3 numbering
            Last edited by Sally Furniss; November 15th, 2006, 05:51 PM.

            Comment


            • #7
              MDCK Cells

              Note that all H5N1 was isolated in MDCK cells dog (as in MAMMAL) kidney cells.

              Comment


              • #8
                Re: MDCK Cells

                This is an important paper.
                21st Century Omega Man

                Comment


                • #9
                  Re: Nature - HA mutations responsible for the binding of H5N1 A viruses

                  I believe this is a related story....
                  http://www.webmd.com/content/article/129/117539.htm

                  Two Threatening Bird Flu Mutations

                  Scientists ID Bird Flu Mutations Likely to Trigger Human Pandemic

                  <TABLE cellSpacing=0 cellPadding=0 width="90%" border=0><TBODY><TR><TD vAlign=top width="45%">By Daniel DeNoon
                  WebMD Medical News </TD><TD vAlign=top width="55%">Reviewed By Louise Chang, MD
                  on Wednesday, November 15, 2006 </TD></TR></TBODY></TABLE>


                  Nov. 15, 2006 - Either of two simple bird flu virus mutations could trigger a deadly pandemic, Japanese scientists warn.

                  Both mutations already have popped up in humans infected with the H5N1 bird fluflu virus.

                  They've been seen in bird flu viruses isolated from two people in Azerbaijan and from one person in Iraq, according to the Japanese scientists. Neither mutation has been seen among the more than 600 H5N1 viruses isolated from birds.

                  The two human mutations give the bird flu virus the ability to attach to human cells. It's the kind of mutation seen early in the 1918, 1957, and 1968 flu pandemics, warn Shinya Yamada of the University of Tokyo and colleagues.

                  Fortunately, the H5N1 viruses carrying these mutations do not appear to have caused any outbreaks of human-to-human transmission.

                  But these mutants seem capable of replicating in humans -- "an essential indicator of pandemic potential," the researchers report.

                  Flu viruses attach to receptor molecules on the outside of cells that line the airway.

                  Bird flu viruses use a receptor called SAalpha2,3Gal. Human flu viruses use a closely related receptor called SAalpha2,6Gal.

                  Previous flu pandemics came from bird flu viruses. Each time, the pandemic took off when the viruses learned to attach to human airway cells.

                  Yamada and colleagues manipulated H5N1 viruses in the laboratory to see what it would take to make a bird flu virus do this. They found that either of two mutations -- single amino-acid changes at specific places in the viral DNA -- did the trick.

                  The researchers suggest that health authorities look for these mutations in bird flu viruses isolated from humans. If found, they could be an early warning of a budding pandemic.

                  Yamada and colleagues report their findings in the Nov. 16 issue of the journal Nature.
                  <HR>SOURCE: Yamada, S. Nature, Nov. 16, 2006; vol 444: pp 378-382.
                  "The next major advancement in the health of American people will be determined by what the individual is willing to do for himself"-- John Knowles, Former President of the Rockefeller Foundation

                  Comment


                  • #10
                    VN/JP12-2/05

                    Ealier, VN/JP12-2/05, was said to have S227N, so now it has been identified in two patients from Vietnam, two from Hong Kong, two from Turkey, and one from Egypt.

                    Comment


                    • #11
                      Re: MDCK Cells

                      Originally posted by Gnosis
                      This is an important paper.
                      The 182 change (which corresponds to 186 in H3 numbering) matches the receptor binding domain of Influenza B.

                      Comment


                      • #12
                        Cumlative Effect

                        Henry – Please comment in lay-persons terms the cumulative effect of the following information released in the last week:<o:p></o:p>
                        <o:p> </o:p>
                        “…The acquisition of the mammalian polymorphism, M230I, produced a match with the sequence adjacent to the human receptor binding domain of influenza A (H3N2 and H1N1) and influenza B. The change creates identity between positions 226-230 (QSGRI) in the receptor binding domain of influenza B…”<o:p></o:p>
                        <o:p> </o:p>
                        “….Now a study of H5N1 isolates from some of the few humans that have been infected (from <st1:country-region><st1:place>Vietnam</st1:place></st1:country-region> and <st1:country-region><st1:place>Thailand</st1:place></st1:country-region>) has identified two mutations in a viral haemagglutinin that allow it to bind to both human and avian receptors. These mutations might be of use as molecular markers for assessing the pandemic potential of H5N1 field isolates….”<o:p></o:p>
                        <o:p> </o:p>
                        “…Today, sequences from an isolate from Shanxi were released. As indicated in the quote above, this isolate was distinct from the <st1:State><st1:place>Fujian</st1:place></st1:State> or <st1:City><st1:place>Shantou</st1:place></st1:City> strains described above. In addition to a novel HA cleavage site, REGRRKKR, the isolate also had a number of changes in the receptor binding domain, A188E, A189T, T192I, L194I, R220K, K222E. Moreover, two related sequences from Hunan had two of the changes found in the <st1:State><st1:place>Shanxi</st1:place></st1:State> isolate, T192I and L194I as well as two more changes, D187N and A189E….”<o:p></o:p>
                        <o:p> </o:p>
                        “…The recent H5N1 sequences from China include four sequences from geese in Shantou (A/goose/Shantou/2086/2006, A/goose/Shantou/239/2006, A/goose/Shantou/2104/2006, A/goose/Shantou/7775/2006) that have four non-synonomous changes in or near the receptor binding domain (V214M, K222R, V223I, and S227R). Changes at position 227 (S227N) have been associated with increased affinity for human 2,6 receptors and decreased affinity for avian 2,3 receptors…”<o:p></o:p>
                        <o:p> </o:p>
                        “..HA sequences from 8 human H5N1 isolates from <st1:country-region><st1:place>Egypt</st1:place></st1:country-region> were released today by the US Naval Medical Research Unit 3 in <st1:City><st1:place>Cairo</st1:place></st1:City> (see list here). These sequences were from the H5N1 outbreak in the spring. Previously, one human sequence from a mild case in the spring, as well as a fatal case who died this month have been released. The recent case has a change, M230I, near the receptor binding domain…”<o:p></o:p>
                        <o:p> </o:p>
                        To the non-geneticist this appears that the chances for H5N1, or a derivative thereof, to become "pandemic" has increased.<o:p></o:p>
                        <o:p> </o:p>
                        "May the long time sun
                        Shine upon you,
                        All love surround you,
                        And the pure light within you
                        Guide your way on."

                        "Where your talents and the needs of the world cross, lies your calling."
                        Aristotle

                        “In a gentle way, you can shake the world.”
                        Mohandas Gandhi

                        Be the light that is within.

                        Comment


                        • #13
                          Re: Nature - HA mutations responsible for the binding of H5N1 A viruses

                          Bloomberg's take with Osterhaus & Palese interviews....
                          (with Bloomberg's usual easily-understood writing)
                          http://www.bloomberg.com/apps/news?p...vQ&refer=japan

                          Two Bird Flu Gene Mutations Might Lead to Faster Human Spread

                          By John Lauerman
                          Nov. 15 (Bloomberg) -- Two mutations in a gene of the bird flu virus may have put it on the path to becoming more contagious among humans, scientists said.

                          The gene controls how the virus attaches to cells in breathing passages, said researchers led by Shinya Yamada, a University of Tokyo virologist who led the research, published today in the journal Nature. The gene changes allow the virus to attach more easily to surface molecules, called receptors, on human cells, the researchers said.

                          Yamada's team found the mutations in strains of the virus taken from chickens and people in 2004, 2005 and early 2006 that didn't cause widespread human outbreaks of flu. The bird virus, called H5N1, will have to undergo more adaptation to start spreading quickly in people, said study co-author John Skehel, retired director of the National Institute for Medical Research in London.

                          ``We need to follow these mutations and see whether they come up again in other viruses isolated from humans,'' Skehel said yesterday in a telephone interview. ``That might give some indication as to whether they were involved in transfer of the virus to humans.''

                          The researchers analyzed variations in the H5N1 hemagglutinin gene, which makes a protein that allows the virus to enter cells and is responsible for its most lethal effects. The protein fuses the influenza virus to the surface of the cell, rips open the cell wall and allows the viral genetic material to flow in and take over the cell.

                          Pandemics

                          Most H5N1 viruses prefer to bind to a bird receptor molecule. The U.K. and Japan researchers found that the mutations at two places in the gene, identified as 182 and 192, allow the virus to bind to both bird and human receptors.

                          Human flu viruses that caused pandemics in 1957 and 1968 also differed from closely related bird viruses at just two points on a similar gene, Skehel said. More study is needed to determine how much difference the changes in the H5N1 gene make in its ability to attack human cells, he said.

                          ``We know they can bind human receptors,'' Skehel said. ``The question is how well they can bind.''

                          Monitoring such hot spots in samples of H5N1 may help give early warning of a pandemic threat, said Albert Osterhaus, head of virology at the Erasmus Medical Center in Rotterdam.

                          ``It's definitely something to be on the lookout for,'' said Nov. 13 in a telephone interview. Still, he said, more genetic changes will be necessary to allow the virus to begin spreading in people.

                          ``The receptor usage is not the only thing,'' Osterhaus said. ``Probably more than that needs to happen for the virus to become pandemic.''

                          Infections

                          H5N1 has infected 258 people and killed 153 of them since late 2003, the World Health Organization said Nov. 13. The virus might kill millions worldwide if it gains the ability to spread quickly in people, like the virus that caused the 1918 pandemic that killed as many as 50 million people.

                          Almost half the total human deaths from H5N1, 75, have occurred this year alone. Still, researchers have said there is no evidence the virus has become contagious among people. That makes it difficult to know whether the mutations found by the Japan and U.K. researchers make a pandemic more likely, said Peter Palese, a microbiologist at Mount Sinai School of Medicine in New York who studies bird flu.

                          ``These viruses are mutating all the time and it's not clear they've gained any ability to be better transmitted,'' Palese said Nov. 13 in a telephone interview. ``These viruses have had a chance, and they haven't done it.''
                          To contact the reporter on this story: John Lauerman in Boston at

                          jlauerman@bloomberg.net .
                          Last Updated: November 15, 2006 13:07 EST
                          "The next major advancement in the health of American people will be determined by what the individual is willing to do for himself"-- John Knowles, Former President of the Rockefeller Foundation

                          Comment


                          • #14
                            Re: MDCK Cells

                            We note that they isolated many variants strains from each individuals samples. Each variant is isolated by a separate culture plate.

                            Typically, a sample is a mix of variant strains.

                            If they do not find a perticular mutation in a samples, it did'nt mean it's not there, remember the Turkish outbreak case.

                            Also, the two most importants mutations they identify (N186R, Q196R), where present in Azerbaijan & Iraq.

                            We have now an explanation of why thoses outbreak where so specials.

                            With S227N in turkey, we see how this region was near the border last year.

                            Note also the presence of Dr.Chairul A. Nidom<sup minmax_bound="true">7
                            we all remember his misterious comment about Indonesian receptor affinity, he probably have access to other non published data from the same serie of experiments.
                            </sup>
                            Last edited by Mingus; November 15th, 2006, 10:23 PM.

                            Comment


                            • #15
                              Re: Nature - HA mutations responsible for the binding of H5N1 A viruses

                              http://www.news.wisc.edu/13198.html





                              Scientists find mutations that let bird flu adapt to humans

                              November 15, 2006
                              by Terry Devitt


                              By comparing influenza viruses found in birds with those of the avian virus that have also infected human hosts, researchers have identified key genetic changes required for pandemic strains of bird flu.

                              Kawaoka



                              The new work, reported in the Nov. 16 issue of the journal Nature, illustrates the genetic changes required for the H5N1 avian influenza virus to adapt to easily recognize the receptors that are the gateway to human cells.


                              "We identified two changes that are important," says Yoshihiro Kawaoka, the senior author of the Nature paper and a virologist at the University of Wisconsin-Madison School of Veterinary Medicine. "Both changes are needed for the H5N1 virus to recognize human receptors."


                              The new report provides a molecular blueprint for the genetic changes required to transform a virus that only infects birds to a virus capable of easily recognizing human receptors. Receptors are molecules on the surface of cells that permit the virus to dock with the cell and commandeer it to initiate a cascade of infection. By knowing what genetic changes are required for the virus to easily infect human cells, it may be possible to detect the emergence of pandemic strains earlier, providing public health officials and vaccine manufacturers with precious time to prepare for a global outbreak of highly pathogenic influenza.


                              To be successful, a virus must be able to recognize and attach to a host cell. But human and avian influenza viruses recognize different cell receptors. Avian flu viruses have demonstrated an ability to evolve to easily infect humans by exchanging genes with human viruses that subsequently permit them to recognize human receptor molecules and gain easy access to cells, typically in the human respiratory system.


                              The change is thought to occur when human patients are exposed at the same time to a human flu virus and an avian flu virus. Most viruses, including influenza, readily swap genes with one another.


                              In the new study, conducted by an international team of researchers, the viruses isolated from human patients in Vietnam and Thailand could recognize both human and avian cell receptors. By contrast, the viruses found in chickens and ducks could recognize the receptors only on avian cells.


                              The work helps flesh out the changes that have occurred in the worrisome strain of avian influenza virus known as H5N1, a strain some fear could be the organism that will trigger a pandemic of virulent human influenza. The avian virus has already changed dramatically from when it was first identified in 1997, says Kawaoka, who also holds an appointment at the University of Tokyo.


                              "There are big differences between the virus first found in 1997 and the virus we see now," Kawaoka explains. "We are watching this virus turn itself into a human pathogen."


                              The mutations found by Kawaoka's group have not yet conferred a complete ability on avian flu to easily recognize the topography of human cells, but they are key steps on that pathway. More mutations, says Kawaoka, will be required for the virus to fully adapt to humans, but it is not known how many mutations are needed for such a change.


                              However, if scientists are able to continue to monitor and secure viral isolates from humans infected with bird flu, they may be able to map a mutation trajectory that will help predict when the avian virus will cross the threshold to become a human pathogen.


                              The last two flu pandemics in 1957 and 1968 were caused by avian viruses that had accumulated enough genetic mutations to be considered hybrids of animal and human viruses, Kawaoka notes.


                              The new work was supported by grants from the Japan Science and Technology Agency; the Japan Ministry of Education, Culture, Sports, Science and Technology; the Japan Ministry of Health Labor and Welfare; and the U.S. National Institutes of Health.
                              "We are in this breathing space before it happens. We do not know how long that breathing space is going to be. But, if we are not all organizing ourselves to get ready and to take action to prepare for a pandemic, then we are squandering an opportunity for our human security"- Dr. David Nabarro

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