Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
Structure and Receptor Specificity of the Hemagglutinin from an H5N1 Influenza Virus
James Stevens,1*Ola Blixt,1,2Terrence M. Tumpey,4Jeffery K. Taubenberger,5James C. Paulson,1,2Ian A. Wilson1,3*
The hemagglutinin (HA) structure at 2.9 angstrom resolution,from a highly pathogenic Vietnamese H5N1 influenza virus, ismore related to the 1918 and other human H1 HAs than to a 1997duck H5 HA. Glycan microarray analysis of this Viet04 HA revealsan avian 2-3 sialic acid receptor binding preference. Introductionof mutations that can convert H1 serotype HAs to human 2-6 receptorspecificity only enhanced or reduced affinity for avian-typereceptors. However, mutations that can convert avian H2 andH3 HAs to human receptor specificity, when inserted onto theViet04 H5 HA framework, permitted binding to a natural human2-6 glycan, which suggests a path for this H5N1 virus to gaina foothold in the human population.
1 Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 2 Glycan Array Synthesis Core-D, Consortium for Functional Glycomics, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 3 Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 4 Influenza Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. 5 Department of Molecular Pathology, Armed Forces Institute of Pathology, Rockville, MD 20306, USA.
* To whom correspondence should be addressed. E-mail: wilson{at}scripps.edu (I.A.W.) and jstevens{at}scripps.edu (J.S.)
The H5N1 avian influenza virus, commonly called "bird flu,"is a highly contagious and deadly pathogen in poultry. Sincelate 2003, H5N1 has reached epizootic levels in domestic fowlin a number of Asian countries, including China, Vietnam, Thailand,Korea, Indonesia, Japan, and Cambodia, and has now spread towild bird populations. More recently, the H5N1 virus has spreadto infect bird populations across much of Europe and into Africa.However, its spread to the human population has so far beenlimited, with only 191 documented severe infections, but witha high mortality accounting for 108 deaths in Indonesia, Vietnam,Thailand, Cambodia, China, Iraq, Turkey, Azerbaijan, and Egypt[as of 4 April 2006, see the World Health Organization Web site(1)]. Of these, evidence suggests direct bird-to-human transmission,although indirect transmission, perhaps through contaminatedwater supplies, cannot be ruled out.
Of the three influenza pandemics of the last century, the 1957(H2N2) and 1968 (H3N2) pandemic viruses were avian-human reassortmentsin which three and two of the eight avian gene segments, respectively,were reassorted into an already circulating, human-adapted virus(2, 3). The origin of the genes of the 1918 influenza virus(H1N1), which killed about 50 million people worldwide (4),is unknown. The extinct pandemic virus from 1918 has recentlybeen reconstructed in the laboratory and was found to be highlyvirulent in mice and chicken embryos (5, 6). With continuedoutbreaks of the H5N1 virus in poultry and wild birds, furtherhuman cases are likely, and the potential for the emergenceof a human-adapted H5 virus, either by reassortment or mutation,is a threat to public health worldwide.
Hemagglutinin (HA), the principal antigen on the viral surface,is the primary target for neutralizing antibodies and is responsiblefor viral binding to host receptors, enabling entry into thehost cell through endocytosis and subsequent membrane fusion.As such, the HA is an important target for both drug and vaccinedevelopment. Although 16 avian and mammalian serotypes of HAare known, only three (H1, H2, and H3) have become adapted tothe human population. HA is a homotrimer; each monomer is synthesizedas a single polypeptide (HA0) that is cleaved by host proteasesinto two subunits (HA1 and HA2). HA binds to receptors containingglycans with terminal sialic acids, where their precise linkagedetermines species preference. A switch in receptor specificityfrom sialic acids connected to galactose in 2-3 linkages (avian)to 2-6 linkages (human) is a major obstacle for influenza Aviruses to cross the species barrier and to adapt to a new host(7, 8). On H3 and H1 HA frameworks, as few as two amino acidmutations can switch human and avian receptor specificity.
Of the H5N1 viral isolates studied to date, A/Vietnam/1203/2004(Viet04) is among the most pathogenic in mammalian models, suchas ferrets and mice (9, 10). This virus was originally isolatedfrom a 10-year-old Vietnamese boy who died from bird flu. Becauseof the importance of HA in viral pathogenesis and host responseto viral infection, we cloned and expressed the ectodomain (HA0)of its HA gene (fig. S1) in a baculovirus expression system,using the same strategy that led to the crystal structure ofthe 1918 influenza virus HA0 (11, 12). Viet04 HA0 was cleavedduring protein production into its activated form (HA1/HA2)and was crystallized at pH 6.55 (13). Its structure was determinedby molecular replacement (MR) to 2.95 Å resolution (tableS1) (14). In addition, we have investigated the potential ofthis H5 HA to acquire human receptor specificity by introducingmutations known to effect such a specificity switch on H1 andH3 frameworks.
Structural overview. The overall fold of the Viet04 HA trimer(Fig. 1, A and B) is very similar to other published HAs, asexpected, with a globular head containing the receptor bindingdomain (RBD) and vestigial esterase domain, and a membrane proximaldomain with its distinctive, central -helical stalk and HA1/HA2cleavage site (essential for viral pathogenicity). AlthoughViet04 HA and the only other avian H5 HA structure, Sing97 [A/Duck/Singapore/3/1997;Protein Data Bank (PDB) entry 1jsm
[PDB]
(15)], are closely relatedin sequence (HA1, 90%; HA2, 98%), the best molecular replacement(MR) solutions were surprisingly achieved by using the 1918H1 structure (sequence identity: HA1, 58%; HA2, 85%) as a searchmodel (16). Superimposition of human, avian, and swine HA structuresby using their HA2 domains (table S2) or individual domains(table S3) confirms that the Viet04 HA is more closely relatedto human 1918 H1 HA [root mean square deviation (RMSD) 1.2 Å]than to Sing97 H5 HA (RMSD 1.7 Å). For example, an interhelicalloop between the two major helices in HA2 is stabilized by ahydrogen bond between HA2 Arg68 and HA2 Asn81, resulting inits having an overall conformation much more akin to the 1918H1 loop than to that of Sing97 or H3 (Fig. 1C).
Fig. 1. Crystal structure of Viet04 HA and comparison with 1918 human H1, duck H5, and 1968 human H3 HAs. (A) Overview of the Viet04 trimer, represented as a ribbon diagram. For clarity, each monomer has been colored differently. Carbohydrates observed in the electron-density maps are colored orange, and all the asparagines that make up a glycosylation site are labeled. Only Glu20, Glu289, and Phe154 are not labeled, as these are on the back of the molecule. The location of the receptor binding, cleavage, and basic patch sites are highlighted only on one monomer. All the figures were generated and rendered with the use of MacPymol (66). (B) Structural comparison of the Viet04 monomer (olive) with duck H5 (orange) and 1918 H1 (red) HAs. Structures were first superimposed on the HA2 domain of Viet04 through the following residues: Viet04, Gly1 to Pro160; 1918 H1 (PDB: 1rd8), Gly1 to Pro160; H3(PDB:2hmg), Gly1 to Pro160; H5 (PDB: 1jsm
[PDB]
), Gly1 to Pro160. Orientation of the overlay approximates to the blue monomer in (A). (C) Superimposition of the two long -helices of HA2 for 1918 H1 (PDB: 1rd8), avian H5 (PDB: 1jsm
[PDB]
), human H3 (PDB: 2hmg
[PDB]
), and Viet04 reveal that the extended interhelical loop of Viet04 is more similar to the 1918 H1 than to the existing avian H5 structure. The side chain of Phe63 is illustrated as an example of the close proximity of the two structures.
[View Larger Version of this Image (58K GIF file)]
The amino acid sequence of Viet04 HA predicts seven possibleglycosylation sites per monomer, although one is in the cytoplasmictail and unlikely to be glycosylated. Interpretable electrondensity is observed at 16 of the possible 54 glycosylation sitesin the asymmetric unit (nine monomers), which represents carbohydratesat two sites, Asn34 and Asn169 in HA1 (17).
Hemagglutinin is synthesized as a single-chain precursor (HA0)in the endoplasmic reticulum, where it is assembled as a trimer,and is then exported to the cell surface via the Golgi network.On the cell surface, HA0 is cleaved by specific host proteases,such as tryptase Clara (18), into HA1 and HA2 (19). For themajority of HAs, the specific cleavage site (Q/E-X-R) (20) andthe narrow tissue distribution of the relevant proteolytic enzymesrestricts infection to the lung in mammals. However, for H5and H7 subtypes, a polybasic sequence has been associated withhigh virulence in birds (21), because of enhanced cleavage susceptibilityby a broader range of cellular proteases, as seen with our baculovirus-expressedViet04 HA (fig. S1) (22). Consequently, the tissue tropism forH5 viruses in mammals is not restricted to the lungs, but extendsto other organs, including the brain (10). In the Viet04 structure,the C-terminal HA1 cleavage site region could be interpretedonly as far as Pro324 and does not account for the remainingQRERRRKKR residues before Gly1 at the N terminus of HA2 (fig.S3). As in other HAs, the HA2 N terminus is stabilized withinan electronegative cavity by hydrogen bonds from its backboneamide groups to Asp112 and to Ser113 of the adjacent HA2 (fig.S3).
From our previous 1918 HA0 structure, we proposed that a pH-sensitivehistidine patch (HisA18, HisA38, and HisB111) (14), togetherwith the adjacent HA2 TrpB21, could play a role in fusion peptidedestabilization and release (Fig. 1A) (11). This structuralfeature is conserved in other avian and human H1, H2, and H5serotypes, as well as in Viet04 HA (fig. S3). In 1918 HA0, asecond patch of four exposed histidines within the vestigialesterase domain (Fig. 1A and fig. S4A), together with a nearbylysine, was also implicated in pathogenicity via enhanced membranefusion (11). Of the five HA1 residues in this basic patch (His47,Lys50, His275, His285, and His298), only three are conservedin avian H5 structures (His47, Lys50, and His298) (fig. S4,B to D), but Viet04 and Sing97 HAs have an additional lysine(Lys45) and histidine (His295) (fig. S4, B, C, and E). Furthermore,Viet04 has yet another lysine (Lys46), which renders this patcheven more basic and is found in two strains (1203/1204) thatwere isolated from the same patient (10) (fig. S5). The contributionof this region to virulence, if any, is as yet unknown, butis worthy of further investigation.
H5N1 antigenic variation. Phylogenetic analysis of H5 HA genesfrom 2004 and 2005 has revealed two distinct lineages, termedclades 1 and 2 (23); Viet04 belongs to the Indochina peninsulalineage (clade 1). Comparison of their amino acid sequencesidentified 13 positions of antigenic variation that are mainlyclustered around the receptor-binding site; the rest are withinthe vestigial esterase domain (Fig. 2). Escape mutants of H5HAs (24, 25) can be clustered into three epitopes (24), as follows:site 1, an exposed loop (HA1 140 to 145) that overlaps withantigenic sites A (26) of H3 (27) and Ca2 of H1 (28); site 2,HA1 residues 156 and 157, which correspond to antigenic siteB in H3 serotypes; and site 3, HA1 129 to 133, which is restrictedto the Sa site in H1 HAs (28) and H9 serotypes (29). Thus, naturalvariation (yellow in Fig. 2), as well as escape mutants (bluein Fig. 2, green in both 2004 and 2005 viral isolates), suggestscontinued evolution of the virus that impacts decisions on whichstrain should be considered for a bird flu vaccine. One mutationthat has alanine at residue 160 replaced by threonine (A160T),which is present in all 2004–05 strains, introduces anew glycosylation site at Asn158, consistent with a strategycommonly used by influenza viruses to mask and unmask antigenicsites from the immune system (30, 31). This glycosylation likelyresults in steric hindrance to antigenic site 2 (around residues156 and 157), thus reducing the ability of the host to mountan effective immune response to these more recent H5N1 viruses.
Fig. 2. Antigenic variation in recent H5N1 viruses mapped onto the Viet04 structure. (Left) Side view of the Viet04 structure in which natural mutations identified by comparison of 2005 with 2004 isolates (23) are colored yellow; escape mutants (24, 25) are blue; and those that overlap in both analyses are green. All of the 2004 and 2005 strains have a new potential glycosylation site at position 158 in the HA1 chain (orange). The receptor binding site is highlighted with a red oval. (Right) Top view looking down onto the globular membrane distal end of the trimer around the RBD showing that the mutations mainly cluster around the RBD.
[View Larger Version of this Image (76K GIF file)]
Receptor binding domain. The RBD is at the membrane distal end(HA1) of each HA monomer (Fig. 1A) and binds to its sialic acid–containingreceptors with very weak (millimolar) affinity (32). However,influenza virus can increase its avidity to host cells throughmultivalent binding via a high density of HA trimers on thevirus surface. Avian viruses bind to sialosides with an 2-3linkage in the intestinal tract, whereas human-adapted virusesare specific for the 2-6 linkage in the respiratory tract (7),although H5 viruses have also been reported in human intestine(33). A switch from 2-3 to 2-6 receptor specificity is a criticalstep in the adaptation of avian viruses to a human host andappears to be one of the reasons why most avian influenza viruses,including current avian H5 strains, are not easily transmittedfrom human to human after avian-to-human infection.
All HA structures, including Viet04 (Fig. 3A), have similarlyconfigured RBDs. The binding site comprises three structuralelements, namely an -helix (190-helix, HA1 188 to 190) and twoloops (130-loop, HA1 134 to 138, and 220-loop, HA1 221 to 228)(Fig. 3A). A number of conserved residues are involved in receptorbinding, including Tyr98, Trp153, and His183 (Table 1) (19).Superimposition of the RBD structural elements of Viet04 withSing97 H5 reveals a very close relation (RMSD 0.3 Å) (Fig. 3B).Indeed, all key residues implicated in receptor specificity[reviewed in (19)] (Table 1) are conserved between structures,although loop 210 to 221 is displaced 1 Å from its equivalentin Sing97 (Fig. 3B). Otherwise, only two RBD residues differbetween these two H5 HAs (Viet04, Arg216 and Ser221; Dk97, Glu216and Pro221). Thus, the question arises as to how a current H5virus could adapt its HA for binding to human receptors.
Fig. 3. Analysis of Viet04 receptor binding site. (A) The Viet04 receptor-binding domain (RBD) with the side chains of key residues for receptor binding labeled. The binding site comprises three structural elements: an -helix (190-helix) and two loops (130-loop and 220-loop). Residues mutated in this study are labeled red. (B) Overlay of the RBDs of Viet04 with Sing97 structure (PDB: 1jsm
[PDB]
) reveals a similar RBD. The most divergent part of the pocket is the loop made up of residues 210 to 221, in which the Viet04 loop is displaced 1 Å farther away from the binding pocket compared with the 1997 avian H5. Only two residues, at position 216 and 221, differ in these two RBDs.
[View Larger Version of this Image (28K GIF file)]
Table 1. Conserved residues within the RBDs of H1 and H5 serotypes that are implicated in receptor specificity. Accession numbers for each wild-type HA are listed in supporting online material. Residues mutated in this study are highlighted in gray. The last two columns give a qualitative assessment of 2-3/2-6 binding preferences for each mutant with the glycan array. Qualitative binding assessments were based on a combination of the signal strength and the number of glycans bound for a given linkage. The binding of Viet04 was used as a standard for strong binding to the 2-3 linkage (), and the double mutant for Dk76 (E190D,G225D) was used for strong binding () to the 2-6 linkage.
* Although Viet04 mutants (G228S and Q226L,G228S) only bound a limited number of 2-6 ligands, they bound strongly to these glycans and were, therefore, assessed as for 2-6 specificity. No binding is represented by "O"; ND indicates binding to the array was not determined.
Receptor binding specificity of Viet04 HA. Our cloning and expressionstrategy produces HA with a His-tag at the C terminus, whichfacilitates receptor-binding studies using a glycan microarray(34–37). Glycan binding analyses of Viet04 HA reveal anavian 2-3 specificity in which the highest affinity is for glycanswith sulfate on the 6 position of the N-acetylglucosamine (GlcNAc)residue at the third position in the glycan chain (Fig. 4A andtable S4) (38, 39). Considerable binding to only one 2-6–linkedsialoside was observed (6'-sialyllactose, no. 49), but thisglycan is only found in milk and is not a receptor candidatefor influenza (40). We also expressed and investigated the glycan-bindingproperties of A/Duck/Singapore/Q-F119-3/1997 (Dk97), whose sequenceis identical to that of Sing97, for correlation with its structure(15). Binding of glycoproteins (nos. 1 to 6) and sulfated glycanswas comparable to those of Viet04, but binding to other 2-3sialosides was reduced relative to Viet04 (Fig. 4B).
Fig. 4. Glycan microarray analyses of (A) Viet04, (B) Dk97, and (C) an avian H1, Dk76. The Dk97 HA sequence is identical to that in the published structure of duck virus Sing97, so a direct structural comparison can be made. Binding to different types of glycans on the array are highlighted where orange represents glycoproteins; yellow, 2-3 ligands; green, 2-6 ligands; blue, 2-8 ligands; and purple, other ligands such as ß-linkages, modified sialic acid analogs or glycolylsialic acid glycans. Red bars indicate sulfated or additional negatively charged ligands. See table S4 for list and tabulated binding results. Because of continual glycan microarray development, a number of new ligands were printed between analyzing the Dk76 protein (C) and the remaining samples reported in this study. Binding to glycans nos. 37 to 44, 56, 58 to 60, 67, and 70 was not determined for Dk76 and its three mutants in Fig. 5.
[View Larger Version of this Image (28K GIF file)]
Mutational analysis of the RBD. Previous studies using wholevirus identified a number of key RBD mutations that were implicatedin avian-human receptor specificity switching in H1, H2, andH3 serotypes. However, adaptation of avian H1 and H2/H3 serotypesfor human receptor binding occurs by different mechanisms. ForH2 and H3, mutation of Gln226 and Gly228 in avian strains toLeu226 and Ser228 in human viruses correlates with a shift tohuman receptor specificity (41, 42). In H1 serotypes, the avianGln226 and Gly228 framework is maintained and a Glu190 to Asp190mutation now appears critical for adaptation to human 2-6 receptors(43, 44). Indeed, glycan microarray and cell-based assays revealedthat the 1918 HA could be readily converted from classic 2-6receptor specificity to classic avian 2-3 specificity by onlytwo mutations (D190E and D225G) (35, 45). Here, the reverseexperiment was performed with an avian H1 virus [A/Duck/Alberta/35/1976(Dk76)] in which the same two residues were mutated to the "human"sequences (E190D and G225D), which completely converted Dk76to exclusive 2-6 specificity, similar to that seen for the SouthCarolina 1918 virus (Figs. 4C and 5, A to C; and table S4) (11,46).
Fig. 5. Glycan microarray analysis of mutants of Viet04 and Dk76. Mutations of an avian H1, Dk76: (A) E190D, (B) G225D, and (C) E190D and G225D were generated and subjected to glycan microarray analysis. Both positions were reported to be important for conversion of 2-6 receptor specificity of the human 1918 virus HA to avian 2-3 specificity (35, 45). These mutations did indeed result in exclusive 2-6 specificity for this avian H1 HA. (D to F) Consequently, Viet04 mutations were generated at the same positions, but did not result in a switch of receptor specificity, except to 6'-sialyllactose, although they did result in decreased 2-3 binding, particularly to nonsulfated glycans (compare Fig. 4A). (G to I) Viet04 was mutated at positions 226 and 228, known to be important for H3 HA 2-6 receptor adaptation. Again, no clear switch in receptor specificity was observed, although binding to biantennary 2-6 moieties was observed, as well as reduced 2-3 binding in the double and single (Q226L) mutant. Graphs are generated as described in the legend for Fig. 4 and labels to the introduced mutations.
[View Larger Version of this Image (42K GIF file)]
However, which mutations are likely to modulate receptor specificityin the H5 serotype is not so obvious. Based on sequence similarity,H5 is in the same clade as H1, H2, and H6 serotypes (47). So,to address that issue, we analyzed glycan binding of Viet04HA (Fig. 5 and fig. S6) by generating a panel of mutants (Fig. 3Aand Table 1) in and around the RBD to explore whether this H5HA can readily become adapted to humans through mutations thatare known to change receptor specificity in H1 and H3 serotypes.Mutations at positions 190 and 225 did not reveal any adaptationof Viet04 to human receptor analogs (Fig. 5, D to F) (48), incontrast to H1 Dk76 (Fig. 5, A to C) and 1918 HAs (35). Indeed,the single E190D mutation on the Viet04 framework reveals markedlyreduced affinity to 2-3 sialosides (Fig. 5D), whereas the doublemutant (E190D,G225D) did not interact at all with the glycanmicroarray (Fig. 5F) (49, 50). However, sulfated glycans boundequally well to the single E190D mutant and to the wild type(Figs. 4A and 5D), which suggests that other residues withinthe Viet04 RBD, such as Lys193 or Lys222 (Fig. 3A), may enhanceinteraction with charged glycans.
Mutation of residues 226 and 228, which enable H3 viruses toswitch from avian to human specificity, was also evaluated asa potential route for H5 viruses to acquire human receptor specificity.Although a dramatic switch to a classic 2-6 human receptor binderwas not observed (51), the double mutant (Q226L,G228S) showedsubstantially reduced affinity to 2-3 sialosides, as noted formutants of the H3 A/Hong Kong/156/1997 virus (52). But it wasnotable that significant binding to a natural, branched 2-6biantennary glycan (nos. 56 and 57) was observed for both thedouble mutant and the single G228S mutant (Fig. 5H). Althoughthe glycan composition of lung epithelia have not been analyzedin detail, the mammalian sialyl-transferase that produces 2-6–linkedstructures on many human tissues (53, 54) is found in lung epithelialcells (55–57). Thus, these two effects could offer advantagesfor an H5N1 virus to adapt to a human host. Decreased bindingto 2-3–linked glycans would help circumvent the inhibitoryeffects of respiratory mucins (58), whereas increased bindingto biantennary N-linked glycans with 2-6–linked sialicacids would allow the virus to attach to the surface of epithelialcells that express this carbohydrate receptor (55–57).In this regard, human H1 viruses before 1957 were reported tobind sialic acid receptors with both 2-3 and 2-6 linkages; post1957 viruses were specific only for 2-6 linkages (37). Thesebinding patterns suggest that, once a foothold in a new hostspecies is made, the virus HA optimizes its specificity to thenew host. It is noteworthy that, of the HAs tested on the array,the humanized avian H1 (Dk76) double mutant (E190D,G225D) (Fig. 5C)and the human H3 HA (A/Moscow/10/1999) (35) did not bind 2-6biantennary glycans, in contrast to 1918 South Carolina H1 HAand human H1, A/Texas/36/1991 (35). Therefore, the HAs of someviruses may be able to increase avidity through interactionwith such bivalent structures on N-linked glycans, whereas,for others, the geometry of the bivalent structure appears torestrict binding to linear sequences containing 2-6 linkages.Thus, although human viral HAs have a primary specificity for2-6 linkages, each may use a different spectrum of glycan receptorsfor cell entry.
All key residues within the RBD are conserved in the majorityof H5 strains that have infected humans (fig. S5). However,two A/Hong Kong/2003 (HK2003) isolates acquired a S227N mutationwithin the binding site, whereas a double mutation (E216R,P221S)in the 220-loop is observed in all 2003–05 isolates (fig.S5). The possible effect of these natural mutations on Viet04HA binding specificity (Table 1) was, therefore, assessed. TheS227N mutation had comparable specificity to that of Viet04,with the exception of increased binding, particularly for branched2-3 fucosylated glycans (nos. 26 to 29) and for 6-sialylatedN-acetylgalactosamine (GalNAc) (no. 20) (fig. S6A) (59, 60),contrary to previous reports that HK2003 isolates had increasedaffinity toward 2-6 analogs, but decreased affinity toward 2-3analogs (39). However, in a previous study from a 1997 isolate,such changes were also not observed (52), although Viet04 differsat a number of other positions around the RBD compared withthe Hong Kong isolates that could account for this difference(61) (Fig. 3A). Reverse R216E and S221P mutants were also generated,as well as the double mutant (R216E,S221P), but the R216E mutantexpressed poorly and could not be analyzed. However, only thedouble mutant is found in natural isolates, suggesting a pressureto select for both mutations, which possibly are related tothe HA stability. Whereas Viet04 HA binds to branched fucosylatedsialosides (nos. 26 to 29) (Fig. 4A), the S221P mutation showedweaker binding, whereas the double mutant abrogated bindingto all branched fucosylated glycans unless sulfated (no. 25)(fig. S6, B and C). In the Viet04 HA structure, these residueshydrogen bond to an adjacent monomer in the trimer (Arg216 withAsn210 and Ser221 with Asp241) (15) and stabilize the displaced210 to 229 loop (Fig. 3B), which, therefore, could possiblyenhance binding to branched fucosylated glycans.
So how might H5 avian HA adapt to human receptors? Knowledgeof genetic changes in circulating viral isolates (39) by themselvesobviously cannot be used to predict the impact on receptor specificity,let alone predict the effect of future mutations. Here, we usea completely recombinant system for structural and functionalanalyses that enables such investigation in the laboratory.Our conclusion is that the mutations that cause a shift fromthe avian-type to human-type specificity on the H1 and H3 frameworksdo not cause an equivalent shift in specificity on the H5 frameworkof the Viet04 isolate. However, the mutations that give riseto 2-6 specificity in H3 HAs do in fact reduce avidity to 2-3sialosides and increase specificity for 2-6–linked biantennaryN-linked glycans that could serve as receptors for the viruson lung epithelial cells. These combined effects could allowthe Viet04 virus to escape entrapment by mucins and increasethe likelihood of binding to and infection of susceptible epithelialcells (52). Thus, such mutations provide one possible routeby which H5 viruses could gain a foot-hold in the human population,although it is possible that other, as yet unidentified, mutationsmay allow the H5N1 virus to effect a switch in receptor specificity.
This glycan microarray technology can, therefore, be used toanalyze not only existing viral HAs, but as we show here, toidentify mutations that enable adaptation of the remaining influenzaserotypes into the human population. Monitoring such changesin the "receptor binding footprint" in the field on whole virusesusing the glycan microarray could be invaluable in the identificationof emerging viruses that could cause new pandemics or epidemics.
12. Materials and Methods are available as supporting material on Science Online.
13. Viet04 HA at a concentration of 9 mg/ml was used to grow crystals in sitting drops with a precipitant solution of 22% polyethylene glycol 2000 and 0.1 M Hepes, pH 6.55 (see also supporting online material).
14. 1918 H1 HA0 (PDB: 1rd8), truncated to remove residues around the cleavage site, was used as the initial MR model. The final Rcryst and Rfree values are 26.9 and 31.9% respectively, at 2.9 Å resolution. The crystal asymmetric unit contains nine hemagglutinin monomers (six HA monomers in two noncrystallographic trimers and three HA monomers that each form one-third of three crystallographic trimers) with an estimated solvent content of 57% based on a Matthews' coefficient (Vm) of 2.9 Å3/dalton (fig. S2). For comparison with previous structures, the Viet04 sequences are numbered as for the H3 subtype. A, C, E, G, I, K, M, O, and Q refer to the nine HA1 subunits in the asymmetric unit, and B, D, F, H, J, L, N, P, and R refer to the nine HA2 subunits; e.g., HisA18 refers to HA1 residue 18 in the A subunit and HisB11 refers to HA2 residue 111 in the B subunit of the same monomer. Insertions in Viet04 relative to H3 are labeled by the preceding residue with a letter (e.g., Asn19A).
16. Scores from the molecular replacement program PHASER revealed superior scores for the 1918 H1 structure (Z score: 37.2; and log-likelihood gain, 3412), as compared with the Sing97 structure (Z scores, 33.8; and log-likelihood gain, 768).
17. Two N-acetyl glucosamines were interpretable at 13 of these sites (AsnA34, AsnC34, AsnC169, AsnE34, AsnG34, AsnI34, AsnI169, AsnK34, AsnK169, AsnM34, AsnM169, AsnO34, AsnO169), but an additional mannose residue could be interpreted at a further three sites (AsnA169, AsnE169, AsnG169). The glycans are stabilized at Asn34 by a neighboring residue (Gln24) in the same chain, whereas at Asn169, an additional mannose was visualized because of stabilization with Lys56 and main-chain amide of Val57, in a symmetry-related monomer.
20. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; X, any amino acid; and Y, Tyr.
21. D. J. Hulse, R. G. Webster, R. J. Russell, D. R. Perez, J. Virol.78, 9954 (2004).[Abstract/Free Full Text]
25. M. Philpott, C. Hioe, M. Sheerar, V. S. Hinshaw, J. Virol.64, 2941 (1990).[Abstract/Free Full Text]
26. Regions of antigenic variation have been identified in H1 and H3 serotypes. For H1, these sites were designated Sa, Sb, Ca, and Cb; for H3, sites were designated A, B, C, and D.
27. D. C. Wiley, I. A. Wilson, J. J. Skehel, Nature289, 373 (1981). [CrossRef] [Medline]
36. HA binding can be analyzed not only for sialic acid–linkage preference, but also for additional features, such as charge; glycan length; or additional sulfation, fucosylation, and sialylation. Of the 265 glycans currently imprinted on the array, 6 are glycoproteins; 38 have sialic acids with 2-3 linkages; 16 have 2-6 linkages; 7 have 2-8 linkages; and a further 16 are ß-linkages, modified sialic acid analogs, or glycolylsialic acid glycans. (See table S4 for the glycans analyzed in this study. Of the 2-6 sialosides, only natural full-sized N-linked glycans represented on the array are the biantennary structures (nos. 56 and 57). The remaining sialosides are fragments or terminal sequences found on glycoproteins. For full information on the array, contact the Consortium for Functional Glycomics (62). Previous binding data using this technology and cell-based assays with whole viruses show that N-linked glycans close to the receptor-binding site can affect receptor binding through steric hindrance (35, 63). Insect cells do not produce complex glycans containing terminal galactose and/or sialic acids, as seen in mammalian cells, although high-mannose glycans are produced (64). However, because of the presence of the influenza sialidase, complex glycans of influenza HAs usually terminate only in galactose, and thus the size of the N-glycans elaborated by insect cells approximate to the size of the complex N-glycans in mammalian host cells. Thus, any importance of complex glycans for HA function is still unknown. Indeed, results for the avian H3 HA (A/Duck/Ukraine/1/1963), published recently (35), are in agreement with previous whole viral studies (65). However, independent studies are ongoing to develop the array for whole-virus analyses so that a direct comparison can be made. Such initial experiments are promising, because the strict 2-3 specificity observed here for Dk76 is also seen with whole-virus studies (37) and preliminary experiments with A/Puerto Rico/8/1934 virus that reveal both 2-3 and 2-6 specificity (34), in agreement with experiments from cell-based assays (37).
38. Whole-virus studies, including those for Viet04 virus, also revealed 2-3 specificity with a preference for sulfation in current H5N1 strains (39). However, this assay used only seven ligands (one 2-6 and six 2-3), which is considerably fewer than the 84 sialosides, sialoside analogs, and glycoproteins analyzed here. In our glycan array, sulfation on the second galactose was not tolerated (no. 37) for Viet04, although binding was apparent for sialosides with Gal in either ß1-3 or ß1-4 linkage to a GlcNAc or GalNAc (nos. 21 to 23, 32, 33), as well as to fucosylated glycans (nos. 26 to 29).
46. Avian H1 bound only to nonbranched glycans and to sulfated and/or negatively charged glycans (Figs. 4C and 5, A to C). The single E190D mutation reduced binding to most 2-3 glycans, except to sulfated sialosides (Fig. 5A). These results suggest mutation at both 190 and 225 positions is always a requirement for H1 serotypes to adapt to a human host.
48. The E190D mutation (Fig. 5D) reduced overall binding of 2-3 ligands and glycoproteins, except for the sulfated and/or negatively charged glycans (nos. 18, 20, 24, 25, and 38). The G225D mutation (Fig. 5E) appeared to have little effect on the binding profile, in contrast to avian H1, where binding was not detected (Fig. 5B). The double mutant (E190D,G225D) did not bind to any glycan on the array (see Fig. 5F).
50. For the human H1 HA from A/Puerto Rico/8/1934, the longer side chain of Glu190 can form hydrogen bonds to sialic acid of both 2-6 and 2-3 sialosides, whereas for structures of A/Swine/Iowa/1930, H1 HA bound to human receptor analogs, the shorter side chain of Asp190 can only interact with the GlcNAc to stabilize the 2-6 conformation (49). Binding data, with the 1918 South Carolina H1 HA (35) and the Dk76 double mutation (E190D,G225D) (Fig. 5C), show that some sulfated glycans with 2-6 sialic acid linkages can bind. However, this situation does not arise for the Viet04 double mutant. Although the G225D mutation would have been expected to enhance 2-6 specificity, the additional stabilizing influence of the E190D mutation toward the GlcNAc may not be possible because of the neighboring Lys193, which could inhibit interaction of Asp190 with the glycan either by steric hindrance or by direct interaction with Asp190. Experiments are in progress to test this notion.
51. The Q226L mutation eliminated binding to the microarray, except for two negatively charged 2-3 glycans [with either an extra sialic acid on the 6-position of a GalNAc (no. 20) or 6-sulfation on GlcNAc with a branched fucose (no. 25)]. The G228S mutation did not have any significant effect compared with Viet04, except that sialosides with sulfation on the 6-position of the galactose, with or without branched fucosylation on the GlcNAc (nos. 12, 37) were tolerated. Stronger binding was observed for fucosylated glycans (nos. 26 to 29), and reduced binding was observed for sialosides with ß1-3 linkages between the galactose and GlcNAc/GalNAc (nos. 21 to 23) (Fig. 5H). In addition to 6'-sialyllactose (no. 49), as seen for Viet04, binding was observed for 2-6 biantennary structures (nos. 56 and 57). The double mutant (Q226L,G228S) showed reduced binding to 2-3 sialosides. Only sulfated and long-chain glycans were tolerated (nos. 16, 20, 24, 25, 35), but binding to 2-6 biantennary structures (nos. 56 and 57), as with the G228S mutation, was also maintained.
52. R. Harvey, A. C. Martin, M. Zambon, W. S. Barclay, J. Virol.78, 502 (2004).[Abstract/Free Full Text]
59. Attenuated viruses with a S227N mutation led to higher hemagglutinin inhibition titers in ferrets (60). Thus, enhanced binding to 2-3 ligands, especially to 6-sulfated GalNAc, could lead to an increased uptake into antigen-presenting cells and subsequent antibody production.
60. E. Hoffmann, A. S. Lipatov, R. J. Webby, E. A. Govorkova, R. G. Webster, Proc. Natl. Acad. Sci. U.S.A.102, 12915 (2005).[Abstract/Free Full Text]
61. The 2003 isolates contain Ala160, Arg193, Lys216 and Asn227, whereas Viet04 has Thr160 (which introduces a glycosylation site at Asn158), Lys193, Arg216, and Ser227.
67. The work was supported in part by National Institute of Allergy and Infectious Diseases grant AI058113 (I.A.W., T.T., J.K.T.); National Institute of General Medical Sciences grants GM062116 (to J.C.P., I.A.W.) and GM060938 (to J.C.P.); and partial support from NIH grants to I.A.W. (CA55896 and AI42266). We thank P. Palese and L. Glaser (Mount Sinai School of Medicine, New York) for providing the full-length clone of A/Vietnam/1203/2004; the staff of the Advanced Light Source Beamline 8.2.2 for the beamline assistance; X. Dai, S. Ferguson, P. Carney, and J. Vanhnasy (The Scripps Research Institute) for expert technical assistance; and R. Stanfield and M. Elsliger (The Scripps Research Institute) for helpful discussions. This is publication 17916-MB from The Scripps Research Institute. Coordinates and structure factors have been deposited in the Protein Data Bank (code 2FK0) and will be released on publication.
S. M. Rich, F. H. Leendertz, G. Xu, M. LeBreton, C. F. Djoko, M. N. Aminake, E. E. Takang, J. L. D. Diffo, B. L. Pike, B. M. Rosenthal, et al. (2009)
PNAS
106, 14902-14907
|Abstract »|Full Text »|PDF »
Historical Perspective -- Emergence of Influenza A (H1N1) Viruses.
S. M. Zimmer and D. S. Burke (2009)
N. Engl. J. Med.
361, 279-285
|Full Text »|PDF »
Ocular Infection of Mice with Influenza A (H7) Viruses: a Site of Primary Replication and Spread to the Respiratory Tract.
J. A. Belser, D. A. Wadford, J. Xu, J. M. Katz, and T. M. Tumpey (2009)
J. Virol.
83, 7075-7084
|Abstract »|Full Text »|PDF »
Influenza Exerts Continued Pressure in an Era of Modern Medicine.
J. W. Noah, D. L. Noah, and S. Matalon (2009)
Am. J. Respir. Cell Mol. Biol.
41, 3-7
|Full Text »|PDF »
Different Evolutionary Trajectories of European Avian-Like and Classical Swine H1N1 Influenza A Viruses.
E. J. Dunham, V. G. Dugan, E. K. Kaser, S. E. Perkins, I. H. Brown, E. C. Holmes, and J. K. Taubenberger (2009)
J. Virol.
83, 5485-5494
|Abstract »|Full Text »|PDF »
Intranasal Vaccination with 1918 Influenza Virus-Like Particles Protects Mice and Ferrets from Lethal 1918 and H5N1 Influenza Virus Challenge.
L. A. Perrone, A. Ahmad, V. Veguilla, X. Lu, G. Smith, J. M. Katz, P. Pushko, and T. M. Tumpey (2009)
J. Virol.
83, 5726-5734
|Abstract »|Full Text »|PDF »
Rapid Detection of H5N1 Subtype Influenza Viruses by Antigen Capture Enzyme-Linked Immunosorbent Assay Using H5- and N1-Specific Monoclonal Antibodies.
H.-T. Ho, H.-L. Qian, F. He, T. Meng, M. Szyporta, N. Prabhu, M. Prabakaran, K.-P. Chan, and J. Kwang (2009)
Clin. Vaccine Immunol.
16, 726-732
|Abstract »|Full Text »|PDF »
Amino Acid Residues in the Fusion Peptide Pocket Regulate the pH of Activation of the H5N1 Influenza Virus Hemagglutinin Protein.
M. L. Reed, H.-L. Yen, R. M. DuBois, O. A. Bridges, R. Salomon, R. G. Webster, and C. J. Russell (2009)
J. Virol.
83, 3568-3580
|Abstract »|Full Text »|PDF »
Role of Sialic Acid Binding Specificity of the 1918 Influenza Virus Hemagglutinin Protein in Virulence and Pathogenesis for Mice.
L. Qi, J. C. Kash, V. G. Dugan, R. Wang, G. Jin, R. E. Cunningham, and J. K. Taubenberger (2009)
J. Virol.
83, 3754-3761
|Abstract »|Full Text »|PDF »
Antibody Recognition of a Highly Conserved Influenza Virus Epitope.
D. C. Ekiert, G. Bhabha, M.-A. Elsliger, R. H. E. Friesen, M. Jongeneelen, M. Throsby, J. Goudsmit, and I. A. Wilson (2009)
Science
324, 246-251
|Abstract »|Full Text »|PDF »
Helicobacter pylori binding to new glycans based on N-acetyllactosamine.
H. Miller-Podraza, K. Weikkolainen, T. Larsson, P. Linde, J. Helin, J. Natunen, and K.-A. Karlsson (2009)
Glycobiology
19, 399-407
|Abstract »|Full Text »|PDF »
H.-L. Yen, J. R. Aldridge, A. C. M. Boon, N. A. Ilyushina, R. Salomon, D. J. Hulse-Post, H. Marjuki, J. Franks, D. A. Boltz, D. Bush, et al. (2009)
PNAS
106, 286-291
|Abstract »|Full Text »|PDF »
Indigenous sources of 2007-2008 H5N1 avian influenza outbreaks in Thailand.
K. Chaichoune, W. Wiriyarat, A. Thitithanyanont, R. Phonarknguen, L. Sariya, S. Suwanpakdee, T. Noimor, S. Chatsurachai, P. Suriyaphol, K. Ungchusak, et al. (2009)
J. Gen. Virol.
90, 216-222
|Abstract »|Full Text »|PDF »
Structural Characterization of the 1918 Influenza Virus H1N1 Neuraminidase.
X. Xu, X. Zhu, R. A. Dwek, J. Stevens, and I. A. Wilson (2008)
J. Virol.
82, 10493-10501
|Abstract »|Full Text »|PDF »
Chemoenzymatic synthesis, characterization, and application of glycopolymers carrying lactosamine repeats as entry inhibitors against influenza virus infection.
K. I P J Hidari, T. Murata, K. Yoshida, Y. Takahashi, Y.-h. Minamijima, Y. Miwa, S. Adachi, M. Ogata, T. Usui, Y. Suzuki, et al. (2008)
Glycobiology
18, 779-788
|Abstract »|Full Text »|PDF »
A Maximum Likelihood Method for Detecting Directional Evolution in Protein Sequences and Its Application to Influenza A Virus.
S. L. Kosakovsky Pond, A. F.Y. Poon, A. J. Leigh Brown, and S. D.W. Frost (2008)
Mol. Biol. Evol.
25, 1809-1824
|Abstract »|Full Text »|PDF »
Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer.
C.-C. Wang, Y.-L. Huang, C.-T. Ren, C.-W. Lin, J.-T. Hung, J.-C. Yu, A. L. Yu, C.-Y. Wu, and C.-H. Wong (2008)
PNAS
105, 11661-11666
|Abstract »|Full Text »|PDF »
Positive selection at the receptor-binding site of haemagglutinin H5 in viral sequences derived from human tissues.
A. Kongchanagul, O. Suptawiwat, P. Kanrai, M. Uiprasertkul, P. Puthavathana, and P. Auewarakul (2008)
J. Gen. Virol.
89, 1805-1810
|Abstract »|Full Text »|PDF »
Molecular Basis of S-layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus.
K. Steiner, R. Novotny, D. B. Werz, K. Zarschler, P. H. Seeberger, A. Hofinger, P. Kosma, C. Schaffer, and P. Messner (2008)
J. Biol. Chem.
283, 21120-21133
|Abstract »|Full Text »|PDF »
Comparative Efficacy of Neutralizing Antibodies Elicited by Recombinant Hemagglutinin Proteins from Avian H5N1 Influenza Virus.
C.-J. Wei, L. Xu, W.-P. Kong, W. Shi, K. Canis, J. Stevens, Z.-Y. Yang, A. Dell, S. M. Haslam, I. A. Wilson, et al. (2008)
J. Virol.
82, 6200-6208
|Abstract »|Full Text »|PDF »
Contemporary North American influenza H7 viruses possess human receptor specificity: Implications for virus transmissibility.
J. A. Belser, O. Blixt, L.-M. Chen, C. Pappas, T. R. Maines, N. Van Hoeven, R. Donis, J. Busch, R. McBride, J. C. Paulson, et al. (2008)
PNAS
105, 7558-7563
|Abstract »|Full Text »|PDF »
Infectivity Studies of Influenza Virus Hemagglutinin Receptor Binding Site Mutants in Mice.
J. Meisner, K. J. Szretter, K. C. Bradley, W. A. Langley, Z.-N. Li, B.-J. Lee, S. Thoennes, J. Martin, J. J. Skehel, R. J. Russell, et al. (2008)
J. Virol.
82, 5079-5083
|Abstract »|Full Text »|PDF »
Direct Sequence Detection of Structured H5 Influenza Viral RNA.
M. B. Kerby, S. Freeman, K. Prachanronarong, A. W. Artenstein, S. M. Opal, and A. Tripathi (2008)
J. Mol. Diagn.
10, 225-235
|Abstract »|Full Text »|PDF »
Pathology, Molecular Biology, and Pathogenesis of Avian Influenza A (H5N1) Infection in Humans.
Identification of the Progenitors of Indonesian and Vietnamese Avian Influenza A (H5N1) Viruses from Southern China.
J. Wang, D. Vijaykrishna, L. Duan, J. Bahl, J. X. Zhang, R. G. Webster, J. S. M. Peiris, H. Chen, G. J. D. Smith, and Y. Guan (2008)
J. Virol.
82, 3405-3414
|Abstract »|Full Text »|PDF »
Crystal Structure of Unliganded Influenza B Virus Hemagglutinin.
Q. Wang, F. Cheng, M. Lu, X. Tian, and J. Ma (2008)
J. Virol.
82, 3011-3020
|Abstract »|Full Text »|PDF »
Mechanisms of Zoonotic Severe Acute Respiratory Syndrome Coronavirus Host Range Expansion in Human Airway Epithelium.
T. Sheahan, B. Rockx, E. Donaldson, A. Sims, R. Pickles, D. Corti, and R. Baric (2008)
J. Virol.
82, 2274-2285
|Abstract »|Full Text »|PDF »
Avian influenza receptor expression in H5N1-infected and noninfected human tissues.
From the Cover: Quantitative biochemical rationale for differences in transmissibility of 1918 pandemic influenza A viruses.
A. Srinivasan, K. Viswanathan, R. Raman, A. Chandrasekaran, S. Raguram, T. M. Tumpey, V. Sasisekharan, and R. Sasisekharan (2008)
PNAS
105, 2800-2805
|Abstract »|Full Text »|PDF »
Update on Avian Influenza A (H5N1) Virus Infection in Humans.
Writing Committee of the Second World Health Organ (2008)
N. Engl. J. Med.
358, 261-273
|Full Text »|PDF »
Epitope Mapping of the Hemagglutinin Molecule of a Highly Pathogenic H5N1 Influenza Virus by Using Monoclonal Antibodies.
N. V. Kaverin, I. A. Rudneva, E. A. Govorkova, T. A. Timofeeva, A. A. Shilov, K. S. Kochergin-Nikitsky, P. S. Krylov, and R. G. Webster (2007)
J. Virol.
81, 12911-12917
|Abstract »|Full Text »|PDF »
Characterization of Low-Pathogenicity H5N1 Avian Influenza Viruses from North America.
E. Spackman, D. E. Swayne, D. L. Suarez, D. A. Senne, J. C. Pedersen, M. L. Killian, J. Pasick, K. Handel, S. P. S. Pillai, C.-W. Lee, et al. (2007)
J. Virol.
81, 11612-11619
|Abstract »|Full Text »|PDF »
Structural basis for receptor specificity of influenza B virus hemagglutinin.
Viral Tropism and the Pathogenesis of Influenza in the Mammalian Host.
K. G. Mansfield (2007)
Am. J. Pathol.
171, 1089-1092
|Full Text »|PDF »
An Avian Influenza H5N1 Virus That Binds to a Human-Type Receptor.
P. Auewarakul, O. Suptawiwat, A. Kongchanagul, C. Sangma, Y. Suzuki, K. Ungchusak, S. Louisirirotchanakul, H. Lerdsamran, P. Pooruk, A. Thitithanyanont, et al. (2007)
J. Virol.
81, 9950-9955
|Abstract »|Full Text »|PDF »
D. J. Vigerust, K. B. Ulett, K. L. Boyd, J. Madsen, S. Hawgood, and J. A. McCullers (2007)
J. Virol.
81, 8593-8600
|Abstract »|Full Text »|PDF »
Immunization by Avian H5 Influenza Hemagglutinin Mutants with Altered Receptor Binding Specificity.
Z.-Y. Yang, C.-J. Wei, W.-P. Kong, L. Wu, L. Xu, D. F. Smith, and G. J. Nabel (2007)
Science
317, 825-828
|Abstract »|Full Text »|PDF »
Human Parainfluenza Viruses hPIV1 and hPIV3 Bind Oligosaccharides with {alpha}2-3-Linked Sialic Acids That Are Distinct from Those Bound by H5 Avian Influenza Virus Hemagglutinin.
M. Amonsen, D. F. Smith, R. D. Cummings, and G. M. Air (2007)
J. Virol.
81, 8341-8345
|Abstract »|Full Text »|PDF »
Phage escape libraries for checkmate analysis.
T. J. Dickerson, K. M. McKenzie, A. S. Hoyt, M. R. Wood, K. D. Janda, S. B. Brenner, and R. A. Lerner (2007)
PNAS
104, 12703-12708
|Abstract »|Full Text »|PDF »
Avian Influenza Virus (H5N1): a Threat to Human Health.
J. S. M. Peiris, M. D. de Jong, and Y. Guan (2007)
Clin. Microbiol. Rev.
20, 243-267
|Abstract »|Full Text »|PDF »
Genomic Analysis and Geographic Visualization of the Spread of Avian Influenza (H5N1).
D. Janies, A. W. Hill, R. Guralnick, F. Habib, E. Waltari, and W. C. Wheeler (2007)
Syst Biol
56, 321-329
|Full Text »|PDF »
A statistical phylogeography of influenza A H5N1.
R. G. Wallace, H. HoDac, R. H. Lathrop, and W. M. Fitch (2007)
PNAS
104, 4473-4478
|Abstract »|Full Text »|PDF »
A Two-Amino Acid Change in the Hemagglutinin of the 1918 Influenza Virus Abolishes Transmission.
T. M. Tumpey, T. R. Maines, N. Van Hoeven, L. Glaser, A. Solorzano, C. Pappas, N. J. Cox, D. E. Swayne, P. Palese, J. M. Katz, et al. (2007)
Science
315, 655-659
|Abstract »|Full Text »|PDF »
Emergence and predominance of an H5N1 influenza variant in China.
G. J. D. Smith, X. H. Fan, J. Wang, K. S. Li, K. Qin, J. X. Zhang, D. Vijaykrishna, C. L. Cheung, K. Huang, J. M. Rayner, et al. (2006)
PNAS
103, 16936-16941
|Abstract »|Full Text »|PDF »
Natural Variation Can Significantly Alter the Sensitivity of Influenza A (H5N1) Viruses to Oseltamivir.
M. A. Rameix-Welti, F. Agou, P. Buchy, S. Mardy, J. T. Aubin, M. Veron, S. van der Werf, and N. Naffakh (2006)
Antimicrob. Agents Chemother.
50, 3809-3815
|Abstract »|Full Text »|PDF »
Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model.
T. R. Maines, L.-M. Chen, Y. Matsuoka, H. Chen, T. Rowe, J. Ortin, A. Falcon, N. T. Hien, L. Q. Mai, E. R. Sedyaningsih, et al. (2006)
PNAS
103, 12121-12126
|Abstract »|Full Text »|PDF »
Host species barriers to influenza virus infections..
T. Kuiken, E. C. Holmes, J. McCauley, G. F. Rimmelzwaan, C. S. Williams, and B. T. Grenfell (2006)
Science
312, 394-397
|Abstract »|Full Text »|PDF »
2-3 sialic acid receptor binding preference. Introduction of mutations that can convert H1 serotype HAs to human 
1 Å from its equivalent in Sing97 (
