Annotation tree for LABEL's H5N1 annotation module.

<p>Each internal node corresponds to an annotation level (classification step) within the hierarchical annotation process. Accordingly, HMM profiles and SVM classes used by the H5 module are represented by all non-root nodes (color circles). The “c-<i>X</i>” notation stands for “cluster <i>X</i>,” where <i>X</i> is some general group of clades. Exact correspondence with the H5N1 clade clustering <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086921#pone.0086921-WHOOIEFAO1" target="_blank">[5]</a> is not preserved in the annotation tree for the sake of algorithmic simplicity.</p

Hemagglutinin clade annotation for avian influenza subtype A(H9N2) by LABEL.

<p>Start and stop 9-mers with translated amino acids are given for each fragment relative to <i>A/chicken/Beijing/1/94</i> (AF156380/EPI_ISL_1270); accuracy is the number of correct lineage annotations over the total number of tested HA nucleotide sequences; BER is the balanced error rate.</p

Annotation tree for LABEL's H9N2 annotation module.

<p>Each internal node corresponds to an annotation level (classification step) within the hierarchical annotation process. Accordingly, HMM profiles and SVM classes used by the H9 module are represented by all non-root nodes (color circles). The “c-<i>X</i>” notation stands for “cluster <i>X</i>,” where <i>X</i> is some general group of clades. Clades are named according to historical names and for representative sequences.</p

Two-dimensional scatterplots of profile HMM negative log-likelihood scores for H5N1 hemagglutinins in clades <i>1</i> (green circles) and <i>1.1</i> (red triangles) along with those in a <i>1.1-like</i> group (blue stars).

<p>(A) Plot shows scores for the clade <i>1.1</i>-specific pHMM (Y-axis) versus scores for the clade <i>1</i>-specific pHMM (X-axis). (B) As in A, but with the X-axis containing scores for the <i>1.1-like</i> pHMM instead. Smaller (more negative) numbers are considered better fits for that clade or group.</p

Hemagglutinin clade annotation for avian influenza subtype A(H5N1) by LABEL.

*<p>Non-redundant H5N1, GISAID, Min. Len. 1200 bp, excluding previous sets, non-laboratory derived.</p>**<p>The cleavage site is not included.</p><p>Start and stop 9-mers with translated amino acids are given for each fragment relative to A/goose/Guangdong/1/96 (AF144305/EPI_ISL_1254); accuracy is the number of correct clade annotations over the total number of tested HA nucleotide sequences; BER is the balanced error rate.</p

Revised 2012 H9N2 nomenclature.

<p>Average pairwise distance (APD) values within and between closely related clades using a <i>p</i>-distance calculation with pairwise deletion. The C-value is the ratio of the between group APD to the within group APD.</p

Two-dimensional scatterplots of profile HMM negative log-likelihood scores for H5N1 hemagglutinins in clades <i>3</i> (green circles) and <i>4</i> (red triangles) along with those in a clade <i>3-like</i> group (blue stars).

<p>(A) Plot shows scores for the clade <i>3</i>-specific pHMM (Y-axis) versus scores for the clade <i>4</i>-specific pHMM (X-axis). (B) As in A, but with the X-axis containing scores for the <i>3-like</i> pHMM instead. Smaller (more negative) numbers are considered better fits for that clade or group.</p

Revised 2012 H9N2 nomenclature references.

*<p>Named for virus not in our dataset that clusters with clade VN222.</p

Structural and Antigenic Variation among Diverse Clade 2 H5N1 Viruses

<div><p>Antigenic variation among circulating H5N1 highly pathogenic avian influenza A viruses mandates the continuous production of strain-specific pre-pandemic vaccine candidates and represents a significant challenge for pandemic preparedness. Here we assessed the structural, antigenic and receptor-binding properties of three H5N1 HPAI virus hemagglutinins, which were recently selected by the WHO as vaccine candidates [A/Egypt/N03072/2010 (Egypt10, clade 2.2.1), A/Hubei/1/2010 (Hubei10, clade 2.3.2.1) and A/Anhui/1/2005 (Anhui05, clade 2.3.4)]. These analyses revealed that antigenic diversity among these three isolates was restricted to changes in the size and charge of amino acid side chains at a handful of positions, spatially equivalent to the antigenic sites identified in H1 subtype viruses circulating among humans. All three of the H5N1 viruses analyzed in this study were responsible for fatal human infections, with the most recently-isolated strains, Hubei10 and Egypt10, containing multiple residues in the receptor-binding site of the HA, which were suspected to enhance mammalian transmission. However, glycan-binding analyses demonstrated a lack of binding to human α2-6-linked sialic acid receptor analogs for all three HAs, reinforcing the notion that receptor-binding specificity contributes only partially to transmissibility and pathogenesis of HPAI viruses and suggesting that changes in host specificity must be interpreted in the context of the host and environmental factors, as well as the virus as a whole. Together, our data reveal structural linkages with phylogenetic and antigenic analyses of recently emerged H5N1 virus clades and should assist in interpreting the significance of future changes in antigenic and receptor-binding properties.</p></div

Structural comparison between H5 hemagglutinins.

<p>(A) Structural alignment of Anhui05 (green), Egypt10 (blue) and Hubei10 (purple) onto Viet04 (yellow) reveals how structurally related these clades are. (B) Alignment of the receptor-binding site (RBS) reveals conserved structural features and residues. (C) Compared to Viet04, a total of eleven residue differences in and around the RBS are present. Amino acid residues in each structure are numbered consecutively according to the ectodomain fragment of the mature HA1 protein. *Deletion of Leu129 in Egypt10 produces a shift in the numbering of residues 129–324 in Egypt10 relative to structurally equivalent residues in Anhui05 and Hubei10.</p