Protein expression and histochemistry.

<p>(A) Amino acid homology of pigeon CoV S1 and partridge CoV S1 to chicken CoV S1 (strain M41-S1); (B) Purified recombinant proteins analyzed by SDS-PAGE and western blotting using an antibody against the <i>Strep</i>-tag; (C) Schematic presentation of protein/spike histochemistry using TMAs.</p

Development of avian tissue microarrays (TMA).

<p>(A) Tissue cores from similar organs from ten avian species were grouped in multispecies TMAs and tissues from one species were grouped in single-species TMA; (B) representative example of arrangement of tissues in multi-species TMA stained with H&E.</p

Tissue binding profiles of S1 of chicken, pigeon, and partridge CoV to the organ systems of the respective host.

<p>Attachment was graded as follows:—no signal, + mild, ++moderate, +++ strong</p><p>Tissue binding profiles of S1 of chicken, pigeon, and partridge CoV to the organ systems of the respective host.</p

Host specificity of HA of influenza virus H5N1.

<p>HA proteins (1 μg/ml) were applied to multispecies TMA containing respiratory tissues. Binding of HA to trachea of avian species from order <i>Galliformes</i> (A), <i>Anseriformes</i> (B), and <i>Columbiformes</i> (C) is shown.</p

Host tissue specificity of S1 proteins of coronaviruses.

<p>Protein histochemistry was performed using chicken, pigeon and partridge CoV S1 on tissues from their respective hosts.</p

Host binding specificity of chicken CoV M41 S1.

<p>Protein histochemistry was performed by applying S1 proteins onto the trachea in the multispecies TMA. Binding affinity of chicken CoV S1 to different avian species is indicated as—(no signal), + (mild), ++ (moderate), +++ (strong). To elucidate the fine glycan specificity of S1 to sialylated glycans. S1 protein was premixed with either sialic acids (SA) type I or type II lactosamines before applying to tissues as described in materials and methods.</p

Glycan binding specificities of pigeon, partridge and chicken CoV S1.

<p>Before applying S1, TMAs were either treated non treated (S1) or pretreated with neuraminidase (NA), or S1 proteins were mixed with sialic acids (SA) type I or type II lactosamines.</p

Binding profiles of chicken CoV S1 in respiratory tissues of various avian species.

<p>Attachment of S1 was graded as follows:—no signal, + mild, ++moderate, +++ strong</p><p>Binding profiles of chicken CoV S1 in respiratory tissues of various avian species.</p

Specificity of wild type and mutant H7 HAs on glycan arrays and binding to chicken and human trachea epithelium.

<p>Glycan binding analyses of Sh2 H7N9 HA wild type and several mutants that confer human-type receptor binding: G228S, K193T G228S, V186K K193T G228S, V186G K193T G228S, with human Cal/04/09 2009 pandemic H1N1 HA as a control. (A) ELISA-like assay using sialoside polymers. The mean signal and standard error were calculated from six independent replicates; white open circles represent α2–3 linked sialylated di-LacNAc (3’SLNLN), black closed circles represent α2–6 linked sialylated di-LacNAc (6’SLNLN), and non-sialylated di-LacNAc (LNLN) are represented in asterisks. (B) The glycan array mean signal and standard error were calculated from six independent replicates; α2–3 linked sialosides are shown in white bars (glycans 11 to 79 on the x axis) and α2–6 linked sialosides in black (glycans 80 to 135). Glycans 1 to 10 are non-sialylated controls (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006390#ppat.1006390.s001" target="_blank">S1 Table</a>). (C) Tissue binding to either chicken or human tracheal sections is observed by HRP-staining. The sialoside array, ELISA-like assay, and tissue binding experiments are representative of three independent assays performed with different batches of HA proteins.</p

Amino acid variation in the receptor binding pocket of influenza HAs and impact of K193T mutation on receptor conformation.

<p>(A) Variation at HA positions that are known to mediate the switch in receptor binding specificity for human H1, H2 and H3 pandemic viruses and corresponding avian viruses of H1, H2, H3 and H5 subtypes in comparison with human H7N9. Red indicates amino acids involved in either human- or avian-type receptor specificity, blue indicates amino-acid positions that are mutated to the amino acids found in human H3N2 and H2N2 viruses. (B) Projection of the receptor glycan from the binding pocket. The receptor analog 6’SLNLN (α2–6 linked sialylated di-LacNAc; NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc) is modeled in the WT H7 with K193 (dark gray), and the mutant H7 with V186K K193T G228S (light gray). In the WT, K193 causes the receptor to project further away from the 190 helix. Symbols in the sugar rings are the conventions for the Symbol Nomenclature For Glycans (SNFG) where sialic acid is the purple cubic diamond, galactose is the yellow sphere and GlcNAc is the blue cube.</p