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

Avidity of Sh2 (WT) and Sh2 V186K-K193T-G228S variant HA for N-linked glycan receptors assessed by glycan ELISA.

<p>Sh2 (upper panels) binds strongly to avian-type (α2–3) receptors (left, white open shapes) with weaker binding to human-type (α2–6) receptors (right, black closed shapes). Sh2 V186K-K193T-G228S (lower panels) shows vastly reduced avidity for avian N-glycans and increased selectivity for extended glycan receptors to human receptors. Assays are conducted with biantennary, N-linked glycans (N) with one to four LacNAc (LN, Galβ1-4GlcNAc) repeats terminated with sialic acid (S) in α2–3 or α2–6 linkage (SLN_1-4-N). An asialo, mono-LacNAc (LacNAc-biotin, LN-L) was used as a negative binding control.</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

Melting curve of recombinant HA obtained by DSC to determine the thermostability of Sh2, Sh2 V186K K193T G228S, and A/KY/07.

<p>The raw data are depicted in the solid line, while the fitted curve, from which the <i>T</i>_m was derived, is depicted with a dotted line. (A) A summary of the peaks observed during the DSC experiments for each recombinant HA, (B) Sh2, (C) A/KY/07 and (D) Sh2 V186K K193T G228S.</p

H7 Sh2 mutant combinations that also bind to human-type receptors.

<p>Glycan binding analyses of Sh2 H7N9 mutant HAs, V186N G226S (A) and V186N N224K G228S (B). The mean signal and standard error were calculated from six independent replicates on both the PAA (left column) and the sialoside array (right column). Tissue binding to either chicken or human tracheal sections is observed by HRP-staining (right column). In the PAA array, 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. In the sialoside array 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>). The sialoside array, ELISA-like assay and tissue binding experiments shown are representative of three independent assays performed with different batches of HA proteins.</p

Change of intraoperative core temperature during operations.

<p>Patients’ core temperature was measured at the tympanic membrane beginning every 15 minutes after the induction of anesthesia until the end of the operation. A total of 3132 subjects were enrolled in the study; each curve represents a connection of 5 percentiles, 25 percentile, 50 percentile, 75 percentile and 95 percentile of core temperature at a specific time point. The time interval is 15 minutes.</p

Risk factors associated with intraoperative hypothermia.

<p>Risk factors associated with intraoperative hypothermia.</p

Incidence of intraoperative hypothermia, patient warming and clinical outcome (N = 3132).

<p>Incidence of intraoperative hypothermia, patient warming and clinical outcome (N = 3132).</p

Outcome of hypothermia and normothermia.

<p>Outcome of hypothermia and normothermia.</p

SDS-PAGE of A/bat/Peru/10 HA0 (lanes 1 to 4) and its mature HA (lanes 5 to 8) in trypsin susceptibility assay.

<p>A/bat/Peru/10 HA with a monobasic cleavage site was expressed in its HA0 form in a baculovirus expression system. Lanes 1 and 2 show A/bat/Peru/10 native HA0 at pH 8.0 and pH 4.9, respectively, while lanes 3 and 4 show the equivalent reducing gel of HA0 treated with trypsin at pH 8.0 and pH 4.9, respectively. Similarly, lanes 5 and 6 show A/bat/Peru/10 mature HA at pH 8.0 and pH 4.9, respectively, while lanes 7 and 8 show the equivalent reducing gel of mature HA treated with trypsin at pH 8.0 and pH 4.9, respectively.</p