Recombinant HA proteins from human and avian influenza viruses bind <i>ex vivo</i> human tracheal epithelium in a sialic acid dependant manner.

<p>Histological sections of <i>ex vivo</i> human tracheal epithelium were probed with human H3 (A), avian H5 (B) or H5 mutants, (C) G228S, and (D) Q226L/G228S, with and without prior neuraminidase (NA) treatment. Slides were pre-incubated for 3 hours with 5 units of recombinant NA cloned from <i>Salmonella typhimurium</i> LT2 (NEBL) that shows a 260-fold preference for α2-3 over α2-6 linked SA or with 15 units of recombinant NA cloned from <i>Clostridium perfringens</i> (NEBL) that cleaves both α2-3 and α2-6 SA linkages. HA was detected as before using anti-human Fc (red) and anti-acetylated α-tubulin was used to indicate ciliated cells (green). White arrows indicate cells that exhibit HA binding. Open arrowheads indicate ciliated cells with HA binding; solid arrowheads indicate non-ciliated cells with HA binding.</p

Nucleotide sequence changes required to switch H5 HA receptor binding preference.

<p>(A) Analysis of codon usage by 1374 H5 influenza viruses indicates that codon 228 is GGA in 93% strains and GGG in 7%. The table illustrates transversions and transitions by which coding capacity at this residue might change from glycine to serine. The ability of the two intermediate mutants 228R (B) and 228A (E) recombinant HA proteins to bind synthetic receptor ligands 3SLN (avian receptor) and 6SLN (human receptor) in a solid phase assay was assessed. The binding phenotype on HAE (C,F) or human tracheal epithelium (D,G) by recombinant proteins with each amino acid sequence changed at residue 228 are shown (C,D) G228R and (F,G) G228A.</p

Expression of recombinant HA-Fc proteins.

<p>(A) Recombinant baculoviruses encoding HA from a recent H3N2 human virus, (A/Panama/2007/99) or from a highly pathogenic H5N1 avian influenza virus (A/Vietnam/1194/04), were generated as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007836#pone.0007836-Barclay1" target="_blank">[32]</a>. The HA proteins were expressed as soluble proteins secreted from infected <i>Sf9</i> cells by removing the HA transmembrane (TM) and cytoplasmic tail (CT) portions of the protein and replacing the HA signal peptide (SP) with the signal peptide of the baculovirus envelope protein gp64 (SP gp64). The HA proteins were tagged at the C-terminus by a human Fc (HuFc) and hexa-histidine (His6) tags. (B) All recombinant HA proteins were expressed at similar levels as determined by western blot analysis using an anti-human Fc antibody.</p

Binding of recombinant HA proteins to synthetic receptor ligands 3SLN (avian receptor) and 6SLN (human receptor) in a solid phase assay.

<p>(A) Synthetic glycans on polyacrylamide linkers were immobilized on 96-well plates following UV treatment. Recombinant HA proteins were preformed into higher order complexes by incubation with anti-human Fc before incubation on the plate and detection with goat anti-human IgG conjugated to horse-radish peroxidise (HRP). (B) Recombinant HA-Fc proteins were adsorbed on 96 well plates coated with anti-human Fc antibody. Synthetic glycans on polyacrylamide linkers with biotin tags (6SLN and 3SL) were incubated on the plates and detected with Streptavidin – HRP conjugate.</p

Recombinant HA proteins bind to human airway epithelial (HAE) cultures.

<p>(A) Morphological features of HAE cultures visualized by H&E counterstain. Cell types present include ciliated cells (C), mucin-secreting cells (MS) and non-ciliated cells (NC). HAE cultures were probed with recombinant HA proteins from human H3 (A/Panama/2007/99) (B,C) or avian H5 (A/Vietnam/1194/04) (D,E) viruses either directly to the apical surface of fixed cultures (B,D) or to histological sections (C,E). Receptor binding site mutants of H5 HA (F–J) were also analysed for binding to histological sections. Ciliated cells were identified using anti acetylated α-tubulin (green) and the HA-Fc proteins were visualized with anti human-Fc (red). Images are representative of cell-type binding seen in experiments. (K) Numbers and types of epithelial cells bound by HA proteins were quantified by counting 100–200 total cells from five different fields of view.</p

Infection of HAE by avian, but not human, influenza viruses is restricted at temperatures of the proximal airways.

<p>(A) Comparison of multi-cycle virus growth in HAE inoculated with either A/Victoria/3/75 at 32°C (<i>closed triangles</i>) or 37°C (<i>open triangles</i>) and A/Dk/Eng/62 at 32°C (<i>closed circles</i>) or 37°C (<i>open circles</i>) both at MOI∼0.01. Apical viral titers at times shown were determined by standard plaque assay on MDCK cells. Data shown represents the mean titer +/−standard error (SE; n = 3–10 cultures). (B) Adenylate kinase activity released into the apical compartment of HAE over time after inoculation with A/Victoria/3/75 or A/Dk/Eng/62 at 32°C and 37°C as a measure of viral-induced CPE. Data shown represents the mean fold change over adenylate kinase activity derived from mock-inoculated HAE +/−SE (n = 3–8). Significance is noted (*p<0.05) where viral titers or AK levels obtained for A/Dk/Eng/62 at 32°C were statistically different from all other titers/AK measurements (Dk/37°C, Vic/32°C and Vic/37°C) at that particular time point. Significance is noted (^†p<0.05) where AK levels obtained for A/Dk/Eng/62 at 32°C and 37°C were statistically different.</p

Cell tropism of human, avian and avianized viruses in HAE.

<p>Representative cross-sections of inoculated HAE, fixed 24 hrs pi, were probed for viral antigen (NP; green) and α−acetylated tubulin, a marker for ciliated cells (red). Notably, the staining pattern for wild-type A/Victoria/3/75 was identical to that of PR8+Vic HA/NA. Arrows mark ciliated cells infected with either wild-type A/Victoria/3/75 or PR8+Vic HA/NA; arrow-head denotes non-ciliated cells infected by these viruses. These data indicate that viruses with Victoria glycoproteins were able to infect both cell types previously shown to express α2,6 SA <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000424#ppat.1000424-Thompson1" target="_blank">[13]</a>. Viral antigen was detected only in ciliated cells in cultures inoculated with Vic-226-228HA (in the Victoria background with either endogenous N2 or avian N1 or PR8+Chick HA/NA). Scale bar equals 20 µm.</p

Spread and histopathology of avian and human influenza viruses in HAE at temperatures of the proximal and distal airway.

<p>(A) Representative <i>en face</i> photomicrographs of HAE inoculated with either A/Victoria/3/75 or A/Dk/Eng/62 at 32°C or 37°C, fixed at 6, 24, 48 and 72 hrs pi and stained for viral nucleoprotein (<i>green</i>) to determine numbers of cells infected. Scale bar equals 100 µm. (B) Representative histological cross-sections of HAE at 24, 72 and 120 hrs after inoculation with A/Victoria/3/75 or A/Dk/Eng/62 at 32°C or 37°C. H&E counterstain. Scale bar equals 20 µm.</p

Temperature restriction of avian influenza viruses at 32°C can be mimicked by inserting avian envelope glycoproteins into human influenza viruses.

<p>(A) Multi-step growth kinetics initiated in HAE over time with PR8+Vic HA/NA at 32°C (<i>closed triangles</i>) or 37°C (<i>open triangles</i>) and PR8+Chick HA/NA at 32°C (<i>closed circles</i>) or 37°C (<i>open circle</i>s) in HAE. Apical viral titers were determined at the times shown by standard plaque assay. Data shown represents mean titer across 4–8 cultures +/−SE. (B) Adenylate kinase activity in apical washes of virus-infected HAE expressed as fold-change over adenylate kinase activity in mock-inoculated HAE +/−SE (n = 4–8). Significance is noted (*p<0.05) where viral titers or AK levels obtained for PR8+Chick HA/NA at 32°C were statistically different from all other titers/AK measurements (Chick/37°C, Vic/32°C and Vic/37°C) at that particular time point. Significance is noted (^†p<0.05) where AK levels obtained for PR8+Chick HA/NA at 32°C and 37°C were statistically different. (C,D) Representative <i>en face</i> photomicrographs of viral nucleoprotein immunoreactivity (<i>green</i>) in HAE inoculated with (C) PR8+Vic HA/NA or (D) PR8+Chick HA/NA, at 24, 48 and 72 hrs pi at 32°C (lower rows) or 37°C (upper rows).</p

Temperature-dependent growth of different serotypes of influenza viruses in HAE.

<p>Multi-step growth kinetics of (A) human influenza virus A/Eng/26/99 or (C) avian influenza virus A/Dk/Sing/97 (MOI∼0.1) at 32°C (<i>open circles</i>, <i>dashed line</i>) or 37°C (<i>closed circles</i>, <i>solid line</i>) in HAE +/−SE (n = 3 cultures). Multi-step growth kinetics in HAE inoculated with an MOI∼0.03 of (B) A/Udorn/307/72 (H3N2) or (D) A/VN/1203/04 (H5N1) at 33°C (<i>open circles</i>, <i>dashed line</i>) or 37°C (<i>closed circles</i>, <i>solid line</i>). Data represents mean titer across two different donors, each performed in duplicate +/−SE. Viral titers were determined by plaque assay in (A) and (B) and by TCID₅₀ assay for (C) and (D). No significant differences in growth between temperatures were found for either A/Eng/26/99 or A/Udorn/307/72. A/VN/1203/04 was significantly restricted for growth at 24, 48 and 72 hrs pi (*p<0.05). (E) Representative histological cross-sections of HAE infected for 72 hrs at 37°C with A/Udorn/307/72 or A/VN/1203/04 and compared to mock-inoculated HAE. H&E counterstain. Scale bar equals 20 µm.</p