Analysis of empty IAPV capsids.

<p><b>A</b>. Sucrose gradient analysis of Sf9 cell extracts infected with recombinant baculovirus ORF2-5TFS-3C. Each fraction represents a 5% step from 30% to 60% sucrose w/v and is blotted with the IAPV anti VP2 serum. <b>B</b>. EM analysis of peak fractions from the sucrose gradient shown in A. Empty capsids are indicated. A damaged particle is labelled D. Baculovirus nucleocapsids are labelled B. The bar is 100 nm.</p

Design and test of baculovirus expression cassettes.

<p><b>A</b>. Cartoon representations of the various genetic constructs used to assess IAPV antigen expression in infected Sf9 cells. The sequences used are identified. FS- the HIV-1 frameshift site, the polypyrimidine tract of which is indicated. 3C-pro – the IAPV 3C like protease. <b>B</b>. Western blots of Sf9 cells infected for 2 days with recombinant baculoviruses constructed with the cassettes shown in A. In B the upper panel was blotted with rabbit anti-VP2 serum, the middle panel with anti-gp64 and the lower panel is the relative VP2:gp64 level. Numbers to the left of the blots are protein molecular mass markers (M) and are in kilodaltons. The expected position of the blotted antigens is indicated.</p

The IAPV genome and origin of sequences used for IAPV fragment expression.

<p><b>A</b>. Genome structure of IAPV dicistrovirus with the two open reading frames and their constituent mature proteins shown. IRES – internal ribosome entry site, IGR – intergenic region. <b>B</b>. The fragments targeted for expression in <i>E.coli</i> and their encoded products. The precise endpoints used were: VP2, VTH→MQC; VP2-4, VTH→FGW; VP3, SKP→ELQ; VP1, INI→ISR. Expression screening for the proteins predicted from the fragments shown in B. <b>C</b>- left. Western blot with anti-His antibody. <b>C</b>-right. Purified VP2 used for the generation of a rabbit serum. Numbers to the left of the blot are protein molecular mass markers and are in kilodaltons.</p

Development of an IAPV capture ELISA.

<p><b>A</b>. Pepscan of 4 monoclonal antibodies to IAPV VP2 on an overlapping peptide library. Filled bars IAPVMAb8; stipple bars IAPVMAb12; striped bars IAPVMAb17; open bars IAPVMAb 27. The relationship between peptide identity and VP2 structure is shown. <b>B</b>. Twin site capture ELISA using capture MAbs 8 (triangles) and 27 (diamonds) as capture layer and probing with HRP-labelled IAPV MAb12. The test sample was a lysate of Sf9 cell infected with the baculovirus expressing ORF-2-5TFS-3C.</p

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

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

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

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