Figure 4. Inhibition of HA-mediated syncytia formation by chicken immune sera.

(a) Diagrams of chimeric hemagglutinins. The chimeric H1/H5 hemagglutinin was constructed by swapping the globular head domain located between C42 and C274 of A/chicken/Yamaguchi/8/2004 (H5N1) HA_H5 (grey) with that of the corresponding region from A/duck/Italy/1447/2005 (H1N1) HA_H1 (green). The chimeric H5/H1 hemagglutinin was constructed accordingly by replacing the HA_H1 globular head domain with the corresponding HA_H5 domain. The proteolytic cleavage site (arrow), fusion peptide (FP) and transmembrane (TM) domain are indicated. (b) Flow cytometric analysis of BHK-21 cells expressing parental and chimeric HAs. Cells were infected with either VSV*ΔG (blue graphs) or VRPs expressing the indicated HAs (red graphs). At 6 hours p.i., cells were stained with chicken immune sera specific for either HA_H5 or HA_H1 and goat anti-chicken IgY Alexa-546 conjugates. (c) Inhibition of syncytia formation. Vero cells were infected with VRPs expressing the indicated HAs using an MOI of 5 ffu/cell. At 5 hours p.i., cells were treated for 60 minutes with acetylated trypsin to proteolytically activate HA_H1 and HA_H5/H1. At 6 hours p.i., the cells were incubated for 30 minutes at 37°C with the indicated chicken immune sera (diluted 1∶20 in MEM), exposed for 5 minutes at 37°C to pH 5.4, further incubated in medium for 2 hours, and fixed with paraformaldehyde. The nuclei were stained with DAPI (blue fluorescence). Expression of eGFP is indicated by green fluorescence. Scale bar represents 20 µm.