FIG. 8.

Membrane-bound chicken-derived GM-CSF fused to the viral HA is readily expressed in a bioactive form and is packaged into influenza virions. (A) Chicken-derived GM-CSF was fused inframe to the carboxy-terminal domain encoding for a short stalk, the transmembrane and cytoplasmic tail regions of the viral HA and inserted into pcDNA3.1 expression plasmid (pcDNA3.1-chGM-CSF/HA). Transfected MDCK cells were selected with Geneticin and cloned by limiting dilution. Each subclone was tested for GM-CSF bioactivity by coculturing mitomycin C-pretreated cells with chicken bone-marrow cells for 3 days at 40^°C, the last 20 h with ³H-thymidine and measuring thymidine incorporation (triplicates). Those subclones inducing the greatest uptake of thymidine are compared with nontransfected MDCK (MDCK-wt) and soluble chicken GM-CSF (1/100 dil of supernatant from transfected COS-7 cells). (B) Influenza virus A/PR/8 (H1N1) was harvested from infected (moi = 1) chGM-CSF/HA expressing MDCK (subclone 39) or wild-type MDCK, concentrated and inactivated with β-propiolactone. Dilutions of the virus were evaluated for GM-CSF bioactivity in a chicken bone marrow proliferation assay. Soluble chicken GM-CSF served as a positive control. Samples were tested in triplicate for ³H-thymidine uptake.