PAM were infected with specific viruses at specified MOIs for 2 h in supplemented serum-free RPMI-1640 with 250 ng/ml TPCK trypsin (Sigma). After 2 h, cells were rinsed three times with PBS and incubated in fresh medium for a further 4 h. Quantification of infectious virus in PAM culture supernatants was conducted as previously described21 (link) which was an immuno-cytochemical focus forming assay based on infection of MDCK cells followed by immunodetection of viral nucleoprotein (NP) expression. Briefly, MDCK cells infected for 6 h were fixed in acetone methanol for 10 min followed by peroxidase treatment for 10 min and incubation with a 1:8000 dilution of primary mouse monoclonal antibody to influenza nucleoprotein (Abcam) for 40 min at room temperature. The cells were subsequently rinsed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labelled polymer for 40 min. After gently rinsing with TBS, the cells were incubated with DAB substrate-chromogen solution for 7 min (Envision + system-HRP kit, Dako). Cells positive for viral nucleoprotein were counted with an inverted microscope and the mean of positive cells in four 96-wells was used to calculate infectious focus-forming units of virus per microlitre of infection volume.
Free full text: Click here
