Expression of the H1 or N1 proteins by recombinant vaccinia viruses was detected by Western blotting. Vero cells in case of the VV-L constructs, or the avian cell line DF-1 [15] in case of MVA, were infected at a multiplicity of infection of 0.1 for 48 hours. MVA-H1-Ca or rVVL-H1-Ca infected cells were harvested by scraping or by adding trypsin. MVA-N1-Ca or rVVL-N1-Ca infected cells were harvested by scraping. Sonicated cell lysates were loaded onto 12% polyacrylamide gels and afterwards blotted on nitrocellulose membrane. To detect the H1 protein, a sheep antiserum against the A/California/7/2009 hemagglutinin (NIBSC 09/152) was used. Donkey-anti-sheep alkaline phosphatase-conjugated IgG was used as a secondary antibody. To detect the N1 protein ab21304 was utilized. Goat-anti-rabbit alkaline phosphatase-conjugated IgG was used as a secondary antibody. A whole virus vaccine H1N1 A/California/7/2009 [16] served as positive control.
Neuraminidase expression in chicken cells.