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. 2014 Sep 22;211(5):791–800. doi: 10.1093/infdis/jiu528

Figure 1.

Figure 1.

Construction of the modified vaccinia virus Ankara (MVA) H7-Sh2 vector (A–E) and H7 protein expression (F). The hemagglutinin (HA) gene sequence of influenza virus A/Shanghai/2/2013 (H7-Sh2) underwent codon optimization, obtained as synthetic gene (A), and cloned between the flank 2 and flank 1 regions of the MVA vector plasmid pMKIIIred under control of the PsynII promoter and containing the mCherry sequence as a marker gene, resulting in pMKIIIred-H7 (B). Subsequently, chicken embryo fibroblasts (CEFs) were infected with MVA (C) and transfected with pMKIIIred-H7 DNA, using Fugene HD (Promega, Leiden, the Netherlands) to generate recombinant MVA containing the H7-Sh2 sequence and mCherry as a fluorescent marker, inserted in the MVA genome through homologous recombination (D). Finally, the mCherry marker gene was removed from the viral genome by means of a second step of intragenomic homologous recombination, resulting in the final marker-free recombinant MVA-H7-Sh2 (E). H7 protein expression was confirmed by Western blot analysis of cell lysates from MVA-H7-Sh2–infected baby hamster kidney cells (BHK-21), using a hyperimmune rabbit serum against influenza virus A/Seal/Massachussets/1/80 and a goat anti-rabbit IRDye Infrared antibody (Westburg, Leusden, the Netherlands. F, Lane c, negative control; lane 1, MVA-H7-Sh2 1:50; lane 2, MVA-H7-Sh2 1:10; lane 3, MVA-H7-Sh2 undiluted).