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. 2022 Mar 29;10(4):533. doi: 10.3390/vaccines10040533

Figure 1.

Figure 1

Generation of recombinant MVA-EBOV viruses. (A,B) Schemes of the MVA genome with the intergenic insertion site 069R-070L (A) or the major deletions sites I-VI (B). Flank-1 and flank-2 refer to MVA DNA sequences targeting the intergenic site 069R-070L (A) or deletion site III (B) in the MVA genome for insertion of recombinant genes. MVA vector plasmids contain recombinant EBOV NP or GP gene sequences, respectively, under transcriptional control of the vaccinia virus promoter PmH5 and a marker gene sequence for transient expression of the fluorescent protein GFP (A) or mCherry (B). Short repetitive sequences of MVA DNA (del) served to remove the marker genes by intragenomic homologous recombination (marker gene deletion). (C,D) Multiple-step growth analysis of recombinant MVA-EBOV viruses. Growth of recombinant viruses MVA-EBOV-NP (C) or MVA-EBOV-GP (D) was monitored upon infection of chicken fibroblast cells (C,E,F); hpi: hours post infection. (E,F) Synthesis of recombinant EBOV NP (E) and EBOV GP (F) was tested by Western blot analysis using cell lysates and supernatants from infected CEF cells. Polypeptides were separated by SDS–PAGE and tested by immunoblotting using either EBOV NP- or GP-specific antibodies. Uninfected cells (mock) or non-recombinant MVA-infected cells (MVA) served as controls.