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. 2023 Jul 7;14:1177324. doi: 10.3389/fimmu.2023.1177324

Figure 1.

Figure 1

In vitro characterization of MVA-NS1. (A) Homologous and intragenomic homologous (marker gene deletion) recombinations lead to production of MVA-NS1 with TBEV NS1 expression under transcriptional control of VACV late promotor psynII. (B) PCR products specific for the six major deletion sites inside the MVA genome performed on MVA-NS1 (1% agarose TBE gel) (I: 291 bp, II: 354 bp, III: 447 bp, IV: 502 bp, V: 603 bp, VI: 702 bp). Integration of the NS1 gene in deletion site III was confirmed (III: 1596 bp). (C) Immunostaining of wtMVA or MVA-NS1 infected HeLa cells (MOI 1). 24 hpi, cells were fixed with 4% PFA, for intracellular staining permeabilized with TritonX®-100 and immunostained against VACV D8 and TBEV NS1 (20x magnification). (D) Western blot analysis of whole cell lysate from HeLa cells infected with wtMVA or MVA-NS1 for 24 h (MOI 5). Blots were stained against TBEV NS1. For control, antibodies against VACV D8 and GAPDH were included. (E) Growth curves of wtMVA (black) and MVA-NS1 (gray) on primary CEF cells (dotted line) and HeLa cells (solid line) (MOI 0.05). Mann-Whitney test was used for statistical comparison between CEF and HeLa cells (* p<0.05).