primary chicken embryo fibroblasts (cef)  (Fort Dodge Animal Health)

Journal: Vaccines

Article Title: Protective CD8+ T Cell Response Induced by Modified Vaccinia Virus Ankara Delivering Ebola Virus Nucleoprotein

doi: 10.3390/vaccines10040533

Figure Lengend Snippet: 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.

Article Snippet: Briefly, the MVA clonal isolate F6 [ , ] was used as virus starting material and propagated on primary chicken embryo fibroblasts (CEF) obtained from 10-day old embryonated SPF chicken eggs (Valo BioMedia GmbH, Osterholz-Scharmbeck, Germany).

Techniques: Recombinant, Plasmid Preparation, Control, Virus, Marker, Sequencing, Expressing, Homologous Recombination, Infection, Western Blot, SDS Page

Journal: Molecular Therapy Oncolytics

Article Title: Development of a Safe and Effective Vaccinia Virus Oncolytic Vector WR-Δ4 with a Set of Gene Deletions on Several Viral Pathways

doi: 10.1016/j.omto.2017.12.002

Figure Lengend Snippet: Characterization of In Vitro Infection and Replication of the Mutant Deletion Viruses (A) Plaque size was measured in a BSC-40 monolayer (solid agar medium) at 48 hpi (MOI 0.005 PFU/cell) and the area given in a.u. (B–E) Replication capacity was analyzed through viral growth curves by recovering infected monolayers (MOI 0.01 PFU/cell) at different time points (0, 8, 24, 32, 48, and 72 hpi) followed by titration; the cell lines used were (B) CEF, (C) BSC-40, (D) B16F10, and (E) TRAMP-C1. (F) Spheroids of TRAMP-C1 cells were infected (MOI 2 PFU/cell) and observed after 6 days. Left: white light; center: fluorescence filter for GFP; right: H&E-stained histological sections. Scale bars, 500 μm. Data are shown as mean ± SD. **p < 0.01; ***p < 0.005.

Article Snippet: Cercopithecus aethiops kidney cells (BSC-40; American Type Culture Collection [ATCC]), B16F10 melanoma cells (ATCC, Barcelona, Spain), and chicken embryo fibroblast (CEF) primary cells (Intervet, Salamanca, Spain) were cultured in DMEM supplemented with 10% (v/v) newborn calf serum (NCS), or with fetal calf serum (FCS) for CEF cells.

Techniques: In Vitro, Infection, Mutagenesis, Titration, Fluorescence, Staining