Journal: Virology

Article Title: Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate ☆

doi: 10.1016/j.virol.2013.07.038

Figure Lengend Snippet: (A) Sequence similarity of Marburg virus 371Bat isolate to other known bat (B) and human (H) isolates. Sequences obtained from Genbank (FJ750958, FJ750957, DQ217792, JN408064, JX458853, DQ447649, JX458857, FJ750953) were aligned and the nucleotide similarity was determined using Mega5 (Tamura et al., 2011). (B) Basic organization of the marburgvirus genome. The seven ORFs are depicted in the viral complementary sense (5′ to 3′). (C) 5′ and 3′ terminal sequences currently reported for 371Bat isolate (top, Genbank #FJ750958) compared to sequencing results of 371Bat isolate obtained by RACE (bottom). Chromatograms are displayed below sequences, and blue boxes indicate where RACE sequence diverges from reported sequence. (D) Schematic of GFP mini-genome plasmid with 371Bat 5′ and 3′ UTRs (black line), and viral protein expression plasmids used in both mini- and full-length genome experiments (dashed line indicates pCAGGS plasmid sequence).

Article Snippet: Moreover, the full-length 371Bat Marburg virus genome was cloned into the same T7 transcription plasmid in viral (genome) sense. (C) GFP-expressing full-length clones.

Techniques: Sequencing, Virus, Plasmid Preparation, Expressing

Journal: Virology

Article Title: Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate ☆

doi: 10.1016/j.virol.2013.07.038

Figure Lengend Snippet: Replication of 371Bat-based GFP mini-genome. GFP expression was assessed as an indicator of mini-genome replication in BSR-T7/5 cells using (A) wild type and (B) codon-optimized viral protein support plasmids. Images were taken either four days (A) or two days (B) post-transfection.

Article Snippet: Moreover, the full-length 371Bat Marburg virus genome was cloned into the same T7 transcription plasmid in viral (genome) sense. (C) GFP-expressing full-length clones.

Techniques: Expressing, Transfection

Journal: Virology

Article Title: Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate ☆

doi: 10.1016/j.virol.2013.07.038

Figure Lengend Snippet: Rescue and characterization of recombinant 371Bat MARV using full-length viral complementary genomes. (A) Wild type recombinant MARV (rMbg) was rescued in BHK-21 cells using the full-length genome depicted, T7 polymerase plasmid, and codon-optimized viral support protein plasmids NP, VP35, VP30, and L. Efficiency of rescue in a single experiment is shown as wells positive/total wells, and average titer was assessed by standard TCID50 assay. VeroE6 cells were infected with rMbg virus, fixed 4 days post-infection, and stained for immunofluoresence assay (IFA) using a polyclonal rabbit anti-Marburg antibody followed by anti-rabbit Alexa-Fluor 594. (B) A recombinant MARV containing the GFP ORF in the intergenic region of NP and VP35 (rMbg/GFP) was rescued under similar conditions as wild type. VeroE6 cells were infected with rMbg/GFP virus and visualized for GFP expression (left) or subjected to the same immunofluorescence assay as in (A) (middle, merged images at right). (C) Wild type 371Bat (Mbg (wt)), rMbg, and rMbg/GFP viruses were used to infect VeroE6 cells at an MOI of 0.01 to assess growth kinetics. Adsorption was allowed for 1 h, after which cells were washed with PBS and given complete growth media. At time points indicated, supernatant was taken and titered by TCID50 assay on VeroE6 cells.

Article Snippet: Moreover, the full-length 371Bat Marburg virus genome was cloned into the same T7 transcription plasmid in viral (genome) sense. (C) GFP-expressing full-length clones.

Techniques: Recombinant, Plasmid Preparation, TCID50 Assay, Infection, Virus, Staining, Expressing, Immunofluorescence, Adsorption

Journal: Virology

Article Title: Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate ☆

doi: 10.1016/j.virol.2013.07.038

Figure Lengend Snippet: (A) Infection of human macrophages with wild type or recombinant MARV. CD14+ monocytes were allowed to mature to macrophages for 7 days and infected with indicated viruses at an MOI of 0.1. IFA was performed as in Fig. 3. (B) Growth kinetics of wild type or recombinant MARV in CD14+ macrophages. Titers were obtained on VeroE6 cells as in Fig. 3C. (C) Pro-inflammatory responses of human macrophages infected with wild type or recombinant marburgviruses. Seven days post-maturation, cells were infected with the indicated viruses at an MOI of 2 and total RNA was harvested 24 h later. Expression of inflammatory-related genes was assessed using a custom qRT-PCR array, and plotted as fold induction over mock-infected cells.

Article Snippet: Moreover, the full-length 371Bat Marburg virus genome was cloned into the same T7 transcription plasmid in viral (genome) sense. (C) GFP-expressing full-length clones.

Techniques: Infection, Recombinant, Expressing, Quantitative RT-PCR