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: Synthetic genes containing codon-optimized NP, VP35, VP30, L, and T7 RNA polymerase ORFs were obtained from a commercial source (GenScript) and cloned into pCAGGS similarly to the wild type ORFs.

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

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: a IgM, IgG, and IgA antibody epitope repertoire recognized in the human plasma at different months post-MARV infection (pi) during convalescence (12-, 30-, 43- and 60-months pi are color coded) and their alignment to the whole proteome of MARV (showing different proteins: NP, VP35, VP40, GP, VP30, VP24 and L). Graphical distribution of representative clones with a frequency of ≥2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone to its homologous sequence in the MARV proteome on alignment. The thickness of each bar represents the frequency of repetitively isolated phage, with the scale shown below the alignment. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and similar number of phage clones and epitope repertoire was observed in both phage display analysis. b Structural representations of immunodominant antigenic sites recognized by IgG using MARV-GFPDL on the surface structure of mature trimeric MARV GP solved structure [PDB #5UQY ]. The different domains of mature GP: GP1 (residues 1334-1769 in cyan), receptor binding site (RBS; residues 1372-1522 in orange), GP2 (residues 1842-2015 in blue), fusion loop (FL; residues 1842-1885 in pink) and N-terminal heptad repeat (NHR; residues 1886-1929 in yellow) are shown on the structure of MARV-GP, with the residue numbers corresponding to the complete MARV proteome used for GFPDL. The MARV GP structure used for crystallography encodes for MARV GPΔmuc ectodomain (encompassing amino acid residues 1–636 with a mucin deletion of residues 257–425). The immunodominant antigenic sites recognized by IgG in MVD survivors identified using MARV-GFPDL that can be mapped on the crystal structure are depicted in red.

Article Snippet: Recombinant Marburg virus-like particles (VLPs expressing MARV trimeric GP on surface of particles made of MARV matrix proteins VP40 and NP) , and MARV GP, NP, VP35, VP40, VP24, VP30 and L polymerase proteins were purchased from MyBioSource, IBT Bioservices, and Creative Diagnostics Inc.

Techniques: Infection, Clone Assay, Selection, Sequencing, Isolation, Binding Assay, Residue

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: Serial dilutions of longitudinal plasma samples collected at 12-months through 60-months post-infection from the MVD survivors ( N = 10; in colors) and uninfected controls ( N = 10; in black) were analyzed for antibody binding to purified recombinant MARV proteins by SPR. Total antibody binding is represented in SPR resonance units (RU) for 10-fold dilution of plasma sample binding to Marburg virus-like particles (VLPs expressing trimeric GP on the surface of particles made of MARV matrix proteins VP40 and NP), and recombinant MARV GP, NP, VP35, VP40, VP24, VP30 and L polymerase. The mean half-life (in months) of the polyclonal binding antibodies against various MARV proteins for the convalescent phase in these MVD survivors is shown, except for VP30 and L, which demonstrated weak antibody binding. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variation for each sample in duplicate SPR runs was <7%. The data shown is the average value of two experimental runs. Source data are provided as a Source Data file.

Article Snippet: Recombinant Marburg virus-like particles (VLPs expressing MARV trimeric GP on surface of particles made of MARV matrix proteins VP40 and NP) , and MARV GP, NP, VP35, VP40, VP24, VP30 and L polymerase proteins were purchased from MyBioSource, IBT Bioservices, and Creative Diagnostics Inc.

Techniques: Infection, Binding Assay, Purification, Recombinant, Virus, Expressing

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: Antibody affinity maturation of longitudinal plasma samples collected from MVD survivors to purified recombinant MARV proteins was determined by SPR. Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the interaction of serially diluted post-infection plasma with MARV proteins using SPR in the dissociation phase only for the sensorgrams with maximum RU in the range of 10-100 RU in the optimized SPR. Antibody affinity was not determined for uninfected control samples or against MARV-VP30 or L as most of the post-infection MVD plasma samples showed very low antibody binding ( < 10 RU) against these two proteins. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variation for each sample in duplicate SPR runs was <7%. The data shown is the average value of two experimental runs. Source data are provided as a Source Data file.

Article Snippet: Recombinant Marburg virus-like particles (VLPs expressing MARV trimeric GP on surface of particles made of MARV matrix proteins VP40 and NP) , and MARV GP, NP, VP35, VP40, VP24, VP30 and L polymerase proteins were purchased from MyBioSource, IBT Bioservices, and Creative Diagnostics Inc.

Techniques: Purification, Recombinant, Infection, Control, Binding Assay

Journal: bioRxiv

Article Title: Genome-wide CRISPR knockout screening with viral replicons for identification of host factors involved in viral replication

doi: 10.1101/2025.01.09.632058

Figure Lengend Snippet: A, Graphical overview of the steps to generate a stable Ebola virus (EBOV) replicon cell line. PiggyBac transposition was applied to generate a “4cis” cell line, in which four EBOV proteins essential for viral replication and transcription (NP, VP35, VP30 and L protein) are stably expressed after integration and subsequent selection in 100 ug/mL hygromycin media. Next, an in vitro transcribed EBOV minigenome RNA (in negative-sense orientation), which encodes the 5ʹ and 3ʹ viral UTRs flanking a reporter-selection (Zeocin-eGFP fusion) cassette, is transfected into the 4cis cell line. Stable population of >90% eGFP + Huh7.5.1-Cas9 EBOV replicon cells were selected and expanded in Zeocin media. B, Validation of the responsiveness of the replicon cell line by CRISPR knockout (KO) of viral L protein and known host factors involved in EBOV entry (Niemann-Pick disease, type C1 [NPC1]) or replication and transcription (dynein light chain LC8-type 1 [DYNLL1]). C, Enrichment plot showing host factor genes recovered from the screen. Enrichment scores are represented as -log10 of the RRA scores obtained from MAGeCK analysis. D, Validation of host factors identified from the screen. Independent KOs of the hits identified in the screen were generated in the EBOV replicon cell line in an arrayed format and flow cytometry was used to measure the eGFP signal over the course of 20 d. The fraction of eGFP + cells on Day 20 of the time course is plotted as mean + SD (three independent technical replicates). E, Quantification of the levels of different types of EBOV minigenome RNA in the replicon cells harboring different host factor knockouts. Total RNA was isolated from replicon cells with different host factor knockouts and used as input for strand-specific RT-qPCR assay. Plotted values are fold expression changes when normalized to the cells expressing non-targeting gRNA (three independent technical replicates). F, Validation of phenotypes in transient EBOV replicon assays in independently generated KOs in parental cell lines. Effect of host factor knock outs on EBOV replication and transcription in a transient replicon assay with a minigenome encoding a secreted nano-luciferase (SecNLuc). Host factor knockout cell lines and controls were co-transfected with an EBOV 4cis plasmid encoding EBOV NP, VP35, VP30, and L protein, and an EBOV minigenome luciferase reporter plasmid. The SecNLuc signal was measured 48 h post-transfection. Data plotted here represent the mean + SD (three independent biological replicates). Gene names are abbreviated according to the human standard ( https://www.genenames.org/ ).

Article Snippet: CHIKV nsP2 (Genetex, GTX135188), CHIKV nsP3 (Genetex, GTX135189), CHIKV nsP4 (Thermo Fisher Scientific, PA5-117443), beta tubulin (Cell Signaling, 15115) NP (IBT Bioservices, 0301-012), VP35 (Kerafast, Kf Ab02366-1.1), VP30 (GeneTex, GTX134035), 2A (Novus, NBP2-59627),EHMT1 (Abcam, ab241306), EHMT2 (Thermo Fisher Scientific, MA5-14880), USP7 (Thermo Fisher Scientific, PA5-34911), and beta tubulin (Thermo Fisher Scientific, MA5-16308).

Techniques: Virus, Stable Transfection, Selection, In Vitro, Transfection, CRISPR, Knock-Out, Generated, Flow Cytometry, Isolation, Quantitative RT-PCR, Expressing, Luciferase, Plasmid Preparation

Journal: Journal of Virology

Article Title: Novel Stable Ebola Virus Minigenome Replicon Reveals Remarkable Stability of the Viral Genome

doi: 10.1128/JVI.01316-17

Figure Lengend Snippet: In vitro transcribed EBOV minigenomic RNA can replicate and transcribe with the help of viral proteins. (A) Schematic representation of EBOV full-length genome. (B) Schematic representation of RNA-based EBOV minigenome transient-transfection system. (C) GLuc activities in the culture supernatants of Huh7 cells transfected with the EBOV minigenomic vRNA alone or together with NP, VP35, and VP30 or with NP, VP35, VP30, and L. The error bars represent standard deviations of results of two independent experiments.

Article Snippet: The pcDNA3.1 vectors carrying EBOV NP, VP35, VP30, and L genes of the Zaire Mayinga strain ( {"type":"entrez-nucleotide","attrs":{"text":"NC_002549","term_id":"10313991","term_text":"NC_002549"}} NC_002549 ) were codon optimized, synthesized, and cloned by GenScript (Nanjing, China).

Techniques: In Vitro, Transfection

Journal: Journal of Virology

Article Title: Novel Stable Ebola Virus Minigenome Replicon Reveals Remarkable Stability of the Viral Genome

doi: 10.1128/JVI.01316-17

Figure Lengend Snippet: Huh7-4P cells efficiently support EBOV minigenome replication and transcription. (A) The protocol to construct Huh7-4P cells stably expressing NP, VP35, VP30, and L proteins. (B) Western blot analysis of NP, VP35, and VP30 proteins. (C) RT-PCR analysis of L gene mRNA expression. (D and E) GLuc activities in the culture supernatants of Huh7-4P cells electroporated with in vitro transcribed minigenomic vRNA. The error bars represent standard deviations of results of two independent experiments.

Article Snippet: The pcDNA3.1 vectors carrying EBOV NP, VP35, VP30, and L genes of the Zaire Mayinga strain ( {"type":"entrez-nucleotide","attrs":{"text":"NC_002549","term_id":"10313991","term_text":"NC_002549"}} NC_002549 ) were codon optimized, synthesized, and cloned by GenScript (Nanjing, China).

Techniques: Construct, Stable Transfection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, In Vitro

Journal: Journal of Virology

Article Title: Novel Stable Ebola Virus Minigenome Replicon Reveals Remarkable Stability of the Viral Genome

doi: 10.1128/JVI.01316-17

Figure Lengend Snippet: Establishment of an EBOV minigenome replicon system. (A) Schematic representation of an EBOV minigenome (EBOV-GLuc-Hyg) that harbors a gene encoding a fusion protein of GLuc and a hygromycin-resistant gene separated by an autocleavable 2A peptide derived from FMDV. (B) Crystal violet staining of Huh7-4P and Huh7-L cells transfected with EBOV-GLuc-Hyg and selected with hygromycin for more than 3 weeks. (C) GLuc activities in the culture supernatants of Huh7-4P cells electroporated with in vitro transcribed EBOV-GLuc-Hyg. The error bars represent standard deviations of results of two independent experiments. (D) Protein levels of NP, VP35, and VP30 were analyzed by Western blotting. (E) L mRNA levels were measured by RT-qPCR and expressed as the RNA level relative to the GAPDH level. The error bars represent standard deviations of results of two independent experiments.

Article Snippet: The pcDNA3.1 vectors carrying EBOV NP, VP35, VP30, and L genes of the Zaire Mayinga strain ( {"type":"entrez-nucleotide","attrs":{"text":"NC_002549","term_id":"10313991","term_text":"NC_002549"}} NC_002549 ) were codon optimized, synthesized, and cloned by GenScript (Nanjing, China).

Techniques: Derivative Assay, Staining, Transfection, In Vitro, Western Blot, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: a IgM, IgG, and IgA antibody epitope repertoire recognized in the human plasma at different months post-MARV infection (pi) during convalescence (12-, 30-, 43- and 60-months pi are color coded) and their alignment to the whole proteome of MARV (showing different proteins: NP, VP35, VP40, GP, VP30, VP24 and L). Graphical distribution of representative clones with a frequency of ≥2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone to its homologous sequence in the MARV proteome on alignment. The thickness of each bar represents the frequency of repetitively isolated phage, with the scale shown below the alignment. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and similar number of phage clones and epitope repertoire was observed in both phage display analysis. b Structural representations of immunodominant antigenic sites recognized by IgG using MARV-GFPDL on the surface structure of mature trimeric MARV GP solved structure [PDB #5UQY ]. The different domains of mature GP: GP1 (residues 1334-1769 in cyan), receptor binding site (RBS; residues 1372-1522 in orange), GP2 (residues 1842-2015 in blue), fusion loop (FL; residues 1842-1885 in pink) and N-terminal heptad repeat (NHR; residues 1886-1929 in yellow) are shown on the structure of MARV-GP, with the residue numbers corresponding to the complete MARV proteome used for GFPDL. The MARV GP structure used for crystallography encodes for MARV GPΔmuc ectodomain (encompassing amino acid residues 1–636 with a mucin deletion of residues 257–425). The immunodominant antigenic sites recognized by IgG in MVD survivors identified using MARV-GFPDL that can be mapped on the crystal structure are depicted in red.

Article Snippet: Recombinant Marburg virus-like particles (VLPs expressing MARV trimeric GP on surface of particles made of MARV matrix proteins VP40 and NP) , and MARV GP, NP, VP35, VP40, VP24, VP30 and L polymerase proteins were purchased from MyBioSource, IBT Bioservices, and Creative Diagnostics Inc.

Techniques: Clinical Proteomics, Infection, Clone Assay, Selection, Sequencing, Isolation, Binding Assay, Residue

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: Serial dilutions of longitudinal plasma samples collected at 12-months through 60-months post-infection from the MVD survivors ( N = 10; in colors) and uninfected controls ( N = 10; in black) were analyzed for antibody binding to purified recombinant MARV proteins by SPR. Total antibody binding is represented in SPR resonance units (RU) for 10-fold dilution of plasma sample binding to Marburg virus-like particles (VLPs expressing trimeric GP on the surface of particles made of MARV matrix proteins VP40 and NP), and recombinant MARV GP, NP, VP35, VP40, VP24, VP30 and L polymerase. The mean half-life (in months) of the polyclonal binding antibodies against various MARV proteins for the convalescent phase in these MVD survivors is shown, except for VP30 and L, which demonstrated weak antibody binding. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variation for each sample in duplicate SPR runs was <7%. The data shown is the average value of two experimental runs. Source data are provided as a Source Data file.

Article Snippet: Recombinant Marburg virus-like particles (VLPs expressing MARV trimeric GP on surface of particles made of MARV matrix proteins VP40 and NP) , and MARV GP, NP, VP35, VP40, VP24, VP30 and L polymerase proteins were purchased from MyBioSource, IBT Bioservices, and Creative Diagnostics Inc.

Techniques: Clinical Proteomics, Infection, Binding Assay, Purification, Recombinant, Virus, Expressing

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: Antibody affinity maturation of longitudinal plasma samples collected from MVD survivors to purified recombinant MARV proteins was determined by SPR. Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the interaction of serially diluted post-infection plasma with MARV proteins using SPR in the dissociation phase only for the sensorgrams with maximum RU in the range of 10-100 RU in the optimized SPR. Antibody affinity was not determined for uninfected control samples or against MARV-VP30 or L as most of the post-infection MVD plasma samples showed very low antibody binding ( < 10 RU) against these two proteins. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variation for each sample in duplicate SPR runs was <7%. The data shown is the average value of two experimental runs. Source data are provided as a Source Data file.

Article Snippet: Recombinant Marburg virus-like particles (VLPs expressing MARV trimeric GP on surface of particles made of MARV matrix proteins VP40 and NP) , and MARV GP, NP, VP35, VP40, VP24, VP30 and L polymerase proteins were purchased from MyBioSource, IBT Bioservices, and Creative Diagnostics Inc.

Techniques: Clinical Proteomics, Purification, Recombinant, Infection, Control, Binding Assay

Journal: Cell reports

Article Title: A Chimeric Lloviu Virus Minigenome System Reveals that the Bat-Derived Filovirus Replicates More Similarly to Ebolaviruses than Marburgviruses

doi: 10.1016/j.celrep.2018.08.008

Figure Lengend Snippet: EGFP-expressing 3L5E (A), 3L5R (B), or 3L5M (C) minigenomes were transfected into HEK293T cells along with LLOV system components, including LLOV VP30. As a negative control, polymerase L was omitted (–L). LLOV VP30 was either omitted (–VP30) or replaced with the VP30 proteins of EBOV, RESTV, or MARV. Minigenome activity was visualized 3 DPT. Experiment was repeated three times, and representative fluorescence images are shown.

Article Snippet: Plasmid: pCAGGS-EBOV VP30 , Mühlberger lab , Addgene Plasmid #103051.

Techniques: Expressing, Transfection, Negative Control, Activity Assay, Fluorescence

Journal: Cell reports

Article Title: A Chimeric Lloviu Virus Minigenome System Reveals that the Bat-Derived Filovirus Replicates More Similarly to Ebolaviruses than Marburgviruses

doi: 10.1016/j.celrep.2018.08.008

Figure Lengend Snippet:

Article Snippet: Plasmid: pCAGGS-EBOV VP30 , Mühlberger lab , Addgene Plasmid #103051.

Techniques: Recombinant, Transfection, Luciferase, Enzymatic Assay, Lysis, Plasmid Preparation, Software