Journal: PLoS ONE

Article Title: Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

doi: 10.1371/journal.pone.0150110

Figure Lengend Snippet: A . Conservation of immunogenic epitopes of recombinant proteins was verified through the recognition of immobilized surface proteins by vaccinia immunoglobulin (VIG) employing an indirect ELISA. Lysates from vaccinia virus infected (VACV NYCBOH ) or non-infected HEp-2 cells were used as the positive or negative control. B . The reactivity of anti-A27, -L1, -D8 and -H3 antibodies (1:1000) against VACV-infected (strain NYCBOH) or non-infected (n-) HEp-2 cells (40 μg lysate per lane) by western immunoblotting. HRP-labelled goat anti-rabbit or rabbit anti-goat IgG antibodies (1:5000) were used for detection. C . Percentage of plaque reduction (PRNT; ranging from 0% to 100%) for two-fold serial dilutions of anti-A27, -L1, -D8 and -H3 antibodies. VIG was included as the positive control. D . IFA of VACV LE infected Hep-2 cells with different combinations of polyclonal antibodies targeting surface proteins (A27, L1, D8 and H3) or vaccinia virus (VACV). Compared to the other antibodies, for the analysis of anti-H3 stained infected cells, the detector gain had to be increased due to the lower signal intensity. Antibodies were either stained with species-specific FITC-labelled secondary antibodies (green) or labeled directly with DyLight 649 (red). Cell nuclei were counterstained with DAPI (blue). Scale bar = 100 μm.

Article Snippet: A polyclonal rabbit anti-VACV LE antibody was obtained from Acris Antibodies (Herford, Germany).

Techniques: Recombinant, Indirect ELISA, Virus, Infection, Negative Control, Western Blot, Positive Control, Staining, Labeling

Journal: PLoS ONE

Article Title: Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

doi: 10.1371/journal.pone.0150110

Figure Lengend Snippet: A . Immobilized recombinant proteins or purified UV-inactivated VACV NYCBOH particles were incubated with a dilution series of purified biotinylated anti-A27, -D8, -H3 and -L1 antibodies in an indirect ELISA. Detection was done with SA-pHRP (1:5000). B . Representative electron microscopic pictures of anti-A27, -D8, or -H3 antibodies binding to purified VACV NYCBOH . Biotinylated antibodies were detected using 5 nm immunogold labelled streptavidin. Scale bars = 100 nm. C . Box-and-whisker plot for quantification of the number of gold particles per viral particle (whiskers: min to max; A27 n = 111; D8 n = 109 viral particles analyzed).

Article Snippet: A polyclonal rabbit anti-VACV LE antibody was obtained from Acris Antibodies (Herford, Germany).

Techniques: Recombinant, Purification, Incubation, Indirect ELISA, Binding Assay, Whisker Assay

Journal: PLoS ONE

Article Title: Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

doi: 10.1371/journal.pone.0150110

Figure Lengend Snippet: A . Binding epitopes and multiple sequence alignment (NCBI BLink) of different OPV strains. Identical sequences with the prominent strain are denoted in brackets, followed by the number of identical sequences deposited in GenBank. The binding epitopes for mAb A3/710 from amino acid 13 to 27 (A) and all other anti-A27 mAbs from amino acid 24 to 38 (B) border the heparin binding site (HBS) on A27. Both the VACV NYCBOH strain and E . coli derived A27 based on CPXV calpox DNA show 100% sequence identity with the reference strain VACV WR in this region. B . Compatibility of all eight anti-A27 mAbs for antigen capture ELISA. All possible combinations of coating antibodies (x-axis) and biotinylated detection were paired and tested for the detection of 5×10 6 PFU/ml UV-inactivated VACV NYCBOH . Shown are representative results for the highest virus concentration tested. The results from the titration series are shown in . C . SPR sensorgrams to determine the binding kinetics of anti-A27 mAbs. Measured responses (double referenced resonance units RU; difference between flow cell 2 minus flow cell 1) are shown as red lines whereas black lines represent results from fitting a 1:1 Langmuir interaction.

Article Snippet: A polyclonal rabbit anti-VACV LE antibody was obtained from Acris Antibodies (Herford, Germany).

Techniques: Binding Assay, Sequencing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Virus, Concentration Assay, Titration

Journal: PLoS ONE

Article Title: Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

doi: 10.1371/journal.pone.0150110

Figure Lengend Snippet: A . Titration curve of rA27 with the antigen capture ELISA. B . To check for cross-reactivity, PPXV, HSV-1 and tanapox virus were tested. C . Detection of different VACV strains by the newly developed assay. D . Detection of different OPV.

Article Snippet: A polyclonal rabbit anti-VACV LE antibody was obtained from Acris Antibodies (Herford, Germany).

Techniques: Titration, Enzyme-linked Immunosorbent Assay, Virus

Journal: PLoS ONE

Article Title: Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

doi: 10.1371/journal.pone.0150110

Figure Lengend Snippet: Limits of detection (LOD) of the newly developed A27-specific antigen sandwich ELISA for different OPV strains.

Article Snippet: A polyclonal rabbit anti-VACV LE antibody was obtained from Acris Antibodies (Herford, Germany).

Techniques: Sandwich ELISA

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 1. The miR-17-92 cluster members are dysregulated upon MMTV expression. (A) Heat-map of miR-17- 92 cluster upon small miRNAseq analysis of the normal mouse mammary epithelial cell lines, HC11 and HC11- MMTV. The miRNAseq was conducted on two biological replicates shown as 1 and 2. The numbers in the table represent the normalized reads observed upon miRNAseq. (B) Fold-change analysis of mature miR-17-92 cluster member expression using the miRNAseq data from HC11 cells. The Q-value (equivalent of p-value corrected for multiple test hypothesis) for each of the miRNA shown was statistically significant (Q < 0.005). (C) Western blot analysis of HEK293 and HEK293T cells expressing MMTV using an anti-MMTV Gag antibody. An antibody against a housekeeping gene (GAPDH) was used as a loading control. (D) Quantitative RT-PCRs (RT-qPCRs) to assess endogenous levels of the primary form of miR-17-92 cluster upon MMTV expression in HEK293T cells. b-Actin was used as the endogenous control. (E) RT-qPCR analysis of mature miR-17, miR-19a and miR-92a in HEK293T cells. U6 was used as the endogenous control. All experiments were carried out in triplicates. Statistical significance is shown as * where *p 0.05; **p 0.01, and ***p 0.001.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 2. Over-expression of the miR-17-92 cluster down-regulates MMTV expression. (A) An illustration of the miR-17-92 cluster, its truncated form miR-19a-20a-19b, and miR-92a over-expressed (OE) exogenously in the HEK293T-MMTV cells. (B) GFP-positive stably-transfected cells selected in puromycin and visualized using a fluorescent microscope (10x). (C) Expression of pri-miR-17-92 in miR-17-92 OE cell line quantified using a SYBR Green qPCR assay. (D) TaqMan miRNA assays used to quantify expression of mature miR-17, miR-19a, miR-92a in the miR17-92 OE cell line, (E) miR-19/20 OE cell line, and (F) miR-92a OE cell line. U6 snRNA was used as the endogenous control in these assays. (G) Western blot analysis of MMTV Gag expression in cell lysates. (H) MMTV RNA-specific TaqMan assay to quantify all MMTV messages in the three OE cell lines. (I) MMTV genomic RNA was assessed using gRNA-specific MMTV TaqMan assay in the miR-17-92 cluster OE and the miR-92a OE cells and virus particles isolated from culture supernatants (sup) via ultracentrifugation. The empty vector (EV) was designated as 1 in all analyses. Mean ± SD (n = 3). Statistical significance is shown as * where *p 0.05; **p 0.01, ***p 0.001, ****p 0.0001. (J) Western blot analysis of viral particles harvested from culture supernatants in the respective miRNA OE stable cell lines.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Over Expression, Expressing, Stable Transfection, Transfection, Microscopy, SYBR Green Assay, Control, Western Blot, TaqMan Assay, Virus, Isolation, Plasmid Preparation

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 3. Mir-17-92 cluster and miR-92a inhibition rescues MMTV genomic RNA expression. TaqMan miRNA assays were used to quantify: (A) miR-17, miR-19a and miR-92a in miR-17-92 over expression (OE) cell line, (B) miR-19a in the miR-19a-20a-19b OE cell line, and (C) miR-92a in the miR-92a OE cell line. U6 snRNA was used as the endogenous control in the miRNA RT-qPCR assays. The error bars represent ± SD. NC, negative control scrambled oligo specific for each miRNA inhibitor used. (D) Western blot analysis of whole cell lysates (40 mg) from the miRNA OE cell lines treated with miRNA inhibitors for MMTV Gag expression. GAPDH was used as the internal control. Expression of the MMTV genomic RNA was quantified in the: (E) miR-17-92 OE cells treated with a cocktail of anti-miR-17, -19, & -92a oligos, (F) miR-19a- 20a-19b OE cells treated with anti-miR-19a oligos, and (G) miR-92a OE cells treated with anti-miR-92a oligos. b-Actin was used as the internal control in the MMTV TaqMan RT-qPCR assays. The empty vector (EV) was designated as 1 in all analyses. Mean ± SD (n = 3). (H) Western Blot analysis of whole cell lysates (50 mg) from the miR-92a OE cell line treated with anti-miR-92a oligos at varying concentrations. GAPDH was used as the internal control. Statistical significance is shown as * where *p 0.05; **p 0.01, ***p 0.001, ****p 0.0001. ns, not significant; ns (P > 0.05).

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Inhibition, RNA Expression, Over Expression, Control, miRNA RT, Negative Control, Western Blot, Expressing, Quantitative RT-PCR, Plasmid Preparation

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 4. Rescue of MMTV expression upon plasmid-based miRNA inhibition. (A) Mature miR-17 and (B) miR-92a levels were quantified in their respective PMIS inhibitor expressing cell lines using TaqMan miRNA RT- qPCRs. U6 snRNA was used as the endogenous control. (C) Expression of miR-17 target, PTEN was quantified using RT-qPCR; b-Actin was used as the internal control. (D) Western blot analysis of MMTV Gag expression across the three cell lines using 50 mg protein. GAPDH served as the endogenous control. (E) The expression of all MMTV transcripts were quantified using TaqMan RT-qPCRs with b-Actin as the internal control. The empty vector (EV; the PMIS backbone without any insert) was designated as unit 1 in all experiments with means represented as ±SD (n = 3). Statistical significance is shown as * * where *p 0.05 and **p 0.01.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Expressing, Plasmid Preparation, Inhibition, miRNA RT, Control, Quantitative RT-PCR, Western Blot

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 5. MMTV expression rescued upon deletion or mutation of miR-92a seed sequence. (A) Schematic representation of the full miR-17-92 cluster (WT), or cluster with deletion of miR-92a (D92a) indicated as a dashed line, or a substitution mutation of miR-92a (92aMUT) within the cluster, both highlighted in red text. (B) Expression of MMTV Gag quantified by western blot analysis using 40 mg lysate from the different transiently transfected cell lines; GAPDH was used as the internal control. (C) Quantification of all MMTV mRNAs by RT-qPCR. b-Actin used as the internal control. The empty vector (EV) was designated as unit 1 with means represented as ± SD (n = 3). Statistical significance is shown as * where *p 0.05; **p 0.01, ***p 0.001, and ****p 0.0001.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Expressing, Mutagenesis, Sequencing, Western Blot, Transfection, Control, Quantitative RT-PCR, Plasmid Preparation

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 6. An in-silico analysis of the potential of the miR-17-92 cluster members to target the MMTV genomic RNA using STarMir bioinformatic tool. (A) Illustration of the MMTV genome and its various mRNAs along with the potential miR-17-92 cluster member predicted binding sites. The shaded box in blue highlights the region of the MMTV genome exclusively found in the full-length genomic RNA which is the same as the mRNA for Gag/Pro/Pol viral proteins. (B) Characterization of the miRNAs predicted to target the MMTV mRNAs. The target RNA regions highlighted in red belong exclusively to the full-length Gag/Pro/Pol mRNA or the genomic RNA (gRNA) with the number of target sites observed in parenthesis. (C) Illustration of the highest probable binding site of miR-92a on MMTV Gag.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: In Silico, Binding Assay

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 8. Expression of miR-17-92 cluster members in MMTV-induced tumors. Data representing the mean expression of miR-17-92 cluster members in MMTV-induced tumors (n = 2) and infected mammary glands (n = 2) obtained by small RNAseq analysis.58

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Expressing, Infection

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 9. Schematic representation of model representing miR-17-92-mediated regulation of MMTV gene expression. MMTV entry via murine transferrin receptor 1 (mTfR1) is followed by fusion of the viral capsid in a late endosomal compartment under low pH, uncoating/reverse transcription, and integration of the viral genome into the host chromosome. The cellular/viral factor-assisted transcriptional activation of the miR-17-92 cluster leads to up- regulation of the pri-miR-17-92. Once activated, due to the differential processing of the cluster, there is an increase in the expression levels of the mature miR-92a cluster member, which in turn, targets the MMTV unspliced genomic RNA. With more than 10 statistically significant miR-92a binding sites on the viral gag region, down-regulation of the genomic RNA takes place, resulting in suppression of MMTV replication. This is because the unspliced genomic RNA serves as the mRNA for the translation of the viral structural and enzymatic proteins (Gag/Pro/Pol) as well as the source of genomic RNA for encapsidation into the newly forming viral particles.Illustration created on BioRender.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Gene Expression, Reverse Transcription, Activation Assay, Expressing, Binding Assay