Journal: bioRxiv

Article Title: Multiple cell types support productive infection and dynamic translocation of infectious Ebola virus to the surface of human skin

doi: 10.1101/2024.07.19.604135

Figure Lengend Snippet: a ) Generation of skin explant cultures from human skin. Full-thickness skin punch biopsies (3-10 mm) were placed dermal side down on a transwell insert in a well containing sufficient media to maintain the explant at the air-liquid interface. Within 24 hours of culture initiation, the virus was inoculated into the culture media, and explants were incubated for up to 2 weeks with media changed every other day. Specifics regarding the virus type, dosages used, and inoculation timing are described in legends for each experiment. b ) Infectious virus was evaluated in culture media after EBOV-GFP infection of explants over time. Media was inoculated with 10 2 , 10 4 , or 10 6 FFU of EBOV-GFP for 24 hours and then the explants were washed and moved to a fresh insert/well. Supernatants were collected daily starting on day 3 and infectious virus titers were determined on Vero cells as previously described. Shown is one of 5 independent experiments and data is expressed as mean ± SD. c-d ) Formalin-fixed and paraffin-embedded (FFPE) skin explants from day 12 of EBOV infection were stained with antibodies specific for EBOV GP (green) and mounted with DAPI nuclear stain. The junction of the epidermal (epi) and dermal skin layers is indicated by the dashed line. EBOV replication was observed in epidermal keratinocytes ( c ) and dermal cells ( d ) with a stellate morphology suggestive of fibroblasts (inset). Scale bar = 25 μM.

Article Snippet: Vero cells (ATCC CCL-81) or Vero E6 (ATCC CRL-1586) were used to generate EBOV and rVSV/EBOV GP stocks.

Techniques: Virus, Incubation, Infection, Staining

Journal: bioRxiv

Article Title: Multiple cell types support productive infection and dynamic translocation of infectious Ebola virus to the surface of human skin

doi: 10.1101/2024.07.19.604135

Figure Lengend Snippet: a) iters of rVSV/EBOV GP in basal supernatants of foreskin explants over the course of a 10-day infection in the presence or absence of B18R. Explants were infected in the basal media with the dose of virus noted. Media was collected on the days noted and refreshed. Titers of virus were assessed by TCID 50 assays on Vero cells. b) LDH was measured in basal supernatants of uninfected (open circles) and rVSV/EBOV GP-infected skin explants (black circles) cultured for 12 days with media changes every other day. Media for both conditions contained rux. c-h) FFPE explants were sectioned and immunostained with antibodies for EBOV GP (green) or lineage-specific antibodies (red) and mounted with DAPI (blue). Lineage markers included CK5 (epidermis), CD140b (fibroblast/smooth muscle cells/pericytes), CD31 (endothelia), and IBA1 (monocyte/macrophage). Scale bars = 10 μM in d, e, f, h and 25 μM in c, g.

Article Snippet: Vero cells (ATCC CCL-81) or Vero E6 (ATCC CRL-1586) were used to generate EBOV and rVSV/EBOV GP stocks.

Techniques: Infection, Virus, Cell Culture

Journal: bioRxiv

Article Title: Multiple cell types support productive infection and dynamic translocation of infectious Ebola virus to the surface of human skin

doi: 10.1101/2024.07.19.604135

Figure Lengend Snippet: Human skin explants were generated and maintained as in . Explants were inoculated with 10 7 iu of rVSV/EBOV GP per explant. a-b) After washing twice to remove input virus, explants were maintained in the presence or absence of 0.1 mg/mL B18R. a ) Viral titers present in basal supernatants were collected on days indicated and assessed on Vero cells. b ) In parallel studies, explants that were infected with 10 7 iu of rVSV/EBOV GP were assessed for virus load by qRT-PCR. RNA was extracted from replicate explants on days indicated and viral load was normalized using the GAPDH housekeeping gene. Data is expressed as mean ± SEM. n = 6 replicates per condition from three donors. c-h ) Replicate explants were collected on the days indicated and FFPE sections were immunostained for virus (EBOV GP in green), and cell lineage markers CK5, CD31, CD140ab, or IBA1 (red) and mounted with DAPI (blue). Viral staining was strongly expressed by the CK5 + epidermal keratinocytes c ) and surrounded CD31 + or CD140ab + dermal structures/cell populations. h ) IBA1 + dermal macrophages were also virus positive. Scale bar = 25 uM for panels d, f and 10 μM for the remaining panels.

Article Snippet: Vero cells (ATCC CCL-81) or Vero E6 (ATCC CRL-1586) were used to generate EBOV and rVSV/EBOV GP stocks.

Techniques: Generated, Virus, Infection, Quantitative RT-PCR, Staining

Journal: bioRxiv

Article Title: Multiple cell types support productive infection and dynamic translocation of infectious Ebola virus to the surface of human skin

doi: 10.1101/2024.07.19.604135

Figure Lengend Snippet: a) Infectious viral titers in the dermis and epidermis over infection. Skin explants were inoculated basally with rVSV/EBOV GP (10 7 iu) in the presence of ruxolitimib and input virus was removed at 16 hpi. After input virus was removed, explants were washed twice, and then moved to a fresh insert/well with media containing ruxolitimib. On the day of harvest, explants were treated with dispase to separate the dermis and epidermis. Individual tissues were homogenized in PBS, filtered and infectious virus was evaluated on Vero cells (n=4 independent experiments). Data are shown as individual points with the mean ± SEM. Student’s t test, *p<0.05 ***p<0.001 ****p<0.0001. b ) Infectious virus present in basal supernatants (left panel) or the apical surface of explants (right panel) over the course of infection. Skin explants were infected basally with rVSV/EBOV GP (10 7 iu) in the presence of ruxolitimib. Input virus was removed, explants were washed twice, and then moved to a fresh insert/well with media containing ruxolitimib. Media was collected and refreshed every other day. At the time indicated, basal supernatants were collected and 10 μL of sterile PBS was placed on the explant stratum corneal surface for one minute and PBS was collected. This was repeated, PBS samples were pooled and titered on Vero cells (n=2 Independent experiments). Data are shown as individual points with the mean ± SEM.

Article Snippet: Vero cells (ATCC CCL-81) or Vero E6 (ATCC CRL-1586) were used to generate EBOV and rVSV/EBOV GP stocks.

Techniques: Infection, Virus, Sterility

Journal: bioRxiv

Article Title: Multiple cell types support productive infection and dynamic translocation of infectious Ebola virus to the surface of human skin

doi: 10.1101/2024.07.19.604135

Figure Lengend Snippet: a-c ) EBOV-GFP infection of primary skin keratinocytes. Keratinocytes infected with 10 4 -10 6 iu of EBOV-GFP were imaged on day 5 of infection (a) and quantitated (b). Scale bar = 1 mm. Supernatants were collected every other day on days 3-7 and titered for infectious virus on Vero cells (c). d-h ) Utilization of receptors by EBOV in the immortalized human skin keratinocyte line, NHSK-1. Cell surface staining of known cell surface receptors utilized by filoviruses (d). Inhibition of rVSV/EBOV GP infection by the Axl signaling inhibitor bemcentinib in a 24 h infection. Data is shown as the mean ± SD of three independent experiments (e). Knock down of NPC1 expression in NHSK-1 cells as evaluated in an immunoblot (f). Loss of NPC1 expression results in decreased rVSV/EBOV GP infection in the presence or absence of rux. Data is shown as individual points with the mean ± SEM (g). Ability of the NPC1 inhibitor 3.47 to block rVSV/EBOV GP infection. Data is shown as the mean ± SD of two or three independent experiments (h).

Article Snippet: Vero cells (ATCC CCL-81) or Vero E6 (ATCC CRL-1586) were used to generate EBOV and rVSV/EBOV GP stocks.

Techniques: Infection, Virus, Staining, Inhibition, Knockdown, Expressing, Western Blot, Blocking Assay

Journal: bioRxiv

Article Title: Multiple cell types support productive infection and dynamic translocation of infectious Ebola virus to the surface of human skin

doi: 10.1101/2024.07.19.604135

Figure Lengend Snippet: a-c ) EBOV-GFP infection of primary skin fibroblasts infected with 10 2 - 10 4 iu of EBOV-GFP were imaged on day 5 of infection (a) and quantitated (b). Scale bar = 1 mm. Supernatants were collected every other day on days 3-7 and titered for infectious virus on Vero cells (c). d-h ) Utilization of receptors by EBOV in the immortalized human skin fibroblast line, NHSF-2. Gene expression of characterized cell surface receptors utilized by filoviruses (d). Inhibition of rVSV/EBOV GP infection by the Axl signaling inhibitor bemcentinib in a 24 h infection. Data is shown as the mean ± SD of three independent experiments (e). Knock down of NPC1 expression in NHSF-2 cells as evaluated in an immunoblot (f). Loss of NPC1 expression results in decreased rVSV/EBOV GP infection in the presence or absence of ruxolitimib. Data is shown as individual points with the mean ± SEM (g). Ability of the NPC1 inhibitor 3.47 to block rVSV/EBOV GP infection. Data are shown as the mean ± SD of three independent experiments (h).

Article Snippet: Vero cells (ATCC CCL-81) or Vero E6 (ATCC CRL-1586) were used to generate EBOV and rVSV/EBOV GP stocks.

Techniques: Infection, Virus, Gene Expression, Inhibition, Knockdown, Expressing, Western Blot, Blocking Assay

Journal: Journal of Virology

Article Title: Mumps Virus SH Protein Inhibits NF-κB Activation by Interacting with Tumor Necrosis Factor Receptor 1, Interleukin-1 Receptor 1, and Toll-Like Receptor 3 Complexes

doi: 10.1128/JVI.01037-17

Figure Lengend Snippet: Generation of rMuV with mutated SH genes. (A) Scheme of the MuV genome and the modified SH fragments of the generated rMuVs. For the SH-deficient viruses, the positions of introduced stop codons starting at the ATG of SH (rMuV-SHstop-C) and the FLAG epitope sequence (rMuV-SHstop) are stated. (B) BSR-T7 cells were transfected with pG-MuV-FL_SH-N-flag, pG-MuV-FL_SH-3stop-N-flag, pG-MuV-FL_SH-C-flag, or pG-MuV-FL_SH-3stop-C-flag plus pMuV-L, pMuV-N, and pMuV-P. As a negative control (mock), cells were transfected with pMuV-EGFP. Pictures of syncytia were taken at 7 days posttransfection. (C) Vero76 cells were infected with rMuV-SH, rMuV-SHstop, rMuV-SH-C, or rMuV-SHstop-C at an MOI of 0.01 or left untreated (mock). At 2 days p.i., cells were fixed and stained using an MuV F-specific Cy3-coupled antibody and DAPI. Images were generated using a Zeiss cLSM780 confocal laser scanning microscope. (D) At 3 days p.i., rMuV-infected Vero76 cells (MOI, 0.01) were lysed and subjected to Western blot analysis. MuV N and GAPDH were detected by specific monoclonal antibodies, and MuV SH was detected by an anti-FLAG antibody.

Article Snippet: 293G (kindly provided by C. Uhlenhaut, Berlin, Germany), Vero76, and A549 cells (both from ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 5% fetal bovine serum (FBS; PAN Laboratories GmbH), 2 mM l -glutamine (Lonza), 100 U/ml penicillin (Gibco), and 100 μg/ml streptomycin (Gibco).

Techniques: Modification, Generated, FLAG-tag, Sequencing, Transfection, Negative Control, Infection, Staining, Laser-Scanning Microscopy, Western Blot, Bioprocessing

Journal: Journal of Virology

Article Title: Mumps Virus SH Protein Inhibits NF-κB Activation by Interacting with Tumor Necrosis Factor Receptor 1, Interleukin-1 Receptor 1, and Toll-Like Receptor 3 Complexes

doi: 10.1128/JVI.01037-17

Figure Lengend Snippet: rMuV replication and SH protein expression. (A) Vero76 cells were infected with rMuV-SH, rMuV-SHstop, rMuV-SH-C, or rMuV-SHstop-C at an MOI of 0.01. Supernatants were collected at the indicated time points and titrated in triplicate on Vero76 cells. The detection limit due to assay procedure is 102 (dashed line). The graph depicts means ± standard deviations (SD) from one representative experiment for which three independent infections were performed at the same time. A549 (B and D) and Vero76 (C and E) cells were infected with rMuVs at an MOI of 5. Cells were harvested at the indicated time points and analyzed by flow cytometry. Total SH protein (B and C) was detected using an APC-conjugated FLAG-specific antibody. The values of rMuV-SHstop or rMuV-SHstop-C were subtracted from the values of rMuV-SH or rMuV-SH-C. Total N protein (D and E) was detected using an N-specific primary antibody and an Alexa Fluor 488-conjugated secondary antibody. The values of mock-infected cells were subtracted from the values of rMuV-infected cells. Graphs depict means ± SD from three individual experiments. *, P < 0.05; results were calculated by unpaired t tests with a two-tailed P value. (F) A549 cells were infected with rMuVs at an MOI of 5 and harvested at 20 h postinfection. Cells were treated with 0.5% saponin (permeabilized) for intra- and extracellular SH detection or not treated (nonpermeabilized) for extracellular SH detection by flow cytometry. Staining and calculation for total SH protein were carried out as described for panels B and C. The graph depicts means ± SD from three individual experiments. ***, P < 0.001; results were calculated by unpaired t tests with a two-tailed P value.

Article Snippet: 293G (kindly provided by C. Uhlenhaut, Berlin, Germany), Vero76, and A549 cells (both from ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 5% fetal bovine serum (FBS; PAN Laboratories GmbH), 2 mM l -glutamine (Lonza), 100 U/ml penicillin (Gibco), and 100 μg/ml streptomycin (Gibco).

Techniques: Expressing, Infection, Flow Cytometry, Two Tailed Test, Staining

Journal: Infection and Immunity

Article Title: Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

doi: 10.1128/IAI.00231-15

Figure Lengend Snippet: Cytotoxic effects of SubAB2-2. Vero B4 cells were incubated at 37°C with different concentrations of the single SubA2-2 (squares) and SubB2-2 (triangles) toxin subunits and their combination, SubAB2-2 (diamonds), in the medium. For a control, cells were left untreated. After 72 h, the amount of viable cells was determined. The values of the amount of viable cells were plotted against cell viability of the control cells in percentages. The standard errors are shown in the graph.

Article Snippet: To determine the cytotoxicity of the recombinant proteins, Vero B4 cells (DSMZ no. ACC 33) were cultured in RPMI 1640 cell culture medium with stable glutamine (Merck, formerly Biochrome, Berlin, Germany) supplemented with 10% (vol/vol) fetal calf serum (FCS) and incubation conditions at 37°C with 5% (vol/vol) CO 2 .

Techniques: Incubation, Control

Journal: Infection and Immunity

Article Title: Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

doi: 10.1128/IAI.00231-15

Figure Lengend Snippet: Comparison of the cytotoxic effects caused by different SubAB variants on Vero cells. (A) Cytotoxic effects of the different SubAB variants on Vero B4 cells were measured after a 72-h incubation period using the crystal violet staining assay and were compared to each other. Results obtained by the use of different toxin dilutions between 2,500 ng/ml and 20 ng/ml were plotted against the resulting Vero cell viability of the different variants. The amount of viable Vero B4 cells, which were incubated in the absence of SubAB proteins, were calculated as 100%. The standard errors are shown in the graph. (B) Binding of recombinant SubB1 and SubB2-1 proteins to Vero and HeLa cells. Confluently grown Vero or HeLa cells in 12-well plates were incubated for 45 min at 4°C in serum-free medium with 5 μg/ml of either biotin-labeled SubB1 or biotin-labeled SubB2-1 to enable binding of the SubB proteins to the cells. For a control (con), cells were left untreated. Cells were washed 3 times to remove unbound SubB, lysed in 50 μl of a 2.5-fold SDS sample buffer, and heated for 5 min at 95°C. After SDS-PAGE, the cell-bound biotinylated SubB proteins were detected by Western blotting with streptavidin-peroxidase and the ECL system. The biotin-labeled SubB1 and SubB2-1 proteins (0.5 μg each) were included as running controls (right lanes). A cellular protein between a molecular weight (MW) of 70,000 and 100,000 (marked by an asterisk) also was recognized by streptavidin-peroxidase, which indicates comparable protein loading and blotting of all lysate samples.

Article Snippet: To determine the cytotoxicity of the recombinant proteins, Vero B4 cells (DSMZ no. ACC 33) were cultured in RPMI 1640 cell culture medium with stable glutamine (Merck, formerly Biochrome, Berlin, Germany) supplemented with 10% (vol/vol) fetal calf serum (FCS) and incubation conditions at 37°C with 5% (vol/vol) CO 2 .

Techniques: Comparison, Incubation, Staining, Binding Assay, Recombinant, Labeling, Control, SDS Page, Western Blot, Molecular Weight

Journal: Infection and Immunity

Article Title: Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

doi: 10.1128/IAI.00231-15

Figure Lengend Snippet: Cytotoxic effects caused by hybrid SubAB toxins. Recombinant SubA2-1 and SubB2-2 (triangles) subunits, as well as SubA2-2 and SubB2-1 (squares) subunits, were mixed in vitro and incubated with Vero B4 cells (for details, see the text). Cells also were incubated with the toxins SubAB2-1 (diamonds) and SubAB2-2 (circles) using the same approach. The standard errors are shown in the graph.

Article Snippet: To determine the cytotoxicity of the recombinant proteins, Vero B4 cells (DSMZ no. ACC 33) were cultured in RPMI 1640 cell culture medium with stable glutamine (Merck, formerly Biochrome, Berlin, Germany) supplemented with 10% (vol/vol) fetal calf serum (FCS) and incubation conditions at 37°C with 5% (vol/vol) CO 2 .

Techniques: Recombinant, In Vitro, Incubation

Journal: bioRxiv

Article Title: Endogenous retroviruses promote prion-like spreading of proteopathic seeds

doi: 10.1101/2022.05.06.490866

Figure Lengend Snippet: a. Schematic overview. HEK NM-HA agg donor cells were transfected with combinations of for amphotropic MLV env 10A1 vector, gag/pol plasmid, retroviral transfer vector and/or empty vector. Donors were subsequently cocultured with recipient Vero NM-GFP sol cells. b. Western blot analysis of cell lysates from recipient cells expressing Pit-2. c. Percentage of recipient Vero cells with induced NM-GFP agg cocultured with donors transfected with single plasmids. d. Percentage of recipient cells with induced NM-GFP agg cocultured with donors transfected with combinations of plasmids coding for env , gag/pol or non-viral empty plasmid. e. Percentage of recipient cells with induced NM- GFP agg cocultured with donors that were additionally transfected with/without transfer vector (TV) for virus production. f. Donor cells transfected with/ without mCherry-coding retroviral transfer vector and plasmids coding for gag/pol and env produce virus that is infectious to wildtype cells. g. Representative image of HEK Tau-GFP AD donor cell population. h. HEK Tau-GFP AD donor cells were transfected with combinations of plasmids coding for amphotropic MLV env 10A1, gag/pol , retroviral transfer vector and/or non-viral empty vector. Donors were subsequently cocultured with recipient Vero Tau- FR sol cells. Alternatively, donor EVs were added to recipient cells. i. Confocal image of HEK Tau-GFP AD donor cells. i. Percentage of recipient cells with induced Tau-FR agg upon coculture with donors transfected with individual plasmids. j. Percentage of recipient cells with induced Tau-FR agg upon coculture with donors transfected with plasmid combinations. k. Percentage of recipient cells with induced Tau-FR agg upon coculture with donors transfected with plasmid combinations and transfer vector. l. Transfection of plasmids coding for gag/pol and env simultaneously increases particle secretion. m. Percentage of recipient cells with Tau-FR agg exposed to conditioned medium of donors adjusted for comparable particle numbers. All data are shown as the means ± SD from two (m), three (l) or six (c-e, i-k) replicate cell cultures. Three (c-e, i-m) independent experiments were carried out with similar results. P-values calculated by two-tailed unpaired Student’s t-test (e, k-m) or one-way ANOVA. (c, d, i, j, l). Source data are provided as a Source Data file.

Article Snippet: Vero cells were purchased from CLS (Cell lines service) and cultivated as recommended.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Endogenous retroviruses promote prion-like spreading of proteopathic seeds

doi: 10.1101/2022.05.06.490866

Figure Lengend Snippet: a. Experimental workflow. Donor HEK cells stably propagating aggregated Tau-GFP AD were transfected with plasmid coding for V5 epitope-tagged Syncytin-1 (Syn- V5) and were subsequently cocultured with recipient HEK or Vero cells expressing Tau- FR sol . b. Western blot analysis of donor clone transfected with plasmids coding for Syn- V5. c. Coculture of donor and HEK recipient cells. Note that we have not stained the donors in this experiment. d. Quantitative analysis of the percentage of recipient cells with induced aggregates upon coculture. e. Coculture of donor and Vero recipient cells. f. Quantitative analysis of the percentage of recipient cells with induced aggregates. All data are shown as the means ± SD from 6 (d, f) replicate cell cultures. Three (d, f) independent experiments were carried out with similar results. P-values calculated by two-tailed unpaired Student’s t-test. Source data are provided as a Source Data file.

Article Snippet: Vero cells were purchased from CLS (Cell lines service) and cultivated as recommended.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Western Blot, Staining, Two Tailed Test