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