Table 1.
Antibodies for immunohistochemistry and flow cytometry.
Table 2.
Primers used for RT-qPCR analysis.
Fig 1.
Analysis of skin structure and keratin expression.
The epithelium of OTC consist of three distinguishable KC layers: polygonal shaped cells on top of the basal lamina followed by flattened KC in spinous and granular layers, followed by flattened corneocytes (A). Co-localized K1/K10 are located in the suprabasal layer (C and E). K14 is distributed all over the epidermis (G). Foreskin histology shows all four epidermal layers of human skin: stratum basale, stratum spinosum, stratum granulosum and stratum corneum (B). K1/K10 staining is located similar to OTC in the suprabasal layer with a stronger K10 stain in stratum corneum (D and F). K14 is strictly located in the cells of the stratum basale (H). Keratins are displayed in red (Cy3) and nuclei are stained blue (Hoechst 33342). Representative H&E and immunofluorescence from three independent experiments (n = 3).
Fig 2.
Restricted sites of ORFV-induced CPE in OTC and foreskin.
H&E staining of epithelial structure of non-infected OTC (A and A’). ORFV infection (d0-10) causes thickening of epidermis and disorganization of the suprabasal layer at distinct sites of OTC (B) consisting of vacuolated KC (B’), ballooning degeneration of KC, and cytoplasmic eosinophilic inclusion bodies (B”). After a 2-day infection period (d8-10), a similar CPE is caused in OTC (C and C’). A well stratified epithelium with clear shape of KC is observed in non-infected foreskin (D and D’). Similar to OTC, vacuolization and ballooning degeneration of KC is present in the suprabasal layer (E and E’). H&E staining of 4 μm cryosections from three independent experiments (n = 3). Granule clumps in KC marked by open arrows. Eosinophilic inclusion bodies in B” marked by open arrow heads. Enlarged vacuolated KC in C’ and ballooning degeneration in E’ are marked by black arrow heads.
Fig 3.
ORFV envelope detection in ORFV-infected KC.
Representative fluorescence images show the localization of ORFV virus envelope protein (green) in few CPE-affected KC in suprabasal layer of OTC upon infection at day 0 (d0-10) (A and A’). Infection on day 8 (d8-10) produced more ORFV envelope positive cells (B and B’). Negative controls of nonspecific reaction by second antibody staining of OTC infected (d0-10) and (d8-10) are shown in images (C and C’) and (D and D’), respectively. The nuclei were stained with Hoechst 33342 (blue). A phase contrast image is overlaid for orientation (A-D).
Table 3.
CPE caused by ORFV re-isolated from OTC and foreskin tissue.
Fig 4.
Transcription of ORFV-specific VEGF-like protein, IL-10-like protein, VITF-3 and B2L in OTC.
Relative quantification of early (A and B), intermediate (C) and late (D) genes of ORFV by RT-qPCR in OTCs (each dot represents a separate experiment). Induction of VEGF-like protein, IL-10-like protein, VITF-3 and B2L (coding for major envelope protein) in OTCs following ORFV infection was measured by RT-qPCR. OTCs were infected at day 8 and analyzed 2 h and 48 h post infection. Values on the y-axes are calculated relative to human reference gene beta-2-microglobulin (B2M). For statistical analysis the normal distribution was calculated with the Shapiro-Wilk test. The ORFV 2 d data were normally distributed, they were tested for statistical significance in comparison to the reference (data: medium 2 d) with the one sample t test. *, p<0.05.
Fig 5.
Reduced presence of K1 in ORFV-infected OTC, foreskin and KC culture.
Representative fluorescence images showing the localization of K1 (green) and K14 (red) in OTC and foreskin (4 μm cryosections). K1 is localized in the suprabasal layer of non-infected OTC. The K14 signal is distributed all over the OTC epidermis (A and A’). In the long-term infected (d0-10) OTC the K1 signal is marginal, whereas the presence of K14 remains unchanged (B and B’). In the short-term infected OTC (d8-10) the K1 signal is also strongly reduced (C and C’). This K1 reduction seems to affect also the non-lesional area. In non-infected foreskin tissue a strong K1 signal is present in the suprabasal layer, whereas K14 staining is restricted to the stratum basale (D and D’). In the infected, CPE-affected sections, the fluorescence signal for K1 is distinctly reduced, while K14 is also visible in the suprabasal layer (E and E’). Each staining was done with OTC and foreskin from three different donors. Nuclei were stained with Hoechst 33342 (blue). Flow cytometry analysis shows a strong K1 reduction in ORFV-infected 2D-cultured KC (F). One representative flow cytometry plot and the statistical analysis of KCs derived from the same donor of three independent experiments is shown, comparing medium control to ORFV-infected samples, after testing for normal distribution using the one sample t-test.
Fig 6.
Down-regulation of K1/K10 and loricrin in ORFV-infected OTC.
Reduction of K1, K10 and loricrin transcription is demonstrated by means of RT-qPCR and compared with non-infected tissue (A-C). Most importantly, K14 transcription was unaltered in non-infected as well as in infected OTC (D). Transcription was analyzed 2 h and 48 h post infection (d8-10 infection) and calculated relative to human reference gene beta-2-microglobulin (B2M). Each dot represents a separate OTC. Considering the fact that, in contrast to ORFV-specific genes, the differentiation markers K1, K10, and loricrin as well as K14 are expressed both in ORFV-infected KC and in non-infected KC (which comprises the vast majority of the OTC), it is not appropriate to apply statistical analysis of expression strength.