ORF virus causes tumor-promoting inflammation in sheep and goats

Abstract

ORF virus (ORFV) causes contagious ecthyma ("ORF"), a disease of sheep and goats characterized by lesions ranging from vesicles and pustules to atypical papilloma-like and angiomatous lesions in the skin and mucosae. The authors investigated the molecular factors leading to the ORF-associated atypical tumor-like changes. Fifteen lambs, 15 kids, and an adult ram clinically affected by natural ORFV infection were enrolled in the study and examined by several methods. ORFV was detected by viral culture or real-time polymerase chain reaction (RT-PCR) in the lesioned tissues and in the blood of the clinically affected sheep and goats. Surprisingly, ORFV was also detected in the blood of healthy goats from an affected herd. Microscopically, they found a pseudo-papillomatous proliferation of the epithelium, while the dermis and lamina propria were expanded by a proliferating neovascular component that highly expressed the viral vascular endothelial growth factor (vVEGF) and its host receptor vascular endothelial growth factor receptor 2 (VEGFR2). Immunohistochemistry, immunofluorescence, and in situ hybridization for mRNA showed that epidermal growth factor receptor (EGFR) was expressed in the fibrovascular component, in the infiltrating CD163+ macrophages, and in the basal stratum of the epidermis. Confocal immunofluorescence microscopy demonstrated that CD163+ macrophages were associated with VEGF and VEGFR2. Finally, they found by quantitative RT-PCR the overexpression of the interleukin-6 and VEGFR2 genes in the lesioned tissues. These findings suggest that ORFV activates an inflammatory reaction characterized by CD163+ macrophages expressing EGFR and VEGFR2, which might play an oncogenic role through synergistic action with vVEGF signaling.

Keywords: CD163+ macrophages; epidermal growth factor receptor 2; gene expression; goat; parapoxvirus; sheep; tumor; vascular endothelial growth factor.

Figure 1.

ORF virus infected lambs, kids, and ram. Representative gross and microscopic findings in the buccal mucosae and skin. (a) Well-circumscribed red masses in the gingiva and lips of a lamb. (b, c) Coalescing hyperemic firm nodules with cauliflower-like appearance covering the line of the incisive and molar teeth in a kid and lamb, respectively. (d, e) Single and coalescing nodules in the browsing pad, margins of the lips, palate, and tongue in a kid. (f) Isolated multilobular hyperemic mass arising among the incisors tooth of an adult ram.

Figure 2.

ORFV infected lambs and kids. ORFV, ORF virus. (a) Tumor-like proliferation of the gingival epithelium characterized by hyperkeratosis and elongated rete ridges sustained by fibrovascular structure. Lamb. Hematoxylin and eosin (HE). (b) Keratinocytes with hydropic alterations, formation of koilocytes, and presence of cytoplasmic eosinophilic inclusion bodies (arrow) in the skin of an affected lamb. HE. (c) A representative area of newly formed vessels in the lamina propria. Kid. HE. (d) A different area, from the same case shown in (c), displayed densely packed fibrovascular cells with no new organized vessels. HE. (e) mRNA localization (red) of ORFV is found only in the damaged keratinocytes. Kid. RNA-scope in situ hybridization for ORFV mRNA. (f) Electron micrograph of lesioned mucosa of a lamb shows parapoxvirus particles.

Figure 3.

Representative photomicrographs of the immunohistochemistry (IHC) and immunofluorescene findings. (a–d) ORF virus infected kid. Buccal mucosae. (a) Proliferating fibrovascular component in the lamina propria showed diffuse immunolabeling for vimentin. Vimentin IHC. (b) Immunoreactivity for von Willebrand factor (vWF) outlines the newly formed vessels. vWF IHC. (c) Abundant immunolabeling for vascular endothelial growth factor receptor (2) (VEGFR2) of fibrovascular component in the lamina propria, which contains morphologically distinct vessels. Macrophages (arrow) infiltrating the epithelium show immunohistochemical reactivity for VEGFR2. VEGFR2 IHC. (d) Immunolabeling of vascular endothelial growth factor (VEGF) is observed in the lamina propria and associated with macrophages (arrow) infiltrating the epithelium. VEGF IHC. (e–g) ORF virus infected lamb. Buccal mucosa. (e) Representative area of the mucosa displaying epidermal growth factor receptor (EGFR) immunolabeling in the lamina propria. EGFR labeling in the epithelium is also associated with large macrophages (arrow). EGFR IHC. (f) Representative area of the lamina propria showing densely packed cells with EGFR immunolabeling. EGFR IHC. (g) Newly formed vessels showing immunolabeling for EGFR. EGFR IHC. (h) Healthy control lamb, buccal mucosa. Moderate immunolabeling for EGFR in the basal stratum of the epithelium. (i) EGFR mRNA localization in the endothelial cells of the newly formed vessels, in the epithelium (inset at the top right corner) and, with distinct spots patterns, in the fibrovascular component (inset at the bottom right corner). RNA-scope in situ hybridization for EGFR mRNA. (j–m) Serial sections of the same area of the buccal mucosa of ORF virus-infected lamb. (j) Diffuse immunolabeling of CD163+ macrophages infiltrating the mucosa. Few macrophages also infiltrate the epithelium (arrow). CD163 IHC. (k) Immunolabeling of few CD68+ macrophages. CD68 IHC. (l) IHC for CD3+ T lymphocytes. CD3 IHC. (m) IHC for CD79αcy+ B lymphocytes. CD79αcy IHC. (n–p) Immunofluorescence and confocal microscopy of the buccal mucosa of an ORFV-infected lamb. (n) In the merged image, EGFR is expressed on CD163+ macrophages (arrow) and other cells of the fibrovascular component (arrowhead). The 2 insets on the right represent the single separate images of EGFR (in green) and CD163 marker (in red). DAPI nuclear stain in blue. E, epithelium. (o In a serial section of Fig. 3n, SMA + pericytes surround endothelial cells expressing EGFR in a newly formed vessel containing erythrocytes. SMA, smooth muscle actin. SMA is shown in red, EGFR in green, and DAPI nuclear stain in blue. (p) Immunofluorescence and confocal microscopy of the lamina propria of the lip mucosa from a bluetongue virus sheep. No expression of EGFR was observed in the lamina propria which displays numerous CD163+ macrophages, while the receptor is observed in the basal layer of the epithelium. EGFR is shown in green, CD163 in red, and DAPI nuclear stain in blue. E, epithelium.

Figure 4.

Graphs and photomicrograph representing the relative levels of gene expression by quantitative real-time PCR in ORF virus infected and healthy control lambs and kids. (a–e). Gene expression relative quantification of host vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and interleukin-6 (IL-6). Statistically significant overexpression was found only for VEGFR1, VEGFR2, and IL-6 when compared with the healthy controls. Values were normalized to the expression levels of the sheep 18s rRNA and β-actin (ACTB) and expressed as relative fold change compared to the expression levels of one of the healthy controls. (f) Representative image of the IL-6 mRNA localization in the dermis. In situ hybridization for IL-6 mRNA.

Figure 5.

Confocal microscopy of the lamina propria of the mucosa from an ORF virus infected kid. (a) Vascular endothelial growth factor receptor 2 (VEGFR2) is expressed in CD163+ macrophages (arrows). VEGFR2 is shown in green, CD163+ macrophages in red, and DAPI nuclear stain in blue. (b) Vascular endothelial growth factor (VEGF) immunolabeling is present in CD163+ macrophages (arrows). VEGF is shown in green, CD163+ macrophages in red, and DAPI nuclear stain in blue. E, epithelium.