Orf virus (ORFV) infection in a three-dimensional human skin model: Characteristic cellular alterations and interference with keratinocyte differentiation.

Mahmod Muhsen,Gabriele Köhler,Sabine Siegemund,Uwe Müller,Mathias Büttner,Thomas M Magin,Gottfried Alber,Martina Protschka,Laura E Schneider,Miroslav Blumenberg

doi:10.1371/journal.pone.0210504

Abstract

ORF virus (ORFV) is the causative agent of contagious ecthyma, a pustular dermatitis of small ruminants and humans. Even though the development of lesions caused by ORFV was extensively studied in animals, only limited knowledge exists about the lesion development in human skin. The aim of the present study was to evaluate a three-dimensional (3D) organotypic culture (OTC) as a human skin model for ORFV infection considering lesion development, replication of the virus, viral gene transcription and modulation of differentiation of human keratinocytes by ORFV. ORFV infection of OTC was performed using the ORFV isolate B029 derived from a human patient. The OTC sections showed a similar structure of stratified epidermal keratinocytes as human foreskin and a similar expression profile of the differentiation markers keratin 1 (K1), K10, and loricrin. Upon ORFV infection, OTCs exhibited histological cytopathic changes including hyperkeratosis and ballooning degeneration of the keratinocytes. ORFV persisted for 10 days and was located in keratinocytes of the outer epidermal layers. ORFV-specific early, intermediate and late genes were transcribed, but limited viral spread and restricted cell infection were noticed. ORFV infection resulted in downregulation of K1, K10, and loricrin at the transcriptional level without affecting proliferation as shown by PCNA or Ki-67 expression. In conclusion, OTC provides a suitable model to study the interaction of virus with human keratinocytes in a similar structural setting as human skin and reveals that ORFV infection downregulates several differentiation markers in the epidermis of the human skin, a hitherto unknown feature of dermal ORFV infection in man.

Highlights

Organotypic cultures (OTC) have been established for various purposes and can be a suitable equivalent, e.g. for studies of wound healing or infectious diseases manifesting in the skin [1,2,3,4]
In order to investigate the effects of Orf virus (ORFV) infection in human skin, we established a 3-dimensional (3D) organotypic culture (OTC) as skin model
To analyze differentiation markers of KC in the OTC model, we compared the presence of keratin 1 (K1) and keratin 10 (K10), keratins produced during differentiation in suprabasal layers, and of keratin 14 (K14), a keratin usually found in proliferating basal KC [25,38]

Summary

Introduction

Organotypic cultures (OTC) have been established for various purposes and can be a suitable equivalent, e.g. for studies of wound healing or infectious diseases manifesting in the skin [1,2,3,4]. In contrast to systemic Orthopoxvirus infections, the human zoonotic infection with PPV, especially with Orf virus (ORFV), is a localized event resulting in a normally benign lesion commonly known as milker’s nodule that is completely resolved within a few weeks [5]. Regarding skin-derived primary keratinocytes or dermal fibroblasts, limited studies were undertaken with ORFV to evaluate cytopathic effects or transcriptomic profiles [11,12]. The infection of OTC generated from ovine foreskin primary lamb keratinocytes with ORFV resulted in ballooning degeneration of cells in the superficial layers [1]. In a mouse model virus-encoded VEGF-E induces dermal vascularization and significantly increases the number of keratinocytes within the skin [15]

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Conclusion