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Histopathological and Immunohistochemical Studies of Cowpox Virus Replication in a Three-Dimensional Skin Model
J. Comp. Path. 2016, Vol. 155, 55e61
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
EXPERIMENTALLY INDUCED DISEASE
Histopathological and Immunohistochemical Studies of Cowpox Virus Replication in a Three-Dimensional Skin Model A. Tamosi unaite_ *, D. Hoffmann†, A. Franke†, J. Schluckebier†, K. Tauscher‡, B. K. Tischer*, M. Beer†, R. Klopfleischx and N. Osterrieder* * Institut f€ur Virologie, Freie Universit€at Berlin, Zentrum f€ur Infektionsmedizin, Berlin, † Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, S€udufer 10, Greifswald-Insel Riems, ‡ Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institute, S€udufer 10, Greifswald-Insel Riems and x Institute for Veterinary Pathology, Freie Universit€at Berlin, Berlin, Germany
Summary Human cowpox virus (CPXV) infections are rare, but can result in severe and sometimes fatal outcomes. The majority of recent cases were traced back to contacts with infected domestic cats or pet rats. The aim of the present study was to evaluate a three-dimensional (3D) skin model as a possible replacement for animal experiments. We monitored CPXV lesion formation, viral gene expression and cell cycle patterns after infection of 3D skin cultures with two CPXV strains of different pathogenic potential: a recent pet rat isolate (RatPox09) and the reference Brighton red strain. Infected 3D skin cultures exhibited histological alterations that were similar to those of mammal skin infections, but there were no differences in gene expression patterns and tissue damage between the two CPXV strains in the model system. In conclusion, 3D skin cultures reflect the development of pox lesions in the skin very well, but seem not to allow differentiation between more or less virulent virus strains, a distinction that is made possible by experimental infection in suitable animal models. Ó 2016 Elsevier Ltd. All rights reserved. Keywords: three-dimensional skin culture; cowpox virus; gene expression
Introduction Cowpox virus (CPXV), a member of the Orthopoxvirus (OPV) genus within the Poxviridae family, causes zoonotic infections and is endemic in Europe and Northern and Central Asia (Bennett and Baxby, 1996; Baxby and Bennett, 1997; Vorou et al., 2008). The name cowpox was coined in the 18th century when CPXV was associated with clinical, but mostly localized, skin disease of cows and milkmaids. Today, human CPXV infections acquired from pet rats and cats are reported in increasing numbers Correspondence to: N. Osterrieder (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2016.05.001
(Vorou et al., 2008; Campe et al., 2009; Carletti et al., 2009; Ninove et al., 2009; Elsendoorn et al., 2011; Vogel et al., 2012; Hobi et al., 2015), without evidence for direct human-to-human transmission. Field and experimental studies have indicated that CPXV maintains a reservoir in wild rodents such as bank voles (Myodes glareolus), field voles (Microtus agrestis), wood mice (Apodemus sylvaticus) or common voles (Microtus arvalis) (Bennett et al., 1997; Feore et al., 1997; Chantrey et al., 1999; Hazel et al., 2000; Burthe et al., 2008; Begon et al., 2009; Hoffmann et al., 2015). Multiple divergent strains of CPXV have been identified; the best characterized are Brighton red (BR) and GRI-90 (Shchelkunov et al., Ó 2016 Elsevier Ltd. All rights reserved.
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1998; Gubser et al., 2004; Xu et al., 2014). A number of full-length CPXV genomes are publicly available (www.poxvirus.org). It is also known that the pathogenic potential of different CPXV strains is variable. Infection of mice with BR results in general, but usually mild clinical signs on high titre inoculation (Martinez et al., 2000). Infection of Wistar rats with CPXV strain BR also results in a mild to asymptomatic course of disease (Hoffmann et al., 2015). In contrast, an isolate derived from a pet rat in Southern Germany in 2009, RatPox09, induces severe smallpox-like signs including rash in infected rats (Kalthoff et al., 2011; Breithaupt et al., 2012). Even in the case of intradermal inoculation, RatPox09 caused systemic disease with multiple lesions that range from proliferative to necrotizing and ulcerative, mixed cellular dermatitis and folliculitis with epithelial ballooning degeneration and necrosis as well as parakeratotic hyperkeratosis (Breithaupt et al., 2012). CPXV infection and disease have been reported in various captive mammals in zoological collections, where interspecies transmission was observed repeatedly and likewise with variable clinical signs, indicating different susceptibilities to infection that may also depend on the particular CPXV strain involved (Marennikova et al., 1977; Baxby et al., 1982; Pilaski et al., 1986; Martina et al., 2006; Kurth et al., 2008; Kalthoff et al., 2014). Naturally occurring human infections are of growing concern because the number of people with vaccinia virus (VACV)-induced immunity is declining; even those that were vaccinated in childhood most probably no longer have a significant smallpox immunity (Shchelkunov, 2013). The normal course of infection in man was reconstructed from an overview of 54 cases (Baxby et al., 1994). In contrast to smallpox, CPXV infection in man is due to direct inoculation of CPXV into compromised skin and/or oral mucous membranes via small lesions, after which the characteristic skin lesions develop. Firstly, inflamed macules arise that change to papulonodular or vesicular, then sterile pustules; later, central haemorrhagic necrosis covered by a black eschar and often surrounded by erythema and indurated oedema is formed (Baxby et al., 1994). Fatal disease occurs rarely and is not completely characterized, although isolated cases of heart failure and encephalitis have been reported (Eis-Hubinger et al., 1990). Organotypic epithelial raft cultures are a promising system to recapitulate in vitro the structure, cell cycle status and physiological conditions of skin (Bell et al., 1983); these are also currently being tested as a substitute for animal models (Roguet et al., 2000).
The PhenionÒ full-thickness skin model is a threedimensional (3D) tissue construct that simulates histological and physiological properties of human skin (Dorn et al., 2006; Mewes et al., 2007; Ackermann et al., 2010). The model represents a multilayered epidermis and a dermal compartment, where the epidermis is generated from male neonatal foreskin keratinocyte stem cells and the dermis from fibroblasts embedded in a bovine collagen matrix. Keratinocytes and fibroblasts are obtained from biopsy material of the same healthy human donor (www.phenion.com). The pathogenesis of several different virus infections, including infections with papillomaviruses, adenoviruses, parvoviruses, poxviruses and herpesviruses, have been tested with 3D skin substitutes (Andrei et al., 2010). In the case of poxviruses, they were used to test the pharmacokinetics of antiviral drugs for CPVX and VACV infections (Duraffour et al., 2007; Snoeck et al., 2002). We tested the suitability of a 3D skin model to assess CPXV replication and virulence prediction by examining the gene expression patterns, tissue damage, cell cycle status and regulation in the PhenionÒ fullthickness skin model infected with two different CPXV strains. We used two CPXV strains, BR and RatPox09, which show very different virulence and pathology in vivo. Our results indicate that, while CPXV skin replication can be successfully modelled using this in-vitro system, an assessment of overall virulence was not possible, likely because important steps in the pathogenesis, such as development of viraemia and innate or non-specific immune response, are absent.
Materials and Methods Viruses
CPXV strain BR (AF428758) was kindly provided by Dr. P. Beard, University of Edinburgh, UK, and used as reference strain. CPXV strain RatPox09 was isolated from a diseased pet rat, which had infected two people in Southern Germany in 2009 and was kindly provided by Dr. H. Meyer, Munich, Germany (Kalthoff et al., 2011). Both strains were propagated on Vero76 cells and amplified to stock titres of approximately 107 TCID50/ml. 3D Skin Model Infections
The PhenionÒ full-thickness skin model PhenionÒFT, obtained at the age of 14 days at aireliquid interphase (Ø 1.32 cm; surface area 1.30 cm2), was purchased from Henkel AG & Co. KGaA (D€ usseldorf, Germany). According to instructions of the supplier, the tissue was kept at 37 C under 5% CO2 overnight and fresh, pre-warmed medium was added.
Cowpox Replication in a Skin Model
The PhenionÒ full-thickness skin model was infected with 105 TCID50 of CPXV strains BR or RatPox09 by scarification of the tissue. Cultures were incubated for 7 days at 37 C and lesion development was monitored macroscopically. Eventually, slices were fixed in 10% neutral buffered formalin, embedded in paraffin wax and used for histological analysis. Sections (2 mm) were mounted on adhesive glass slides and stained with haematoxylin and eosin (HE). Immunohistochemistry
Expression of the CPXV protein encoded by the VACV A27L homologous gene, as well as human cyclins B1, D1, E and histone H3 phospho S10, was analyzed using immunohistochemical labelling. For each treatment group, six slides were analyzed. Endogenous peroxidase was blocked with H2O2 3% in phosphate buffered saline (PBS, 0.1 M, pH 7.4) for 20 min. Antigen retrieval was performed by heating in citrate buffer (pH 6.0) for 12 min for detection of cyclin E (mouse monoclonal anti-human cyclin E, dilution 1 in 100, eBiosciences, San Diego, California, USA), cyclin B1 (polyclonal rabbit anti-human cyclin B1, dilution 1 in 100, Abcam, Cambridge, UK), cyclin D1 (monoclonal rabbit anti-human cyclin D1, dilution 1 in 100, Abcam) and histone H3 phospho S10 (polyclonal rabbit anti-human histone H3 phospho S10, dilution 1 in 400, Abcam). Monoclonal mouse anti-vaccinia A27 antibody (dilution 1 in 1,000, kindly provided by Dr. A. Nitsche, Robert Koch Institute, Berlin, Germany) required no antigen retrieval. To block non-specific antibody binding, slides were incubated for 30 min with Roti-ImmunoblockÒ (Roth, Karlsruhe, Germany) and normal goat serum. Sections were incubated overnight at 4 C with the primary antibody. Secondary antibodies were applied as previously described (Werner et al., 2007; Vahlenkamp et al., 2008). In each experiment, slides that were incubated with irrelevant commercial mouse or rabbit immunoglobulins (BioGenex, Fremont, California, USA) instead of primary antibodies served as negative controls.
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ual cells could no longer be distinguished (Fig. 1). The 3D skin tissue infected with CPXV BR or RatPox09 allowed virus replication with evidence of a cytopathic effect. Sections of 3D skin models infected with BR or RatPox09 virus were also stained with HE. Infection led to epithelial necrosis as well as epithelial and stromal proliferation, cytoplasmic swelling and ballooning of keratinocytes (Fig. 1). The necrotic epithelial structures contained intracytoplasmic Guarnieri bodies (B-type inclusions) within the affected keratinocytes and showed hyperplasia of epithelial cells and eosinophilic inclusion bodies in epithelial cells. However, we were unable to detect any differences in the tissue damage between the two CPXV strains (Fig. 1).
Results Histology of the Uninfected and CPXV-Infected Skin Model
Histological images obtained from uninfected rafts stained with HE revealed a fully differentiated epithelium consisting of epidermis and dermis. Four cell layers were distinguished: the stratum basale with keratinocytes arranged in a palisade-like pattern, a thin layer of stratum spinosum, the stratum granulosum keratinocytes with a predominantly flattened appearance and the stratum corneum, where individ-
Fig. 1. Sections of 3D skin models mock-infected (MOCK) and infected with CPXV BR and CPXV RatPox09 and stained with HE. Infection with both virus strains led to epidermal necrosis and the presence of intracytoplasmic inclusion bodies. Bars, 50 mm. Insets show higher magnifications of epidermal cells with ballooning degeneration and intracytoplasmic inclusion bodies (arrows). Bars, 25 mm.
A. Tamosi unaite_ et al.
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Analysis of Cell Cycle and Gene Expression in the Infected 3D Skin Culture Model
The CPXV A27L homologous protein (VACV terminology) was detected in keratinocytes and some fibroblasts in all CPXV-infected skin tissues, while no A27L gene expression was detected in mock-infected skin (Fig. 2). Cyclin E, D1, B1 and histone H3 phospho S10 expression was recognized both in mock- and CPXV-infected skin. Histone H3 phospho S10 was expressed more strongly in infected than in noninfected cells (Fig. 2). Cyclin D1 was expressed at slightly reduced levels in infected tissue when compared with non-infected cells (Fig. 3). However, no differences in the cell cycle patterns between BRand RatPox09-infected raft cultures were detected (Figs. 2 and 3).
Discussion The number of human CPXV infections is growing (Baxby et al., 1994; Baxby and Bennett, 1997; Vorou et al., 2008; Campe et al., 2009; Ninove et al.,
2009; Elsendoorn et al., 2011; Vogel et al., 2012; Hobi et al., 2015; Kinnunen et al., 2015; Switaj et al., 2015), most likely due to the cessation of smallpox vaccination in 1978 (Shchelkunov, 2013). Although infection of people with CPXV generally remains localized, it can be fatal for immunosuppressed individuals (Eis-Hubinger et al., 1990). In the present study, we examined infections of PhenionÒ, a multilayered skin equivalent that resembles human skin under culture conditions, with two CPXV strains (BR and RatPox09) with contrasting virulence in the Wistar rat model. CPXV infection of the 3D skin model caused cytopathic effects, including ballooning of keratinocytes and intracytoplasmic Guarnieri bodies, as seen in histological sections of infected mammalian skin after systemic or local infection in vivo. Productive CPXV infection of the 3D skin model was confirmed by immunohistochemistry (Fig. 2). In addition, RatPox09 and BR caused similar gene expression patterns and tissue damage in the 3D skin model. Both VACV and myxoma virus are known to alter cell cycle progression (Wali and Strayer, 1999; Johnston et al., 2005; Yoo et al.,
Fig. 2. Sections of 3D skin models mock-infected (MOCK) and infected with CPXV BR and CPXV RatPox09 were labelled with antiA27L and anti-histone H3 phospho S10 antibodies. A27L expression was detected in epidermal cells infected with both virus strains. In addition, infected epidermal areas showed an increased expression of phosphorylated histone H3. IHC. Bars, 50 mm.
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Cowpox Replication in a Skin Model
Fig. 3. Sections of 3D skin models, mock-infected (MOCK) and infected with CPXV BR and CPXV RatPox09 were labelled with antibodies against cyclins B1, D1 and E. Infected epidermal areas showed an decreased labelling intensity for cyclin D1, while cyclins B1 and E expression was not altered. IHC. Bar, 50 mm.
2008). To analyze the impact of CPXV BR or RatPox09 infection on cell cycle status, cell cycle markers were identified immunohistochemically. As a marker of M phase cells, anti-histone H3 phospho S10 was used (Hans and Dimitrov, 2001). Histone H3 phospho S10 was highly expressed in infected cells; interestingly, not only in the nucleus, but also in the cytoplasm (Fig. 2). Phosphorylation of the serine residue at position 10 of histone H3 is associated with highly condensed mitotic chromosomes and relaxed chromatin of a number of activated genes during interphase (Prigent and Dimitrov, 2003). In addition, previous reports have shown that H3 S10 phosphorylation also occurs when cells are exposed to various death stimuli (Bode and Dong, 2005). Cyclin B plays a role in control of the M phase of the cell cycle, while cyclin E is important for G1eS phase transition (Evans et al., 1983). Both of these cyclins were expressed similarly in infected and noninfected cells of the 3D skin model (Fig. 3). As a marker for progression from G0 to G1, anti-cyclin D1 was used. Cyclin D1 was slightly downregulated in infected cells (Fig. 3). Although little is known about cyclin D1 downregulation in the course of CPXV infection, previous reports have shown that the short murine schlafen gene, m-slfn1, encodes a protein that causes arrest of fibroblast growth by inhibition of cyclin D1 in vitro (Brady et al., 2005). It has
been demonstrated that a single copy of schlafen is present in all sequenced OPVs, CPXV included (Gubser et al., 2007; Bustos et al., 2009). Hence, the CPXV Schlafen homologue may result in reduced expression of cyclin D1. In conclusion, the present experiments have shown that a 3D skin model can be used to examine the development of pox lesions, CPXV gene expression and cell cycle progression of infected cells, but it seems not to allow for the distinction between the pathogenicity of more or less virulent isolates.
Acknowledgments The authors are grateful to H. Meyer, Institut f€ ur Mikrobiologie der Bundeswehr, M€ unchen, for kindly providing the pet rat CPXV strain RatPox09. This project was funded by a project awarded to MB and NO by the German Research Foundation (DFG) in the framework of the Focus Program SPP1596.
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Cowpox Replication in a Skin Model
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Snoeck R, Holy A, Wolf-Peeters C, Van Den OJ, De CE et al. (2002) Antivaccinia activities of acyclic nucleoside phosphonate derivatives in epithelial cells and organotypic cultures. Antimicrobial Agents and Chemotherapy, 46, 3356e3361. Switaj K, Kajfasz P, Kurth A, Nitsche A (2015) Cowpox after a cat scratch e case report from Poland. Annals of Agriculture and Environmental Medicine, 3, 456e458. Vahlenkamp TW, Harder TC, Giese M, Lin F, Teifke JP et al. (2008) Protection of cats against lethal influenza H5N1 challenge infection. Journal of General Virology, 89, 968e974. Vogel S, Sardy M, Glos K, Korting HC, Ruzicka T et al. (2012) The Munich outbreak of cutaneous cowpox infection: transmission by infected pet rats. Acta Dermatolgica Venereologica, 92, 126e131. Vorou RM, Papavassiliou VG, Pierroutsakos IN (2008) Cowpox virus infection: an emerging health threat. Current Opinion in Infectious Diseases, 21, 153e156. Wali A, Strayer DS (1999) Infection with vaccinia virus alters regulation of cell cycle progression. DNA Cell Biology, 18, 837e843. Werner O, Starick E, Teifke J, Klopfleisch R, Prajitno TY et al. (2007) Minute excretion of highly pathogenic avian influenza virus A/chicken/Indonesia/2003 (H5N1) from experimentally infected domestic pigeons (Columbia livia) and lack of transmission to sentinel chickens. Journal of General Virology, 88, 3089e3093. Xu Z, Zikos D, Osterrieder N, Tischer BK (2014) Generation of a complete single-gene knockout bacterial artificial chromosome library of cowpox virus and identification of its essential genes. Journal of Virology, 88, 490e502. Yoo NK, Pyo CW, Kim Y, Ahn BY, Choi SY (2008) Vaccinia virus-mediated cell cycle alteration involves inactivation of tumour suppressors associated with Brf1 and TBP. Cellular Microbiology, 10, 583e592.
March 10th, 2016 ½ Received, Accepted, May 4th, 2016
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
EXPERIMENTALLY INDUCED DISEASE
Histopathological and Immunohistochemical Studies of Cowpox Virus Replication in a Three-Dimensional Skin Model A. Tamosi unaite_ *, D. Hoffmann†, A. Franke†, J. Schluckebier†, K. Tauscher‡, B. K. Tischer*, M. Beer†, R. Klopfleischx and N. Osterrieder* * Institut f€ur Virologie, Freie Universit€at Berlin, Zentrum f€ur Infektionsmedizin, Berlin, † Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, S€udufer 10, Greifswald-Insel Riems, ‡ Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institute, S€udufer 10, Greifswald-Insel Riems and x Institute for Veterinary Pathology, Freie Universit€at Berlin, Berlin, Germany
Summary Human cowpox virus (CPXV) infections are rare, but can result in severe and sometimes fatal outcomes. The majority of recent cases were traced back to contacts with infected domestic cats or pet rats. The aim of the present study was to evaluate a three-dimensional (3D) skin model as a possible replacement for animal experiments. We monitored CPXV lesion formation, viral gene expression and cell cycle patterns after infection of 3D skin cultures with two CPXV strains of different pathogenic potential: a recent pet rat isolate (RatPox09) and the reference Brighton red strain. Infected 3D skin cultures exhibited histological alterations that were similar to those of mammal skin infections, but there were no differences in gene expression patterns and tissue damage between the two CPXV strains in the model system. In conclusion, 3D skin cultures reflect the development of pox lesions in the skin very well, but seem not to allow differentiation between more or less virulent virus strains, a distinction that is made possible by experimental infection in suitable animal models. Ó 2016 Elsevier Ltd. All rights reserved. Keywords: three-dimensional skin culture; cowpox virus; gene expression
Introduction Cowpox virus (CPXV), a member of the Orthopoxvirus (OPV) genus within the Poxviridae family, causes zoonotic infections and is endemic in Europe and Northern and Central Asia (Bennett and Baxby, 1996; Baxby and Bennett, 1997; Vorou et al., 2008). The name cowpox was coined in the 18th century when CPXV was associated with clinical, but mostly localized, skin disease of cows and milkmaids. Today, human CPXV infections acquired from pet rats and cats are reported in increasing numbers Correspondence to: N. Osterrieder (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2016.05.001
(Vorou et al., 2008; Campe et al., 2009; Carletti et al., 2009; Ninove et al., 2009; Elsendoorn et al., 2011; Vogel et al., 2012; Hobi et al., 2015), without evidence for direct human-to-human transmission. Field and experimental studies have indicated that CPXV maintains a reservoir in wild rodents such as bank voles (Myodes glareolus), field voles (Microtus agrestis), wood mice (Apodemus sylvaticus) or common voles (Microtus arvalis) (Bennett et al., 1997; Feore et al., 1997; Chantrey et al., 1999; Hazel et al., 2000; Burthe et al., 2008; Begon et al., 2009; Hoffmann et al., 2015). Multiple divergent strains of CPXV have been identified; the best characterized are Brighton red (BR) and GRI-90 (Shchelkunov et al., Ó 2016 Elsevier Ltd. All rights reserved.
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1998; Gubser et al., 2004; Xu et al., 2014). A number of full-length CPXV genomes are publicly available (www.poxvirus.org). It is also known that the pathogenic potential of different CPXV strains is variable. Infection of mice with BR results in general, but usually mild clinical signs on high titre inoculation (Martinez et al., 2000). Infection of Wistar rats with CPXV strain BR also results in a mild to asymptomatic course of disease (Hoffmann et al., 2015). In contrast, an isolate derived from a pet rat in Southern Germany in 2009, RatPox09, induces severe smallpox-like signs including rash in infected rats (Kalthoff et al., 2011; Breithaupt et al., 2012). Even in the case of intradermal inoculation, RatPox09 caused systemic disease with multiple lesions that range from proliferative to necrotizing and ulcerative, mixed cellular dermatitis and folliculitis with epithelial ballooning degeneration and necrosis as well as parakeratotic hyperkeratosis (Breithaupt et al., 2012). CPXV infection and disease have been reported in various captive mammals in zoological collections, where interspecies transmission was observed repeatedly and likewise with variable clinical signs, indicating different susceptibilities to infection that may also depend on the particular CPXV strain involved (Marennikova et al., 1977; Baxby et al., 1982; Pilaski et al., 1986; Martina et al., 2006; Kurth et al., 2008; Kalthoff et al., 2014). Naturally occurring human infections are of growing concern because the number of people with vaccinia virus (VACV)-induced immunity is declining; even those that were vaccinated in childhood most probably no longer have a significant smallpox immunity (Shchelkunov, 2013). The normal course of infection in man was reconstructed from an overview of 54 cases (Baxby et al., 1994). In contrast to smallpox, CPXV infection in man is due to direct inoculation of CPXV into compromised skin and/or oral mucous membranes via small lesions, after which the characteristic skin lesions develop. Firstly, inflamed macules arise that change to papulonodular or vesicular, then sterile pustules; later, central haemorrhagic necrosis covered by a black eschar and often surrounded by erythema and indurated oedema is formed (Baxby et al., 1994). Fatal disease occurs rarely and is not completely characterized, although isolated cases of heart failure and encephalitis have been reported (Eis-Hubinger et al., 1990). Organotypic epithelial raft cultures are a promising system to recapitulate in vitro the structure, cell cycle status and physiological conditions of skin (Bell et al., 1983); these are also currently being tested as a substitute for animal models (Roguet et al., 2000).
The PhenionÒ full-thickness skin model is a threedimensional (3D) tissue construct that simulates histological and physiological properties of human skin (Dorn et al., 2006; Mewes et al., 2007; Ackermann et al., 2010). The model represents a multilayered epidermis and a dermal compartment, where the epidermis is generated from male neonatal foreskin keratinocyte stem cells and the dermis from fibroblasts embedded in a bovine collagen matrix. Keratinocytes and fibroblasts are obtained from biopsy material of the same healthy human donor (www.phenion.com). The pathogenesis of several different virus infections, including infections with papillomaviruses, adenoviruses, parvoviruses, poxviruses and herpesviruses, have been tested with 3D skin substitutes (Andrei et al., 2010). In the case of poxviruses, they were used to test the pharmacokinetics of antiviral drugs for CPVX and VACV infections (Duraffour et al., 2007; Snoeck et al., 2002). We tested the suitability of a 3D skin model to assess CPXV replication and virulence prediction by examining the gene expression patterns, tissue damage, cell cycle status and regulation in the PhenionÒ fullthickness skin model infected with two different CPXV strains. We used two CPXV strains, BR and RatPox09, which show very different virulence and pathology in vivo. Our results indicate that, while CPXV skin replication can be successfully modelled using this in-vitro system, an assessment of overall virulence was not possible, likely because important steps in the pathogenesis, such as development of viraemia and innate or non-specific immune response, are absent.
Materials and Methods Viruses
CPXV strain BR (AF428758) was kindly provided by Dr. P. Beard, University of Edinburgh, UK, and used as reference strain. CPXV strain RatPox09 was isolated from a diseased pet rat, which had infected two people in Southern Germany in 2009 and was kindly provided by Dr. H. Meyer, Munich, Germany (Kalthoff et al., 2011). Both strains were propagated on Vero76 cells and amplified to stock titres of approximately 107 TCID50/ml. 3D Skin Model Infections
The PhenionÒ full-thickness skin model PhenionÒFT, obtained at the age of 14 days at aireliquid interphase (Ø 1.32 cm; surface area 1.30 cm2), was purchased from Henkel AG & Co. KGaA (D€ usseldorf, Germany). According to instructions of the supplier, the tissue was kept at 37 C under 5% CO2 overnight and fresh, pre-warmed medium was added.
Cowpox Replication in a Skin Model
The PhenionÒ full-thickness skin model was infected with 105 TCID50 of CPXV strains BR or RatPox09 by scarification of the tissue. Cultures were incubated for 7 days at 37 C and lesion development was monitored macroscopically. Eventually, slices were fixed in 10% neutral buffered formalin, embedded in paraffin wax and used for histological analysis. Sections (2 mm) were mounted on adhesive glass slides and stained with haematoxylin and eosin (HE). Immunohistochemistry
Expression of the CPXV protein encoded by the VACV A27L homologous gene, as well as human cyclins B1, D1, E and histone H3 phospho S10, was analyzed using immunohistochemical labelling. For each treatment group, six slides were analyzed. Endogenous peroxidase was blocked with H2O2 3% in phosphate buffered saline (PBS, 0.1 M, pH 7.4) for 20 min. Antigen retrieval was performed by heating in citrate buffer (pH 6.0) for 12 min for detection of cyclin E (mouse monoclonal anti-human cyclin E, dilution 1 in 100, eBiosciences, San Diego, California, USA), cyclin B1 (polyclonal rabbit anti-human cyclin B1, dilution 1 in 100, Abcam, Cambridge, UK), cyclin D1 (monoclonal rabbit anti-human cyclin D1, dilution 1 in 100, Abcam) and histone H3 phospho S10 (polyclonal rabbit anti-human histone H3 phospho S10, dilution 1 in 400, Abcam). Monoclonal mouse anti-vaccinia A27 antibody (dilution 1 in 1,000, kindly provided by Dr. A. Nitsche, Robert Koch Institute, Berlin, Germany) required no antigen retrieval. To block non-specific antibody binding, slides were incubated for 30 min with Roti-ImmunoblockÒ (Roth, Karlsruhe, Germany) and normal goat serum. Sections were incubated overnight at 4 C with the primary antibody. Secondary antibodies were applied as previously described (Werner et al., 2007; Vahlenkamp et al., 2008). In each experiment, slides that were incubated with irrelevant commercial mouse or rabbit immunoglobulins (BioGenex, Fremont, California, USA) instead of primary antibodies served as negative controls.
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ual cells could no longer be distinguished (Fig. 1). The 3D skin tissue infected with CPXV BR or RatPox09 allowed virus replication with evidence of a cytopathic effect. Sections of 3D skin models infected with BR or RatPox09 virus were also stained with HE. Infection led to epithelial necrosis as well as epithelial and stromal proliferation, cytoplasmic swelling and ballooning of keratinocytes (Fig. 1). The necrotic epithelial structures contained intracytoplasmic Guarnieri bodies (B-type inclusions) within the affected keratinocytes and showed hyperplasia of epithelial cells and eosinophilic inclusion bodies in epithelial cells. However, we were unable to detect any differences in the tissue damage between the two CPXV strains (Fig. 1).
Results Histology of the Uninfected and CPXV-Infected Skin Model
Histological images obtained from uninfected rafts stained with HE revealed a fully differentiated epithelium consisting of epidermis and dermis. Four cell layers were distinguished: the stratum basale with keratinocytes arranged in a palisade-like pattern, a thin layer of stratum spinosum, the stratum granulosum keratinocytes with a predominantly flattened appearance and the stratum corneum, where individ-
Fig. 1. Sections of 3D skin models mock-infected (MOCK) and infected with CPXV BR and CPXV RatPox09 and stained with HE. Infection with both virus strains led to epidermal necrosis and the presence of intracytoplasmic inclusion bodies. Bars, 50 mm. Insets show higher magnifications of epidermal cells with ballooning degeneration and intracytoplasmic inclusion bodies (arrows). Bars, 25 mm.
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Analysis of Cell Cycle and Gene Expression in the Infected 3D Skin Culture Model
The CPXV A27L homologous protein (VACV terminology) was detected in keratinocytes and some fibroblasts in all CPXV-infected skin tissues, while no A27L gene expression was detected in mock-infected skin (Fig. 2). Cyclin E, D1, B1 and histone H3 phospho S10 expression was recognized both in mock- and CPXV-infected skin. Histone H3 phospho S10 was expressed more strongly in infected than in noninfected cells (Fig. 2). Cyclin D1 was expressed at slightly reduced levels in infected tissue when compared with non-infected cells (Fig. 3). However, no differences in the cell cycle patterns between BRand RatPox09-infected raft cultures were detected (Figs. 2 and 3).
Discussion The number of human CPXV infections is growing (Baxby et al., 1994; Baxby and Bennett, 1997; Vorou et al., 2008; Campe et al., 2009; Ninove et al.,
2009; Elsendoorn et al., 2011; Vogel et al., 2012; Hobi et al., 2015; Kinnunen et al., 2015; Switaj et al., 2015), most likely due to the cessation of smallpox vaccination in 1978 (Shchelkunov, 2013). Although infection of people with CPXV generally remains localized, it can be fatal for immunosuppressed individuals (Eis-Hubinger et al., 1990). In the present study, we examined infections of PhenionÒ, a multilayered skin equivalent that resembles human skin under culture conditions, with two CPXV strains (BR and RatPox09) with contrasting virulence in the Wistar rat model. CPXV infection of the 3D skin model caused cytopathic effects, including ballooning of keratinocytes and intracytoplasmic Guarnieri bodies, as seen in histological sections of infected mammalian skin after systemic or local infection in vivo. Productive CPXV infection of the 3D skin model was confirmed by immunohistochemistry (Fig. 2). In addition, RatPox09 and BR caused similar gene expression patterns and tissue damage in the 3D skin model. Both VACV and myxoma virus are known to alter cell cycle progression (Wali and Strayer, 1999; Johnston et al., 2005; Yoo et al.,
Fig. 2. Sections of 3D skin models mock-infected (MOCK) and infected with CPXV BR and CPXV RatPox09 were labelled with antiA27L and anti-histone H3 phospho S10 antibodies. A27L expression was detected in epidermal cells infected with both virus strains. In addition, infected epidermal areas showed an increased expression of phosphorylated histone H3. IHC. Bars, 50 mm.
59
Cowpox Replication in a Skin Model
Fig. 3. Sections of 3D skin models, mock-infected (MOCK) and infected with CPXV BR and CPXV RatPox09 were labelled with antibodies against cyclins B1, D1 and E. Infected epidermal areas showed an decreased labelling intensity for cyclin D1, while cyclins B1 and E expression was not altered. IHC. Bar, 50 mm.
2008). To analyze the impact of CPXV BR or RatPox09 infection on cell cycle status, cell cycle markers were identified immunohistochemically. As a marker of M phase cells, anti-histone H3 phospho S10 was used (Hans and Dimitrov, 2001). Histone H3 phospho S10 was highly expressed in infected cells; interestingly, not only in the nucleus, but also in the cytoplasm (Fig. 2). Phosphorylation of the serine residue at position 10 of histone H3 is associated with highly condensed mitotic chromosomes and relaxed chromatin of a number of activated genes during interphase (Prigent and Dimitrov, 2003). In addition, previous reports have shown that H3 S10 phosphorylation also occurs when cells are exposed to various death stimuli (Bode and Dong, 2005). Cyclin B plays a role in control of the M phase of the cell cycle, while cyclin E is important for G1eS phase transition (Evans et al., 1983). Both of these cyclins were expressed similarly in infected and noninfected cells of the 3D skin model (Fig. 3). As a marker for progression from G0 to G1, anti-cyclin D1 was used. Cyclin D1 was slightly downregulated in infected cells (Fig. 3). Although little is known about cyclin D1 downregulation in the course of CPXV infection, previous reports have shown that the short murine schlafen gene, m-slfn1, encodes a protein that causes arrest of fibroblast growth by inhibition of cyclin D1 in vitro (Brady et al., 2005). It has
been demonstrated that a single copy of schlafen is present in all sequenced OPVs, CPXV included (Gubser et al., 2007; Bustos et al., 2009). Hence, the CPXV Schlafen homologue may result in reduced expression of cyclin D1. In conclusion, the present experiments have shown that a 3D skin model can be used to examine the development of pox lesions, CPXV gene expression and cell cycle progression of infected cells, but it seems not to allow for the distinction between the pathogenicity of more or less virulent isolates.
Acknowledgments The authors are grateful to H. Meyer, Institut f€ ur Mikrobiologie der Bundeswehr, M€ unchen, for kindly providing the pet rat CPXV strain RatPox09. This project was funded by a project awarded to MB and NO by the German Research Foundation (DFG) in the framework of the Focus Program SPP1596.
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March 10th, 2016 ½ Received, Accepted, May 4th, 2016