Replication of Varicella-Zoster Virus in Human Ski
http://www.100md.com
病菌学杂志 2005年第17期
Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, New York 13210
ABSTRACT
Varicella-zoster virus (VZV) infection is restricted to humans, which hinders studies of its pathogenesis in rodent models of disease. To facilitate the study of VZV skin tropism, we developed an ex vivo system using human fetal skin organ culture (SOC). VZV replication was analyzed by plaque assay, transmission electron microscopy, and histology. The yield of infectious VZV from SOC increased 100-fold over 6 days, virions were abundant, and lesions developed that contained VZV antigens and resembled varicella and zoster lesions. The SOC system for VZV replication has applications for testing virus mutants and antiviral drugs.
TEXT
The hallmark signs of chicken pox (varicella) and shingles (zoster) are itchy, painful skin lesions, caused by varicella-zoster virus (VZV). Epithelial cells, keratinocytes, and dermal fibroblasts are major targets for VZV replication. It is important to understand the determinants of VZV skin tropism in order to develop new treatments and vaccines. A lack of small-animal models that mimic primary infection, establishment of latency, and reactivation has hindered the study of VZV pathogenesis. However, the development of the SCID-hu mouse model led to advancement over cell culture techniques to study VZV replication (15). SCID-hu mice implanted with human fetal thymus (T cells) or skin xenografts enabled the systematic investigation of VZV replication in fully differentiated human tissues in vivo (16, 17). From its inception, the SCID-hu model has revealed interesting and sometimes unanticipated phenotypes of VZV mutants and variants (2, 3, 7, 9, 13, 20, 23, 24). This body of work underscores the necessity of using intact human tissues, such as skin, for addressing questions about VZV pathogenesis and molecular virulence determinants.
Alternatives to studying VZV in skin implanted in SCID-hu mice are organotypic raft cultures and skin organ culture (SOC). There are few reports of organ cultures being used to study infectious diseases (8, 21, 22). Instead, organotypic raft cultures, composed of fibroblasts embedded in a collagen matrix that feeds overlying keratinocytes, are frequently employed to study human papillomaviruses and less often for herpesviruses (6, 12, 25). Although both SOC and organotypic raft cultures are composed of dermis and epidermis, hair follicles and sebaceous glands are only found in SOC. There is mounting evidence that hair follicles are the site of VZV transfer to the skin from infected T cells, which express skin-homing markers that direct them to endothelial cells in the dermis (10). Thus, SOC appears highly suited for studying VZV in full-thickness skin where all the major cell types are present and are correctly positioned and differentiated.
Given the importance of studying VZV in human skin, we set out to facilitate this in several ways: increase the sampling frequency and number of skin tissues tested, shorten experiments from months to days, and reduce the number of SCID mice used. To achieve these goals, we developed an ex vivo system to study VZV in human fetal skin organ culture, which maintains skin tissues on NetWell supports as opposed to xenografts in SCID mice. Our hypothesis that SOC would be an effective system for investigating VZV skin tropism was supported by the results. We found that VZV replicated in all layers of the skin, causing typical lesions and producing abundant virions.
The first step in development of the SOC model for VZV replication was to determine the proper conditions for tissue sterility, integrity, and differentiation. Human fetal skin tissue (18 to 23 weeks gestational age) was purchased from Advance Biosciences Resources (Alameda, CA), in accordance with all local, state, and federal guidelines. The skin was disinfected by swabbing with Betadyne (10% povidone iodine), dipped in 70% ethanol, and rinsed in tissue culture medium [TCM: minimal essential medium with Earle's salts and L-glutamine, supplemented with 10% heat-inactivated fetal bovine serum, penicillin/streptomycin (5,000 IU/ml), amphotericin B (250 μg/ml), and nonessential amino acids (Mediatech, Washington, D.C.)]. TCM contains 1.4 mM Ca2+, which is needed for preservation of skin integrity since it quickly dedifferentiates in 0.15 mM calcium (26).
Skin tissue was cut into 1.0-cm2 pieces, placed individually in wells of a 24-well plate, and submerged in TCM supplemented with nystatin (10,000 U/ml) and ciprofloxacin (10 μg/ml) (Sigma, St. Louis MO) for 24 h in a tissue culture incubator (humidified 5% CO2 at 37°C). For long-term cultivation at 37°C, the tissues were placed individually in 500-μm mesh NetWell inserts (Corning, Corning, NY) that rested above 1.0 ml of TCM containing nystatin and ciprofloxacin in each well of a 12-well plate, thereby situating the tissues at the air-liquid interface. The skin explant was nourished through the dermis by contact with the TCM, which was refreshed every 2 days.
To observe the architecture of the skin over time, tissues were harvested after 0, 2, 4, and 6 days, then prepared for histological analysis. After 6 days, the skin appeared grossly normal, with only slight contraction from its original size, and hematoxylin and eosin-stained sections indicated that the cellular anatomy was preserved (Fig. 1A to D). Specifically, the dermis was composed of fibroblasts in a collagen matrix, with prominent hair follicles. The basement membrane separated the dermis from the epidermis, where stratified layers of basal cells and flattened keratinocytes were observed. The presence of keratin sheets on the top surface indicated that differentiation was occurring in the epidermis. The histological appearance of tissues from SOC was nearly identical to skin implants from SCID-hu mice (for review see 14) with the exception that SOC did not produce a thick layer of subcutaneous fat.
It was not known whether skin cultured ex vivo would provide growth conditions suitable for VZV replication. A cloned clinical isolate of VZV, rPOka (20), was selected for these studies because of its known virulence for skin in the SCID-hu model (29). To prepare the virus for inoculation, VZV-infected or mock-infected MeWo cells were trypsinized, then washed and resuspended in TCM, and used immediately for direct injection into MeWo cells (human melanoma cell line, kindly provided by Charles Grose, University of Iowa) and VZV rPOka were cultivated as described (27). Each tissue was injected five times with 10 μl of the cell suspension using a 1-ml syringe fitted with a 27-gauge needle attached to a volumetric stepper (Tridak, Brookfield, CT).
Tissues were incubated at 37°C for 2 h to allow the virus to adhere, and then placed individually on NetWells for cultivation as above. On days 0, 2, 4, and 6, VZV-infected skin tissues were harvested and processed for histological analysis to observe the location of VZV replication and any pathological changes. Light microscopy of fixed sections stained with hematoxylin and eosin revealed that mock-infected and VZV-infected tissues were indistinguishable on days 0, 2, and 4 (Fig. 1, A to C and E to G). By 6 days after inoculation, no lesions were visible by gross examination, although VZV-infected explants showed histopathology typical of varicella and zoster lesions, such as thickening of the epidermis, sloughing of keratinocytes (white arrow), and the presence of balloon cells (Fig. 1H). Destruction of hair follicles, as evidenced by loss of hematoxylin staining, was widespread (black arrow, Fig. 1H).
To confirm that lesions and tissue damage were caused by VZV infection, immunohistochemistry was performed. VZV proteins were detected using a polyclonal human immune serum (GK serum, kindly provided by Ann Arvin, Stanford University); a secondary antibody conjugated to alkaline phosphatase (goat anti-human alkaline phosphatase, Jackson ImmunoResearch Laboratories, Inc., Westgrove PA), and the signal was developed with Vector Red (Vector Laboratories, Burlingame CA). Tissues were counterstained with hematoxylin for contrast. After 6 days postinoculation, mock-infected skin reacted slightly with the antibodies in the outer keratinocytes, giving a faint red color (Fig. 2A). A strong red signal in the epidermis of VZV-infected skin indicated where viral antigens were present in a lesion (Fig. 2B). This lesion had been observed in adjacent sections that were stained with hematoxylin and eosin (Fig. 1H).
Higher magnification of the lesion outlined by the white box showed infected cells (arrows) in the epidermis (Fig. 2C). Examination of the entire tissue showed that VZV antigens were present in all skin cell types, particularly keratinocytes of the epidermis and hair follicles, and in scattered dermal fibroblasts (Fig. 2D). Higher magnification of the infected skin (boxes in panel D) revealed spaces forming around cell nuclei, termed balloon cells (arrow, Fig. 2E) and virus spread through the hair follicle (arrow, Fig. 2F). These signs indicate that VZV was replicating in the skin and causing histopathological changes typically seen in biopsies of varicella and zoster lesions (28). To rule out the possibility that VZV infection induced apoptosis, and thus caused indirect damage, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays (Hoffmann-La Roche, Switzerland) were performed on fixed tissue sections. Very few apoptotic cells were observed in VZV-infected tissues compared to positive controls that were treated with camptothecin (2 μg/ml, Sigma), a known inducer of apoptosis (data not shown).
To measure the extent and kinetics of VZV replication in the SOC system, we compared two modes of virus inoculation: intradermal injection and scarification. The rationale was that VZV infects skin from within, via dissemination of infected T cells (1), and externally, via breaks in the epidermis. Tissues were inoculated by injection as described above, or by scarification using a 27-gauge needle dragged across the skin 10 times while flooded in VZV-infected MeWo cells. Since the inoculum was freshly prepared to obtain the highest possible VZV titer, the virus concentration was not calculated before use. Instead, tissues were harvested 2 h after inoculation (time zero) and processed for titration by mincing tissue and placing them in 1 ml of TCM that contained 2.5-mm glass beads. After vortexing for 3 min., an infectious focus assay was performed to assess the actual amount of input virus received by the tissues following the method described (27). The tissues were incubated at 37°C on NetWells, and then four tissues were removed for virus quantitation by infectious focus assay after 2, 4, and 6 days.
Both inoculation methods resulted in a 100-fold increase in virus between days 2 and 6, and the doubling time was 17 h in tissues injected intradermally and 23 h in scarified tissues (brackets, Fig. 3). This is similar to the 16 to 20 h measured in cultured human foreskin fibroblasts (J. Moffat, unpublished observations). Scarification introduced approximately 10 PFU per tissue, whereas intradermal injection introduced over 100 PFU. Another difference was that VZV titer increased exponentially following scarification, but there was a decrease in titer in the first 2 days after intradermal injection. There is a possibility that some of the increase in titer was from VZV replication in the MeWo cells used for the inoculum, so the values stated may slightly overestimate the extent of virus growth in the skin tissue.
Using a cell-associated inoculum raised the question of whether the virus continued to grow in the MeWo cells, or if it spread to the skin cells as expected. Direct examination of virus particles in keratinocytes by transmission electron microscopy was performed to address this issue. Tissues infected with VZV for 6 days were prepared for transmission electron microscopy as described (18). In agreement with the histological analysis, virions and virus components were found widely distributed in all layers of the skin. Many infected cells were observed in the epidermis, where keratinocytes, identifiable by sheets of keratin in the cytoplasm, remained intact and tightly joined to surrounding cells during VZV replication (Fig. 4A).
Several phases of VZV assembly were observed in a single keratinocyte, outlined in boxes numbered 1 to 3, that are shown at higher magnification. Both empty and DNA-containing capsids were seen in the nucleus (Fig. 4A-1). Although capsids budding into the perinuclear space and de-envelopment at the outer nuclear membrane were not observed in this cell, capsids were found near the membrane inside (white arrowhead). Many enveloped virions were in cytoplasmic vesicles (black arrowheads, Fig. 4A-2 and -3), but secondary envelopment was not captured in partial stages. It was possible that these were input virions, since the related virus, herpes simplex virus type 1, can enter cells by endocytosis (5, 19). However, the enveloped virions within vesicles were most likely assembled in the infected cells and were destined for egress by fusion with the plasma membrane. This canonical herpesvirus assembly pathway (11) was supported by the presence of countless VZV particles in the intercellular spaces (white arrowheads, Fig. 4A-2 and -3). Some of these were complete virions while others lacked capsids, which are often scraped away when the tissues are sectioned for transmission electron microscopy.
An additional question was whether VZV replication and assembly would differ in the various skin cell types. Thus, we examined infected dermal fibroblasts, notable for their lack of keratin, by transmission electron microscopy. VZV assembly in fibroblasts proceeded similarly to that observed in keratinocytes with the exception that very large accumulations of virions were seen outside cells (Fig. 4B), which may indicate differences in VZV egress in these cell types. This phenomenon, of cell membranes crowded with particles near masses of virions, was observed throughout the dermis. Of the extracellular virions, defective particles and viral debris were abundant, which is typical of VZV morphology, although many complete virions were also seen (Fig. 4B, white arrowheads). After several different tissues were examined, it was apparent that the predominant skin cell types, keratinocytes and fibroblasts, were susceptible to VZV infection and the assembly patterns were generally similar.
The SOC model presented here demonstrates the convenience and relevance of studying VZV replication in human skin ex vivo. The virus grew rapidly in the tissues, forming typical lesions and clusters of extracellular virions and viral debris. We were hopeful that these virions could be collected from the tissue, however, large amounts of infectious cell-free virus were not obtained using mechanical or enzymatic methods. VZV was difficult to extract due to the architecture of the skin, where cells are tightly joined and embedded in a collagen matrix. The inability to isolate concentrated, infectious VZV virions has been a barrier to using common and powerful techniques such as plaque purification and high-multiplicity infections, and so we continue to explore methods for isolating them in usable quantities.
The success of the SOC system could lead to several new research directions. These include VZV pathogenesis studies that are designed to distinguish the viral genes required in vitro from in vivo, and studying VZV assembly in differentiated skin, specifically the roles of tegument proteins and glycoproteins in this process. The SOC model could also provide a screening tool for antiviral agents because results can be obtained so rapidly, making it a useful first step before confirming results in mice with the SCID-hu model. Lastly, SOC could be applied to other skin-tropic viruses such as papillomaviruses and the human poxvirus molluscum contagiosum virus, which lack good tissue culture and animal models (4).
ACKNOWLEDGMENTS
This work was supported by the Hendricks Research Fund from SUNY Upstate Medical University and by Public Health Service grant AI052168 (J.F.M.).
Transmission electron microscopy was performed with expert technical assistance from Maureen Barcza, Department of Pathology.
REFERENCES
Arvin, A. M. 2001. Varicella-zoster virus, p. 2731-2768. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed., vol. 2. Lippincott-Raven, Philadelphia, Pa.
Baiker, A., C. Bagowski, H. Ito, M. Sommer, L. Zerboni, K. Fabel, J. Hay, W. Ruyechan, and A. M. Arvin. 2004. The immediate-early 63 protein of Varicella-Zoster virus: analysis of functional domains required for replication in vitro and for T-cell and skin tropism in the SCIDhu model in vivo. J. Virol. 78:1181-1194.
Besser, J., M. H. Sommer, L. Zerboni, C. P. Bagowski, H. Ito, J. Moffat, C. C. Ku, and A. M. Arvin. 2003. Differentiation of varicella-zoster virus ORF47 protein kinase and IE62 protein binding domains and their contributions to replication in human skin xenografts in the SCID-hu mouse. J. Virol. 77:5964-5974.
Bugert, J. J., and G. Darai. 1997. Recent advances in molluscum contagiosum virus research. Arch. Virol. Suppl. 13:35-47.
Gianni, T., G. Campadelli-Fiume, and L. Menotti. 2004. Entry of herpes simplex virus mediated by chimeric forms of nectin1 retargeted to endosomes or to lipid rafts occurs through acidic endosomes. J. Virol. 78:12268-12276.
Hukkanen, V., H. Mikola, M. Nykanen, and S. Syrjanen. 1999. Herpes simplex virus type 1 infection has two separate modes of spread in three-dimensional keratinocyte culture. J. Gen. Virol. 80:2149-2155.
Ito, H., M. H. Sommer, L. Zerboni, H. He, D. Boucaud, J. Hay, W. Ruyechan, and A. M. Arvin. 2003. Promoter sequences of varicella-zoster virus glycoprotein I targeted by cellular transactivating factors Sp1 and USF determine virulence in skin and T cells in SCIDhu mice in vivo. J. Virol. 77:489-498.
Jackson, A. D., P. J. Cole, and R. Wilson. 1996. Comparison of haemophilus influenzae type b interaction with respiratory mucosa organ cultures maintained with an air interface or immersed in medium. Infect. Immun. 64:2353-2355.
Jones, J. O., and A. M. Arvin. 2003. Microarray analysis of host cell gene transcription in response to varicella-zoster virus infection of human T cells and fibroblasts in vitro and SCIDhu skin xenografts in vivo. J. Virol. 77:1268-1280.
Ku, C. C., J. A. Padilla, C. Grose, E. C. Butcher, and A. M. Arvin. 2002. Tropism of varicella-zoster virus for human tonsillar CD4+ T lymphocytes that express activation, memory, and skin homing markers. J. Virol. 76:11425-11433.
Mettenleiter, T. C. 2002. Herpesvirus assembly and egress. J. Virol. 76:1537-1547.
Meyers, C., T. J. Mayer, and M. A. Ozbun. 1997. Synthesis of infectious human papillomavirus type 18 in differentiating epithelium transfected with viral DNA. J. Virol. 71:7381-7386.
Moffat, J., H. Ito, M. Sommer, S. Taylor, and A. M. Arvin. 2002. Glycoprotein I of varicella-zoster virus is required for viral replication in skin and T cells. J. Virol. 76:8468-8471.
Moffat, J. F., and A. M. Arvin. 1999. Varicella-zoster virus infection of T cells and skin in the SCID-hu mouse model, p. 973-980. In O. Zak and M. A. Sande (ed.), Handbook of animal models of infection. Academic Press, San Diego, CA.
Moffat, J. F., M. D. Stein, H. Kaneshima, and A. M. Arvin. 1995. Tropism of varicella-zoster virus for human CD4+ and CD8+ T lymphocytes and epidermal cells in SCID-hu mice. J. Virol. 69:5236-5242.
Moffat, J. F., L. Zerboni, P. R. Kinchington, C. Grose, H. Kaneshima, and A. M. Arvin. 1998. Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J. Virol. 72:965-974.
Moffat, J. F., L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin. 1998. The ORF47 and ORF66 putative protein kinases of varicella-zoster virus determine tropism for human T cells and skin in the SCID-hu mouse. Proc. Natl. Acad. Sci. USA 95:11969-11974.
Nagy, G., M. Barcza, N. Gonchoroff, P. E. Phillips, and A. Perl. 2004. Nitric oxide-dependent mitochondrial biogenesis generates Ca2+ signaling profile of lupus T cells. J. Immunol. 173:3676-3683.
Nicola, A. V., and S. E. Straus. 2004. Cellular and viral requirements for rapid endocytic entry of herpes simplex virus. J. Virol. 78:7508-7517.
Niizuma, T., L. Zerboni, M. H. Sommer, H. Ito, S. Hinchliffe, and A. M. Arvin. 2003. Construction of varicella-Zoster virus recombinants from parent Oka cosmids and demonstration that ORF65 protein is dispensable for infection of human skin and T cells in the SCID-hu mouse model. J. Virol. 77:6062-6065.
Phanucharas, J. P., and G. L. Gorby. 1997. Differential intracellular efficacy of ciprofloxacin and cefimixe against Neisseria gonorrhoeae in human fallopian tube organ culture. Antimicrob. Agents Chemother. 41:1547-1551.
Rayner, C. F. J., A. D. Jackson, A. Rutman, A. Dewar, T. J. Mitchell, P. W. Andrew, P. J. Cole, and R. Wilson. 1995. Interaction of pneumolysin-sufficient and -deficient isogenic variants of Streptococcus pneumoniae with human respiratory mucosa. Infect. Immun. 63:442-447.
Santos, R. A., C. C. Hatfield, N. L. Cole, J. A. Padilla, J. F. Moffat, A. M. Arvin, W. T. Ruyechan, J. Hay, and C. Grose. 2000. Varicella-zoster virus gE escape mutant VZV-MSP exhibits an accelerated cell-to-cell spread phenotype in both infected cell cultures and SCID-hu mice. Virology 275:306-317.
Sato, B., H. Ito, S. Hinchliffe, M. H. Sommer, L. Zerboni, and A. M. Arvin. 2003. Mutational analysis of open reading frames 62 and 71, encoding the varicella-Zoster virus immediate-early transactivating protein, IE62, and effects on replication In vitro and in skin xenografts in the SCID-hu mouse In vivo. J. Virol. 77:5607-5620.
Syrjanen, S., H. Mikola, M. Nykanen, and V. Hukkanen. 1996. In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture. J. Virol. 70:6524-6528.
Tavakkol, A., J. Varani, J. T. Elder, and C. C. Zouboulis. 1999. Maintenance of human skin in organ culture: role for insulin-like growth factor-1 receptor and epidermal growth factor receptor. Arch. Dermatol. Res. 291:643-651.
Taylor, S. L., P. R. Kinchington, A. Brooks, and J. F. Moffat. 2004. Roscovitine, a cyclin dependent kinase inhibitor, prevents replication of varicella-zoster virus. J. Virol. 78:2853-2862.
Unna, P. G. 1896. The histopathology of the diseases of the skin. Macmillan & Co., New York, NY.
Zerboni, L., S. Hinchliffe, M. H. Sommer, H. Ito, J. Besser, S. Stamatis, J. Cheng, D. Distefano, N. Kraiouchkine, A. Shaw, and A. M. Arvin. 2005. Analysis of varicella zoster virus attenuation by evaluation of chimeric parent Oka/vaccine Oka recombinant viruses in skin xenografts in the SCIDhu mouse model. Virology 332:337-346.(Shannon L. Taylor and Jen)
ABSTRACT
Varicella-zoster virus (VZV) infection is restricted to humans, which hinders studies of its pathogenesis in rodent models of disease. To facilitate the study of VZV skin tropism, we developed an ex vivo system using human fetal skin organ culture (SOC). VZV replication was analyzed by plaque assay, transmission electron microscopy, and histology. The yield of infectious VZV from SOC increased 100-fold over 6 days, virions were abundant, and lesions developed that contained VZV antigens and resembled varicella and zoster lesions. The SOC system for VZV replication has applications for testing virus mutants and antiviral drugs.
TEXT
The hallmark signs of chicken pox (varicella) and shingles (zoster) are itchy, painful skin lesions, caused by varicella-zoster virus (VZV). Epithelial cells, keratinocytes, and dermal fibroblasts are major targets for VZV replication. It is important to understand the determinants of VZV skin tropism in order to develop new treatments and vaccines. A lack of small-animal models that mimic primary infection, establishment of latency, and reactivation has hindered the study of VZV pathogenesis. However, the development of the SCID-hu mouse model led to advancement over cell culture techniques to study VZV replication (15). SCID-hu mice implanted with human fetal thymus (T cells) or skin xenografts enabled the systematic investigation of VZV replication in fully differentiated human tissues in vivo (16, 17). From its inception, the SCID-hu model has revealed interesting and sometimes unanticipated phenotypes of VZV mutants and variants (2, 3, 7, 9, 13, 20, 23, 24). This body of work underscores the necessity of using intact human tissues, such as skin, for addressing questions about VZV pathogenesis and molecular virulence determinants.
Alternatives to studying VZV in skin implanted in SCID-hu mice are organotypic raft cultures and skin organ culture (SOC). There are few reports of organ cultures being used to study infectious diseases (8, 21, 22). Instead, organotypic raft cultures, composed of fibroblasts embedded in a collagen matrix that feeds overlying keratinocytes, are frequently employed to study human papillomaviruses and less often for herpesviruses (6, 12, 25). Although both SOC and organotypic raft cultures are composed of dermis and epidermis, hair follicles and sebaceous glands are only found in SOC. There is mounting evidence that hair follicles are the site of VZV transfer to the skin from infected T cells, which express skin-homing markers that direct them to endothelial cells in the dermis (10). Thus, SOC appears highly suited for studying VZV in full-thickness skin where all the major cell types are present and are correctly positioned and differentiated.
Given the importance of studying VZV in human skin, we set out to facilitate this in several ways: increase the sampling frequency and number of skin tissues tested, shorten experiments from months to days, and reduce the number of SCID mice used. To achieve these goals, we developed an ex vivo system to study VZV in human fetal skin organ culture, which maintains skin tissues on NetWell supports as opposed to xenografts in SCID mice. Our hypothesis that SOC would be an effective system for investigating VZV skin tropism was supported by the results. We found that VZV replicated in all layers of the skin, causing typical lesions and producing abundant virions.
The first step in development of the SOC model for VZV replication was to determine the proper conditions for tissue sterility, integrity, and differentiation. Human fetal skin tissue (18 to 23 weeks gestational age) was purchased from Advance Biosciences Resources (Alameda, CA), in accordance with all local, state, and federal guidelines. The skin was disinfected by swabbing with Betadyne (10% povidone iodine), dipped in 70% ethanol, and rinsed in tissue culture medium [TCM: minimal essential medium with Earle's salts and L-glutamine, supplemented with 10% heat-inactivated fetal bovine serum, penicillin/streptomycin (5,000 IU/ml), amphotericin B (250 μg/ml), and nonessential amino acids (Mediatech, Washington, D.C.)]. TCM contains 1.4 mM Ca2+, which is needed for preservation of skin integrity since it quickly dedifferentiates in 0.15 mM calcium (26).
Skin tissue was cut into 1.0-cm2 pieces, placed individually in wells of a 24-well plate, and submerged in TCM supplemented with nystatin (10,000 U/ml) and ciprofloxacin (10 μg/ml) (Sigma, St. Louis MO) for 24 h in a tissue culture incubator (humidified 5% CO2 at 37°C). For long-term cultivation at 37°C, the tissues were placed individually in 500-μm mesh NetWell inserts (Corning, Corning, NY) that rested above 1.0 ml of TCM containing nystatin and ciprofloxacin in each well of a 12-well plate, thereby situating the tissues at the air-liquid interface. The skin explant was nourished through the dermis by contact with the TCM, which was refreshed every 2 days.
To observe the architecture of the skin over time, tissues were harvested after 0, 2, 4, and 6 days, then prepared for histological analysis. After 6 days, the skin appeared grossly normal, with only slight contraction from its original size, and hematoxylin and eosin-stained sections indicated that the cellular anatomy was preserved (Fig. 1A to D). Specifically, the dermis was composed of fibroblasts in a collagen matrix, with prominent hair follicles. The basement membrane separated the dermis from the epidermis, where stratified layers of basal cells and flattened keratinocytes were observed. The presence of keratin sheets on the top surface indicated that differentiation was occurring in the epidermis. The histological appearance of tissues from SOC was nearly identical to skin implants from SCID-hu mice (for review see 14) with the exception that SOC did not produce a thick layer of subcutaneous fat.
It was not known whether skin cultured ex vivo would provide growth conditions suitable for VZV replication. A cloned clinical isolate of VZV, rPOka (20), was selected for these studies because of its known virulence for skin in the SCID-hu model (29). To prepare the virus for inoculation, VZV-infected or mock-infected MeWo cells were trypsinized, then washed and resuspended in TCM, and used immediately for direct injection into MeWo cells (human melanoma cell line, kindly provided by Charles Grose, University of Iowa) and VZV rPOka were cultivated as described (27). Each tissue was injected five times with 10 μl of the cell suspension using a 1-ml syringe fitted with a 27-gauge needle attached to a volumetric stepper (Tridak, Brookfield, CT).
Tissues were incubated at 37°C for 2 h to allow the virus to adhere, and then placed individually on NetWells for cultivation as above. On days 0, 2, 4, and 6, VZV-infected skin tissues were harvested and processed for histological analysis to observe the location of VZV replication and any pathological changes. Light microscopy of fixed sections stained with hematoxylin and eosin revealed that mock-infected and VZV-infected tissues were indistinguishable on days 0, 2, and 4 (Fig. 1, A to C and E to G). By 6 days after inoculation, no lesions were visible by gross examination, although VZV-infected explants showed histopathology typical of varicella and zoster lesions, such as thickening of the epidermis, sloughing of keratinocytes (white arrow), and the presence of balloon cells (Fig. 1H). Destruction of hair follicles, as evidenced by loss of hematoxylin staining, was widespread (black arrow, Fig. 1H).
To confirm that lesions and tissue damage were caused by VZV infection, immunohistochemistry was performed. VZV proteins were detected using a polyclonal human immune serum (GK serum, kindly provided by Ann Arvin, Stanford University); a secondary antibody conjugated to alkaline phosphatase (goat anti-human alkaline phosphatase, Jackson ImmunoResearch Laboratories, Inc., Westgrove PA), and the signal was developed with Vector Red (Vector Laboratories, Burlingame CA). Tissues were counterstained with hematoxylin for contrast. After 6 days postinoculation, mock-infected skin reacted slightly with the antibodies in the outer keratinocytes, giving a faint red color (Fig. 2A). A strong red signal in the epidermis of VZV-infected skin indicated where viral antigens were present in a lesion (Fig. 2B). This lesion had been observed in adjacent sections that were stained with hematoxylin and eosin (Fig. 1H).
Higher magnification of the lesion outlined by the white box showed infected cells (arrows) in the epidermis (Fig. 2C). Examination of the entire tissue showed that VZV antigens were present in all skin cell types, particularly keratinocytes of the epidermis and hair follicles, and in scattered dermal fibroblasts (Fig. 2D). Higher magnification of the infected skin (boxes in panel D) revealed spaces forming around cell nuclei, termed balloon cells (arrow, Fig. 2E) and virus spread through the hair follicle (arrow, Fig. 2F). These signs indicate that VZV was replicating in the skin and causing histopathological changes typically seen in biopsies of varicella and zoster lesions (28). To rule out the possibility that VZV infection induced apoptosis, and thus caused indirect damage, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays (Hoffmann-La Roche, Switzerland) were performed on fixed tissue sections. Very few apoptotic cells were observed in VZV-infected tissues compared to positive controls that were treated with camptothecin (2 μg/ml, Sigma), a known inducer of apoptosis (data not shown).
To measure the extent and kinetics of VZV replication in the SOC system, we compared two modes of virus inoculation: intradermal injection and scarification. The rationale was that VZV infects skin from within, via dissemination of infected T cells (1), and externally, via breaks in the epidermis. Tissues were inoculated by injection as described above, or by scarification using a 27-gauge needle dragged across the skin 10 times while flooded in VZV-infected MeWo cells. Since the inoculum was freshly prepared to obtain the highest possible VZV titer, the virus concentration was not calculated before use. Instead, tissues were harvested 2 h after inoculation (time zero) and processed for titration by mincing tissue and placing them in 1 ml of TCM that contained 2.5-mm glass beads. After vortexing for 3 min., an infectious focus assay was performed to assess the actual amount of input virus received by the tissues following the method described (27). The tissues were incubated at 37°C on NetWells, and then four tissues were removed for virus quantitation by infectious focus assay after 2, 4, and 6 days.
Both inoculation methods resulted in a 100-fold increase in virus between days 2 and 6, and the doubling time was 17 h in tissues injected intradermally and 23 h in scarified tissues (brackets, Fig. 3). This is similar to the 16 to 20 h measured in cultured human foreskin fibroblasts (J. Moffat, unpublished observations). Scarification introduced approximately 10 PFU per tissue, whereas intradermal injection introduced over 100 PFU. Another difference was that VZV titer increased exponentially following scarification, but there was a decrease in titer in the first 2 days after intradermal injection. There is a possibility that some of the increase in titer was from VZV replication in the MeWo cells used for the inoculum, so the values stated may slightly overestimate the extent of virus growth in the skin tissue.
Using a cell-associated inoculum raised the question of whether the virus continued to grow in the MeWo cells, or if it spread to the skin cells as expected. Direct examination of virus particles in keratinocytes by transmission electron microscopy was performed to address this issue. Tissues infected with VZV for 6 days were prepared for transmission electron microscopy as described (18). In agreement with the histological analysis, virions and virus components were found widely distributed in all layers of the skin. Many infected cells were observed in the epidermis, where keratinocytes, identifiable by sheets of keratin in the cytoplasm, remained intact and tightly joined to surrounding cells during VZV replication (Fig. 4A).
Several phases of VZV assembly were observed in a single keratinocyte, outlined in boxes numbered 1 to 3, that are shown at higher magnification. Both empty and DNA-containing capsids were seen in the nucleus (Fig. 4A-1). Although capsids budding into the perinuclear space and de-envelopment at the outer nuclear membrane were not observed in this cell, capsids were found near the membrane inside (white arrowhead). Many enveloped virions were in cytoplasmic vesicles (black arrowheads, Fig. 4A-2 and -3), but secondary envelopment was not captured in partial stages. It was possible that these were input virions, since the related virus, herpes simplex virus type 1, can enter cells by endocytosis (5, 19). However, the enveloped virions within vesicles were most likely assembled in the infected cells and were destined for egress by fusion with the plasma membrane. This canonical herpesvirus assembly pathway (11) was supported by the presence of countless VZV particles in the intercellular spaces (white arrowheads, Fig. 4A-2 and -3). Some of these were complete virions while others lacked capsids, which are often scraped away when the tissues are sectioned for transmission electron microscopy.
An additional question was whether VZV replication and assembly would differ in the various skin cell types. Thus, we examined infected dermal fibroblasts, notable for their lack of keratin, by transmission electron microscopy. VZV assembly in fibroblasts proceeded similarly to that observed in keratinocytes with the exception that very large accumulations of virions were seen outside cells (Fig. 4B), which may indicate differences in VZV egress in these cell types. This phenomenon, of cell membranes crowded with particles near masses of virions, was observed throughout the dermis. Of the extracellular virions, defective particles and viral debris were abundant, which is typical of VZV morphology, although many complete virions were also seen (Fig. 4B, white arrowheads). After several different tissues were examined, it was apparent that the predominant skin cell types, keratinocytes and fibroblasts, were susceptible to VZV infection and the assembly patterns were generally similar.
The SOC model presented here demonstrates the convenience and relevance of studying VZV replication in human skin ex vivo. The virus grew rapidly in the tissues, forming typical lesions and clusters of extracellular virions and viral debris. We were hopeful that these virions could be collected from the tissue, however, large amounts of infectious cell-free virus were not obtained using mechanical or enzymatic methods. VZV was difficult to extract due to the architecture of the skin, where cells are tightly joined and embedded in a collagen matrix. The inability to isolate concentrated, infectious VZV virions has been a barrier to using common and powerful techniques such as plaque purification and high-multiplicity infections, and so we continue to explore methods for isolating them in usable quantities.
The success of the SOC system could lead to several new research directions. These include VZV pathogenesis studies that are designed to distinguish the viral genes required in vitro from in vivo, and studying VZV assembly in differentiated skin, specifically the roles of tegument proteins and glycoproteins in this process. The SOC model could also provide a screening tool for antiviral agents because results can be obtained so rapidly, making it a useful first step before confirming results in mice with the SCID-hu model. Lastly, SOC could be applied to other skin-tropic viruses such as papillomaviruses and the human poxvirus molluscum contagiosum virus, which lack good tissue culture and animal models (4).
ACKNOWLEDGMENTS
This work was supported by the Hendricks Research Fund from SUNY Upstate Medical University and by Public Health Service grant AI052168 (J.F.M.).
Transmission electron microscopy was performed with expert technical assistance from Maureen Barcza, Department of Pathology.
REFERENCES
Arvin, A. M. 2001. Varicella-zoster virus, p. 2731-2768. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed., vol. 2. Lippincott-Raven, Philadelphia, Pa.
Baiker, A., C. Bagowski, H. Ito, M. Sommer, L. Zerboni, K. Fabel, J. Hay, W. Ruyechan, and A. M. Arvin. 2004. The immediate-early 63 protein of Varicella-Zoster virus: analysis of functional domains required for replication in vitro and for T-cell and skin tropism in the SCIDhu model in vivo. J. Virol. 78:1181-1194.
Besser, J., M. H. Sommer, L. Zerboni, C. P. Bagowski, H. Ito, J. Moffat, C. C. Ku, and A. M. Arvin. 2003. Differentiation of varicella-zoster virus ORF47 protein kinase and IE62 protein binding domains and their contributions to replication in human skin xenografts in the SCID-hu mouse. J. Virol. 77:5964-5974.
Bugert, J. J., and G. Darai. 1997. Recent advances in molluscum contagiosum virus research. Arch. Virol. Suppl. 13:35-47.
Gianni, T., G. Campadelli-Fiume, and L. Menotti. 2004. Entry of herpes simplex virus mediated by chimeric forms of nectin1 retargeted to endosomes or to lipid rafts occurs through acidic endosomes. J. Virol. 78:12268-12276.
Hukkanen, V., H. Mikola, M. Nykanen, and S. Syrjanen. 1999. Herpes simplex virus type 1 infection has two separate modes of spread in three-dimensional keratinocyte culture. J. Gen. Virol. 80:2149-2155.
Ito, H., M. H. Sommer, L. Zerboni, H. He, D. Boucaud, J. Hay, W. Ruyechan, and A. M. Arvin. 2003. Promoter sequences of varicella-zoster virus glycoprotein I targeted by cellular transactivating factors Sp1 and USF determine virulence in skin and T cells in SCIDhu mice in vivo. J. Virol. 77:489-498.
Jackson, A. D., P. J. Cole, and R. Wilson. 1996. Comparison of haemophilus influenzae type b interaction with respiratory mucosa organ cultures maintained with an air interface or immersed in medium. Infect. Immun. 64:2353-2355.
Jones, J. O., and A. M. Arvin. 2003. Microarray analysis of host cell gene transcription in response to varicella-zoster virus infection of human T cells and fibroblasts in vitro and SCIDhu skin xenografts in vivo. J. Virol. 77:1268-1280.
Ku, C. C., J. A. Padilla, C. Grose, E. C. Butcher, and A. M. Arvin. 2002. Tropism of varicella-zoster virus for human tonsillar CD4+ T lymphocytes that express activation, memory, and skin homing markers. J. Virol. 76:11425-11433.
Mettenleiter, T. C. 2002. Herpesvirus assembly and egress. J. Virol. 76:1537-1547.
Meyers, C., T. J. Mayer, and M. A. Ozbun. 1997. Synthesis of infectious human papillomavirus type 18 in differentiating epithelium transfected with viral DNA. J. Virol. 71:7381-7386.
Moffat, J., H. Ito, M. Sommer, S. Taylor, and A. M. Arvin. 2002. Glycoprotein I of varicella-zoster virus is required for viral replication in skin and T cells. J. Virol. 76:8468-8471.
Moffat, J. F., and A. M. Arvin. 1999. Varicella-zoster virus infection of T cells and skin in the SCID-hu mouse model, p. 973-980. In O. Zak and M. A. Sande (ed.), Handbook of animal models of infection. Academic Press, San Diego, CA.
Moffat, J. F., M. D. Stein, H. Kaneshima, and A. M. Arvin. 1995. Tropism of varicella-zoster virus for human CD4+ and CD8+ T lymphocytes and epidermal cells in SCID-hu mice. J. Virol. 69:5236-5242.
Moffat, J. F., L. Zerboni, P. R. Kinchington, C. Grose, H. Kaneshima, and A. M. Arvin. 1998. Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J. Virol. 72:965-974.
Moffat, J. F., L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin. 1998. The ORF47 and ORF66 putative protein kinases of varicella-zoster virus determine tropism for human T cells and skin in the SCID-hu mouse. Proc. Natl. Acad. Sci. USA 95:11969-11974.
Nagy, G., M. Barcza, N. Gonchoroff, P. E. Phillips, and A. Perl. 2004. Nitric oxide-dependent mitochondrial biogenesis generates Ca2+ signaling profile of lupus T cells. J. Immunol. 173:3676-3683.
Nicola, A. V., and S. E. Straus. 2004. Cellular and viral requirements for rapid endocytic entry of herpes simplex virus. J. Virol. 78:7508-7517.
Niizuma, T., L. Zerboni, M. H. Sommer, H. Ito, S. Hinchliffe, and A. M. Arvin. 2003. Construction of varicella-Zoster virus recombinants from parent Oka cosmids and demonstration that ORF65 protein is dispensable for infection of human skin and T cells in the SCID-hu mouse model. J. Virol. 77:6062-6065.
Phanucharas, J. P., and G. L. Gorby. 1997. Differential intracellular efficacy of ciprofloxacin and cefimixe against Neisseria gonorrhoeae in human fallopian tube organ culture. Antimicrob. Agents Chemother. 41:1547-1551.
Rayner, C. F. J., A. D. Jackson, A. Rutman, A. Dewar, T. J. Mitchell, P. W. Andrew, P. J. Cole, and R. Wilson. 1995. Interaction of pneumolysin-sufficient and -deficient isogenic variants of Streptococcus pneumoniae with human respiratory mucosa. Infect. Immun. 63:442-447.
Santos, R. A., C. C. Hatfield, N. L. Cole, J. A. Padilla, J. F. Moffat, A. M. Arvin, W. T. Ruyechan, J. Hay, and C. Grose. 2000. Varicella-zoster virus gE escape mutant VZV-MSP exhibits an accelerated cell-to-cell spread phenotype in both infected cell cultures and SCID-hu mice. Virology 275:306-317.
Sato, B., H. Ito, S. Hinchliffe, M. H. Sommer, L. Zerboni, and A. M. Arvin. 2003. Mutational analysis of open reading frames 62 and 71, encoding the varicella-Zoster virus immediate-early transactivating protein, IE62, and effects on replication In vitro and in skin xenografts in the SCID-hu mouse In vivo. J. Virol. 77:5607-5620.
Syrjanen, S., H. Mikola, M. Nykanen, and V. Hukkanen. 1996. In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture. J. Virol. 70:6524-6528.
Tavakkol, A., J. Varani, J. T. Elder, and C. C. Zouboulis. 1999. Maintenance of human skin in organ culture: role for insulin-like growth factor-1 receptor and epidermal growth factor receptor. Arch. Dermatol. Res. 291:643-651.
Taylor, S. L., P. R. Kinchington, A. Brooks, and J. F. Moffat. 2004. Roscovitine, a cyclin dependent kinase inhibitor, prevents replication of varicella-zoster virus. J. Virol. 78:2853-2862.
Unna, P. G. 1896. The histopathology of the diseases of the skin. Macmillan & Co., New York, NY.
Zerboni, L., S. Hinchliffe, M. H. Sommer, H. Ito, J. Besser, S. Stamatis, J. Cheng, D. Distefano, N. Kraiouchkine, A. Shaw, and A. M. Arvin. 2005. Analysis of varicella zoster virus attenuation by evaluation of chimeric parent Oka/vaccine Oka recombinant viruses in skin xenografts in the SCIDhu mouse model. Virology 332:337-346.(Shannon L. Taylor and Jen)