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Dengue virus (DENV) is the most disease-causative flavivirus worldwide. DENV as a mosquito-borne virus infects human hosts through the skin; however, the initial target cells in the skin remain unclear. In this study, we have investigated whether epidermal Langerhans cells (LCs) play a role in DENV acquisition and dissemination. We have used a human epidermal ex vivo infection model as well as isolated LCs to investigate infection by DENV. Notably, both immature and mature LCs were permissive to DENV infection in vitro and ex vivo, and infection was dependent on C-type lectin receptor langerin because blocking antibodies against langerin significantly reduced DENV infection in vitro and ex vivo. DENV-infected LCs efficiently transmitted DENV to target cells such as dendritic cells. Moreover, DENV exposure increased the migration of LCs from epidermal explants. These results strongly suggest that DENV targets epidermal LCs for infection and dissemination in the human host. These findings could provide potential drug targets to combat the early stage of DENV infection., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Herpes simplex virus 1 (HSV-1) must overcome epidermal barriers to reach its receptors on keratinocytes and initiate infection in human skin. The cell-adhesion molecule nectin-1, which is expressed in human epidermis, acts as an efficient receptor for HSV-1 but is not within reach of the virus upon exposure of human skin under nonpathological conditions. Atopic dermatitis skin, however, can provide an entry portal for HSV-1 emphasizing the role of impaired barrier functions. Here, we explored how epidermal barriers impact HSV-1 invasion in human epidermis and influence the accessibility of nectin-1 for the virus. Using human epidermal equivalents, we observed a correlation of the number of infected cells with tight-junction formation, suggesting that mature tight junctions prior to formation of the stratum corneum prevent viral access to nectin-1. Consequently, impaired epidermal barriers driven by Th2-inflammatory cytokines interleukin 4 (IL-4) and IL-13 as well as the genetic predisposition of nonlesional atopic dermatitis keratinocytes correlated with enhanced infection supporting the impact of functional tight junctions for preventing infection in human epidermis. Comparable to E-cadherin, nectin-1 was distributed throughout the epidermal layers and localized just underneath the tight-junctions. While nectin-1 was evenly distributed on primary human keratinocytes in culture, the receptor was enriched at lateral surfaces of basal and suprabasal cells during differentiation. Nectin-1 showed no major redistribution in the thickened atopic dermatitis and IL-4/IL-13-treated human epidermis in which HSV-1 can invade. However, nectin-1 localization toward tight junction components changed, suggesting that defective tight-junction barriers make nectin-1 accessible for HSV-1 which enables facilitated viral penetration. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a widely distributed human pathogen which productively infects epithelia. The open question is which barriers of the highly protected epithelia must the virus overcome to reach its receptor nectin-1. Here, we used human epidermal equivalents to understand how physical barrier formation and nectin-1 distribution contribute to successful viral invasion. Inflammation-induced barrier defects led to facilitated viral penetration strengthening the role of functional tight-junctions in hindering viral access to nectin-1 that is localized just underneath tight junctions and distributed throughout all layers. We also found nectin-1 ubiquitously localized in the epidermis of atopic dermatitis and IL-4/IL-13-treated human skin implying that impaired tight-junctions in combination with a defective cornified layer allow the accessibility of nectin-1 to HSV-1. Our results support that successful invasion of HSV-1 in human skin relies on defective epidermal barriers, which not only include a dysfunctional cornified layer but also depend on impaired tight junctions., Competing Interests: The authors declare no conflict of interest.

To infect its human host, herpes simplex virus 1 (HSV-1) must overcome the protective barriers of skin and mucosa. Here, we addressed whether pathological skin conditions can facilitate viral entry via the skin surface and used ex vivo infection studies to explore viral invasion in atopic dermatitis (AD) skin characterized by disturbed barrier functions. Our focus was on the visualization of the onset of infection in single cells to determine the primary entry portals in the epidermis. After ex vivo infection of lesional AD skin, we observed infected cells in suprabasal layers indicating successful invasion in the epidermis via the skin surface which was never detected in control skin where only sample edges allowed viral access. The redistribution of filaggrin, loricrin, and tight-junction components in the lesional skin samples suggested multiple defective mechanical barriers. To dissect the parameters that contribute to HSV-1 invasion, we induced an AD-like phenotype by adding the Th2 cytokines interleukin 4 (IL-4) and IL-13 to healthy human skin samples. Strikingly, we detected infected cells in the epidermis, implying that the IL-4/IL-13-driven inflammation is sufficient to induce modifications allowing HSV-1 to penetrate the skin surface. In summary, not only did lesional AD skin facilitate HSV-1 penetration but IL-4/IL-13 responses alone allowed virus invasion. Our results suggest that the defective epidermal barriers of AD skin and the inflammation-induced altered barriers in healthy skin can make receptors accessible for HSV-1. IMPORTANCE Herpes simplex virus 1 (HSV-1) can target skin to establish primary infection in the epithelium. While the human skin provides effective barriers against viral invasion under healthy conditions, a prominent example of successful invasion is the disseminated HSV-1 infection in the skin of atopic dermatitis (AD) patients. AD is characterized by impaired epidermal barrier functions, chronic inflammation, and dysbiosis of skin microbiota. We addressed the initial invasion process of HSV-1 in atopic dermatitis skin to understand whether the physical barrier functions are sufficiently disturbed to allow the virus to invade skin and reach its receptors on skin cells. Our results demonstrate that HSV-1 can indeed penetrate and initiate infection in atopic dermatitis skin. Since treatment of skin with IL-4 and IL-13 already resulted in successful invasion, we assume that inflammation-induced barrier defects play an important role for the facilitated access of HSV-1 to its target cells.

Herpes simplex virus 1 (HSV-1) enters its human host via the skin and mucosa. The open question is how the virus invades this highly protective tissue in vivo to approach its receptors in the epidermis and initiate infection. Here, we performed ex vivo infection studies in human skin to investigate how susceptible the epidermis and dermis are to HSV-1 and whether wounding facilitates viral invasion. Upon ex vivo infection of complete skin, only sample edges with integrity loss demonstrated infected cells. After removal of the dermis, HSV-1 efficiently invaded the basal layer of the epidermis and, from there, gained access to suprabasal layers. This finding supports a high susceptibility of all epidermal layers which correlated with the surface expression of the receptors nectin-1 and herpesvirus entry mediator (HVEM). In contrast, only single infected cells were detected in the separated dermis, where minor expression of the receptors was found. Interestingly, after wounding, nearly no infection of the epidermis was observed via the skin surface. However, if the wounding of the skin samples led to breaks through the dermis, HSV-1 infected mainly keratinocytes via the damaged dermal layer. The application of latex beads revealed only occasional entry via the wounded dermis; however, it facilitated penetration via the wounded skin surface. Thus, we suggest that although the wounded human skin surface allows particle penetration, the skin still provides barriers that prevent HSV-1 from reaching its receptors. IMPORTANCE The human pathogen herpes simplex virus 1 (HSV-1) invades its host via the skin and mucosa, which leads to primary infection of the epithelium. As the various epithelial barriers effectively protect the tissue against viral invasion, successful infection most likely depends on tissue damage. We addressed the initial invasion process in human skin by ex vivo infection to understand how HSV-1 overcomes physical skin barriers and reaches its receptors to enter skin cells. Our results demonstrate that intact skin samples allow viral access only from the edges, while the epidermis is highly susceptible once the basal epidermal layer serves as an initial entry portal. Surprisingly, mechanical wounding did not facilitate HSV-1 entry via the skin surface, although latex beads still penetrated via the lesions. Our results imply that successful invasion of HSV-1 depends on how well the virus can reach its receptors, which was not accomplished by skin lesions under ex vivo conditions.

Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting., Competing Interests: The authors have declared that no competing interests exist.

Measles is characterized by fever and a maculopapular skin rash, which is accompanied by immune clearance of measles virus (MV)-infected cells. Histopathological analyses of skin biopsies from humans and non-human primates (NHPs) with measles rash have identified MV-infected keratinocytes and mononuclear cells in the epidermis, around hair follicles and near sebaceous glands. Here, we address the pathogenesis of measles skin rash by combining data from experimentally infected NHPs, ex vivo infection of human skin sheets and in vitro infection of primary human keratinocytes. Analysis of NHP skin samples collected at different time points following MV inoculation demonstrated that infection in the skin precedes onset of rash by several days. MV infection was detected in lymphoid and myeloid cells in the dermis before dissemination to the epidermal leukocytes and keratinocytes. These data were in good concordance with ex vivo MV infections of human skin sheets, in which dermal cells were more targeted than the epidermal cells. To address viral dissemination to the epidermis and to determine whether the dissemination is receptor-dependent, we performed experimental infections of primary keratinocytes collected from healthy donors. These experiments demonstrated that MV infection of keratinocytes is mainly nectin-4-dependent, and differentiated keratinocytes, which express higher levels of nectin-4, are more susceptible to MV infection than proliferating keratinocytes. Based on these data, we propose a model to explain measles skin rash: migrating MV-infected lymphocytes initiate the infection of dermal skin-resident CD150+ immune cells. The infection is subsequently disseminated from the dermal papillae to nectin-4+ keratinocytes in the basal epidermis. Lateral spread of MV infection is observed in the superficial epidermis, most likely due to the higher level of nectin-4 expression on differentiated keratinocytes. Finally, MV-infected cells are cleared by infiltrating immune cells, causing hyperemia and edema, which give the appearance of morbilliform skin rash., Competing Interests: The authors have declared that no competing interests exist.

Skin is a major target tissue of herpes simplex virus 1 (HSV-1), and we are only beginning to understand how individual receptors contribute to the initiation of infection in tissue. We recently demonstrated the impact of the receptors nectin-1 and herpesvirus entry mediator (HVEM) for entry of HSV-1 into murine epidermis. Here, we focus on viral invasion into the dermis, a further critical target tissue in vivo In principle, murine dermal fibroblasts are highly susceptible to HSV-1, and we previously showed that nectin-1 and HVEM can act as alternative receptors. To characterize their contribution as receptors in dermal tissue, we established an ex vivo infection assay of murine dermis. Only after separation of the epidermis from the dermis, we observed single infected cells in the upper dermis from juvenile mice at 5 h postinfection with increasing numbers of infected cells at later times. While nectin-1-expressing cells were less frequently detected, we found HVEM expressed on most cells of juvenile dermis. The comparison of infection efficiency during aging revealed a strong delay in the onset of infection in the dermis from aged mice. This observation correlated with a decrease in nectin-1-expressing fibroblasts during aging while the number of HVEM-expressing cells remained stable. Accordingly, aged nectin-1-deficient dermis was less susceptible to HSV-1 than the dermis from control mice. Thus, we conclude that the reduced availability of nectin-1 in aged dermis is a key contributor to a decrease in infection efficiency during aging. IMPORTANCE HSV-1 is a prevalent human pathogen which invades skin and mucocutaneous linings. So far, the underlying mechanisms of how the virus invades tissue, reaches its receptors, and initiates infection are still unresolved. To unravel the mechanical prerequisites that limit or favor viral invasion into tissue, we need to understand the contribution of the receptors that are involved in viral internalization. Here, we investigated the invasion process into murine dermis with the focus on receptor availability and found that infection efficiency decreases in aging mice. Based on studies of the expression of the receptors nectin-1 and HVEM, we suggest that the decreasing number of nectin-1-expressing fibroblasts leads to a delayed onset of infection in the dermis from aged compared to juvenile mice. Our results imply that the level of infection efficiency in murine dermis is closely linked to the availability of the receptor nectin-1 and can change during aging., (Copyright © 2020 American Society for Microbiology.)

Human Papillomavirus infection is highly prevalent worldwide. While most types of HPV cause benign warts, some high-risk types are known to cause cervical cancer, as well as cancer of the oral cavity and head and neck. Persistent cutaneous HPV infection can be particularly problematic in patients with chronic immunosuppression, for example following organ transplantation. Due to unknown mechanisms, these patients may develop numerous warts, as well as present with a dramatically increased skin cancer prevalence. Despite an association between HPV persistence in the epidermis and excessive wart or squamous cancer development, the molecular mechanisms linking immunosuppression, HPV expression and excessive epidermal proliferation have not been determined, largely due to low-sensitivity methodology to capture rare viral transcription events. Here, we use single-cell RNA sequencing to profile HPV-positive skin lesions from an immunosuppressed patient that were found to express the alphapapillomavirus HPV78 in basal keratinocytes, suprabasal keratinocytes and hair follicle stem cells. This method can be applied to detect and investigate HPV transcripts in cutaneous lesions, allowing mechanistic links between immunosuppression-induced HPV life cycle and epidermal hyperproliferation to be uncovered., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Myxomatosis is an infectious disease caused by myxoma virus (MYXV; genus Leporipoxvirus), which affects the European wild rabbit (Oryctolagus cuniculus) and sporadically brown hares (Lepus europaeus). Here, we describe the first outbreak of myxomatosis in Iberian hares (Lepus granatensis). Between mid-July and the end of September 2018, around 530 dead animals were detected in Iberian hare populations in southern Spain. The apparent mean mortality rate was 56.7%, and the estimated mean case fatality rate was 69.2%. Histopathological and molecular results confirmed MYXV infections in all hares analysed. To the authors' knowledge, this is the first myxomatosis outbreak causing a high mortality in hares and the first detailed characterization of a myxomatosis outbreak in the Iberian hare. The absence of cases in sympatric wild rabbits suggests differences in the susceptibility between both lagomorph species to the virus strain implicated in the outbreak. After the first case, the number of affected areas increased sharply affecting most of the Iberian Peninsula where the Iberian hare is present. Further studies are required to elucidate the origin of the implicated MYXV strain as well as to assess the impact of this outbreak on the Iberian hare populations., (© 2019 Blackwell Verlag GmbH.)

Destruction of epidermal barrier function associated with atopic dermatitis or Darier's disease often causes severe secondary skin infections. Patients with skin barrier disorders often repeatedly acquire Kaposi varicelliform eruption, which is caused by herpes simplex virus, but the underlying mechanisms and effective preventive methods have yet to be found. Viral infection through an impaired epidermal barrier can be prevented by enhancing innate immunity and/or inhibiting viral entry. In this study, we established a three-dimensional skin barrier dysfunction model by silencing ATP2A2, which is mutated in some Darier's disease patients. We confirmed the loss of desmosomes and presence of histopathological clefts in the suprabasal layer. Herpes simplex virus 1 applied to the stratum corneum infected the deep epidermis. An innate immune reaction was assessed by evaluating the expression of IFNB1 and related genes. Pretreatment with polyinosinic-polycytidylic acid alone or plus the antimicrobial peptide, LL37 enhanced IFN-β production and suppressed viral replication. Furthermore, topical application of a white petrolatum ointment containing heparin, which binds viral glycoproteins related to virus entry, strongly inhibited viral replication, probably by inhibiting invasion. Our human barrier-dysfunctional model will have future application for identifying the mechanism of Kaposi varicelliform eruption onset, preventive methods, and therapies., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

A continuous cell line consisting mostly of epithelioid cells was established from the caudal fin of marbled eels (Anguilla marmorata) and designated as marbled eel caudal fin (MECF)-1. The cells multiplied well in Leibovitz's L-15 medium containing 2% to 15% foetal bovine serum at temperatures of 20°C to 35°C and were subcultured for >90 passages during a 5-year period from 2012 to 2017. Transcripts of ictacalcin, keratin 13, cd146, nestin, ncam1 and myod1 were demonstrated in the cells using reverse transcription polymerase chain reaction. The results indicated that MECF-1 was composed of epidermal and mesenchyme stem and progenitor cells including myoblasts. MECF-1 was susceptible to Japanese eel herpesvirus HVA980811, marbled eel polyoma-like virus (MEPyV), aquabirnavirus MEIPNV1310 and aquareovirus CSV. By contrast, MECF-1 was noted refractory to megalocytiviruses RSIV-Ku and GSIV-K1 infection. Moreover, the cells were resistant to betanodavirus infection. In conclusion, MECF-1 derived from marbled eel is suitable for studies on anguillid viruses and interaction with host cells., (© 2018 John Wiley & Sons Ltd.)

To enter host cells, herpes simplex virus 1 (HSV-1) initially attaches to cell surface glycosaminoglycans, followed by the requisite binding to one of several cellular receptors, leading to viral internalization. Although virus-receptor interactions have been studied in various cell lines, the contributions of individual receptors to uptake into target tissues such as mucosa, skin, and cornea are not well understood. We demonstrated that nectin-1 acts as a major receptor for HSV-1 entry into murine epidermis, while herpesvirus entry mediator (HVEM) can serve as an alternative receptor. Recently, the macrophage receptor with collagenous structure (MARCO) has been described to mediate adsorption of HSV-1 to epithelial cells. Here, we investigated the impact of MARCO on the entry process of HSV-1 into the two major cell types of skin, keratinocytes in the epidermis and fibroblasts in the underlying dermis. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered basal keratinocytes of MARCO -/- epidermis as efficiently as those of control epidermis. In addition, entry into dermal fibroblasts was not impaired in the absence of MARCO. When we treated epidermis, primary keratinocytes, or fibroblasts with poly(I), a ligand for class A scavenger receptors, HSV-1 entry was strongly reduced. As we also observed reducing effects of poly(I) in the absence of both MARCO and scavenger receptor A1, we concluded that the inhibitory effects of poly(I) on HSV-1 infection are not directly linked to class A scavenger receptors. Overall, our results support that HSV-1 entry into skin cells is independent of MARCO. IMPORTANCE During entry into its host cells, the human pathogen herpes simplex virus (HSV) interacts with various cellular receptors. Initially, receptor interaction can mediate cellular adsorption, followed by receptor binding that triggers viral internalization. The intriguing question is which receptors are responsible for the various steps during entry into the natural target tissues of HSV? Previously, we demonstrated the role of nectin-1 as a major receptor and that of HVEM as an alternative receptor for HSV-1 to invade murine epidermis. As MARCO has been described to promote infection in skin, we explored the predicted role of MARCO as a receptor that mediates adsorption to epithelial cells. Our infection studies of murine skin cells indicate that the absence of MARCO does not interfere with the efficiency of HSV-1 entry and that the inhibitory effect on viral adsorption by poly(I), a ligand of MARCO, is independent of MARCO., (Copyright © 2018 American Society for Microbiology.)

T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b -/- CD8 + T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b -/- CD8 + T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b -/- CD8 + T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8 + T cell adaptation from lymphoid to NLT surveillance and the establishment of protective tissue-resident T cell populations., (© 2018 Moalli et al.)

Interleukin (IL)-33 is released on cell injury and activates the immune reaction. IL-33 is involved in antiviral reaction in herpes virus infection, but the source that secretes IL-33 has not been identified. We speculate that keratinocytes injured in herpes virus infection secrete IL-33. In order to detect IL-33 in the lesional epidermis of patients with herpes virus infection, we immunostained several cutaneous herpes virus infection samples with an anti-IL-33 antibody, and compared them with cutaneous human papilloma virus (HPV) infection samples. We observed strong nuclear and mild cytoplasmic staining in epidermal keratinocytes of the lesional skin samples with herpes simplex virus and varicella zoster virus infections. However, staining was not observed in the epidermis of verruca vulgaris (VV) samples. We assumed that the strong immune reaction to herpes virus infection may depend on strong IL-33 expression in the epidermis, while very weak immune reaction in samples from patients with VV may be due to low or no expression of IL-33 in the lesional epidermis., (© 2018 Japanese Dermatological Association.)

Northern pygmy mice from 2 localities in East Central Texas, USA, had proliferative epidermal lesions on the tail and feet. Electron microscopy of lesion tissue revealed poxvirus. Phylogenetic analyses indicated the virus differed 35% from its closest relatives, the Chordopoxvirinae. Future research is needed to determine whether this virus could affect human health.

It has been shown that γδ T cells protect against the formation of squamous cell carcinoma (SCC) in several models. However, the role of γδ T cells in human papillomavirus (HPV)-associated uterine cervical SCC, the third-leading cause of death by cancer in women, is unknown. Here, we investigated the impact of γδ T cells in a transgenic mouse model of carcinogenesis induced by HPV16 oncoproteins. Surprisingly, γδ T cells promoted the development of HPV16 oncoprotein-induced lesions. HPV16 oncoproteins induced a decrease in epidermal Skint1 expression and the associated antitumor Vγ5 + γδ T cells, which were replaced by γδ T-cell subsets (mainly Vγ6 + γδ low CCR2 + CCR6 - ) actively producing IL-17A. Consistent with a proangiogenic role, γδ T cells promoted the formation of blood vessels in the dermis underlying the HPV-induced lesions. In human cervical biopsies, IL-17A + γδ T cells could only be observed at the cancer stage (SCC), where HPV oncoproteins are highly expressed, supporting the clinical relevance of our observations in mice. Overall, our results suggest that HPV16 oncoproteins induce a reorganization of the local epithelial-associated γδ T-cell subpopulations, thereby promoting angiogenesis and cancer development., Competing Interests: The authors declare no conflict of interest., (Published under the PNAS license.)

Hand-foot-and-mouth disease is a self-limiting paediatric infectious disease commonly caused by Enterovirus A71 (Genus: Enterovirus, Family: Picornaviridae). Typical lesions in and around the hands, feet, oral cavity and other places may rarely be complicated by acute flaccid paralysis and acute encephalomyelitis. Although virus is readily cultured from skin vesicles and oral secretions, the cellular target/s of Enterovirus A71 in human skin and oral mucosa are unknown. In Enterovirus A71-infected human skin and oral mucosa organotypic cultures derived from the prepuce and lip biopsies, focal viral antigens and viral RNA were localized to cytoplasm of epidermal and mucosal squamous cells as early as 2 days post-infection. Viral antigens/RNA were associated with cytoplasmic vacuolation and cellular necrosis. Infected primary prepuce epidermal keratinocyte cultures showed cytopathic effects with concomitant detection of viral antigens from 2 days post-infection. Supernatant and/or tissue homogenates from prepuce skin organotypic cultures and primary prepuce keratinocyte cultures showed viral titres consistent with active viral replication. Our data strongly support Enterovirus A71 squamous epitheliotropism in the human epidermis and oral mucosa, and suggest that these organs are important primary and/or secondary viral replication sites that contribute significantly to oral and cutaneous viral shedding resulting in person-to-person transmission, and viraemia, which could lead to neuroinvasion.

Herpes simplex virus 1 (HSV-1) infects humans through stratified epithelia that are composed primarily of keratinocytes. The route of HSV-1 entry into keratinocytes has been the subject of limited investigation, but it is proposed to involve pH-dependent endocytosis, requiring the gD-binding receptor nectin-1. Here, we have utilized the nTERT human keratinocyte cell line as a new model for dissecting the mechanism of HSV-1 entry into the host. Although immortalized, these cells nonetheless retain normal growth and differentiation properties of primary cells. Using short interfering RNA (siRNA) depletion studies, we confirm that, despite nTERT cells expressing high levels of the alternative gD receptor HVEM, HSV-1 requires nectin-1, not HVEM, to enter these cells. Strikingly, virus entry into nTERT cells occurred with unusual rapidity, such that maximum penetration was achieved within 5 min. Moreover, HSV-1 was able to enter keratinocytes but not other cell types at temperatures as low as 7°C, conditions where endocytosis was shown to be completely inhibited. Transmission electron microscopy of early entry events at both 37°C and 7°C identified numerous examples of naked virus capsids located immediately beneath the plasma membrane, with no evidence of virions in cytoplasmic vesicles. Taken together, these results imply that HSV-1 uses the nectin-1 receptor to enter human keratinocyte cells via a previously uncharacterized rapid plasma membrane fusion pathway that functions at low temperature. These studies have important implications for current understanding of the relationship between HSV-1 and its relevant in vivo target cell., Importance: The gold standard of antiviral treatment for any human virus infection is the prevention of virus entry into the host cell. In the case of HSV-1, primary infection in the human begins in the epidermis of the skin or the oral mucosa, where the virus infects keratinocytes, and it is therefore important to understand the molecular events involved in HSV-1 entry into this cell type. Nonetheless, few studies have looked specifically at entry into these relevant human cells. Our results reveal a new route for virus entry that is specific to keratinocytes, involves rapid entry, and functions at low temperatures. This may reflect the environmental conditions encountered by HSV-1 when entering its host through the skin and emphasizes the importance of studying virus-host interactions in physiologically relevant cells., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Cationic antimicrobial peptides (AMPs) are part of the innate immunity, and act against a wide variety of pathogenic microorganisms by perturbation of the microorganism's plasma membrane. Although attractive for clinical applications, these agents suffer from limited stability and activity in vivo, as well as non-specific interaction with host biological membranes, leading to cytotoxic adverse effects. We hypothesized that encapsulation of AMPs within liposomes could result in reduced cytotoxicity, and with enhanced stability as well as bioactivity against herpes simplex virus 1 (HSV-1). We formulated nano-sized liposomal formulations of LL-37 and indolicidin, and their physicochemical properties, cellular uptake, in vitro cytotoxicity and antiviral efficacy have been determined. Lower cytotoxicity of LL-37 liposomes was found in comparison to indolicidin liposomes attributed to the superior physicochemical properties, and to the different degree of interaction with the liposomal membrane. The disc-like shaped LL-37 liposomes (106.8±10.1nm, shelf-life stability of >1year) were taken up more rapidly and to a significantly higher extent than the free peptide by human keratinocyte cell line (HaCaT), remained intact within the cells, followed by release of the active peptide within the cytoplasm and migration of the vesicles' lipids to the plasma membrane. LL-37 liposomes were found significantly less toxic than both the free agent and liposomal indolicidin. In the new 3D epidermis model (immortalized primary keratinocytes) liposomal LL-37 treatment (>20μM), but not free LL-37, efficiently protected the epidermis, inhibiting HSV-1 infection. This positive antiviral effect was obtained with no cytotoxicity even at very high concentrations (400μM). Thus, the antiviral activity of encapsulated LL-37 was significantly improved, expanding its therapeutic window. Liposomal LL-37 appears to be a promising delivery system for HSV therapy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

When dengue virus (DENV)-infected mosquitoes use their proboscis to probe into human skin during blood feeding, both saliva and virus are released. During this process, cells from the epidermis and dermis layers of the skin, along with small blood vessels, may get exposed to or infected with DENV. In these microenvironments of the skin, the presence of DENV initiates a complex interplay among the DENV-infected and non-infected neighboring cells at the initial bite site. Previous studies suggested that DENV-infected human dermal fibroblasts (HDFs) participate in the immune response against DENV by secreting soluble mediators of innate immunity. In the present study, we investigated whether DENV-infected HDFs activate human dermal microvascular endothelial cells (HDMECs) in co-cultures. Our results suggest that co-cultures of DENV-infected HDFs and HDMECs elicit soluble mediators that are sufficient to reduce viral replication, activate HDMECs, and induce leukocyte migration through HDMEC monolayers. These effects were partly dependent on HDF donor and DENV serotype, which may provide novel insights into the natural variation in host susceptibility to DENV disease.

The role of transient receptor potential vanilloid-1 (TRPV1) in the initiation of neurogenic inflammation and transduction of pain is well established. In this study 33 patients with herpes zoster (HZ) were recruited from a single centre and underwent a questionnaire interview at their first visit. Punch biopsies from the HZ lesions and the contralateral unaffected skin were performed to localize and quantify the expression of TRPV1. Immunofluorescent staining for TRPV1 was most prominent in the epidermal keratinocytes. Both TRPV1 mRNA and protein levels were significantly higher in the HZ epidermis than in control epidermis (relative ratio 1.62 ± 0.27, p = 0.033 and 2.55 ± 0.51, p = 0.005, respectively). Protein TRPV1 ratio (HZ lesion/control) correlated with the degree of pain (measured on a visual analogue scale; VAS) (p = 0.017) and was significantly lower in patients who had taken either HZ medication or painkillers prior to their visit. These results suggest that non-neuronal TRPV1 may contribute to acute pain in herpes zoster.

It is well established how effector T cells exit the vasculature to enter the peripheral tissues in which an infection is ongoing. However, less is known regarding how CTLs migrate toward infected cells after entry into peripheral organs. Recently, it was shown that the chemokine receptor CXCR3 on T cells has an important role in their ability to localize infected cells and to control vaccinia virus infection. However, the search strategy of T cells for virus-infected targets has not been investigated in detail and could involve chemotaxis toward infected cells, chemokinesis (i.e., increased motility) combined with CTL arrest when targets are detected, or both. In this study, we describe and analyze the migration of CTLs within HSV-1-infected epidermis in vivo. We demonstrate that activated T cells display a subtle distance-dependent chemotaxis toward clusters of infected cells and confirm that this is mediated by CXCR3 and its ligands. Although the chemotactic migration is weak, computer simulations based on short-term experimental data, combined with subsequent long-term imaging indicate that this behavior is crucial for efficient target localization and T cell accumulation at effector sites. Thus, chemotactic migration of effector T cells within peripheral tissue forms an important factor in the speed with which T cells are able to arrive at sites of infection., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Herpes simplex virus type 1 (HSV-1) invades its human host via the skin or mucosa. We aim to understand how HSV-1 overcomes the barrier function of the host epithelia, and for this reason, we established an ex vivo infection assay initially with murine skin samples. Here, we report how tissue has to be prepared to be susceptible to HSV-1 infection. Most efficient infection of the epidermis was achieved by removing the dermis. HSV-1 initially invaded the basal epidermal layer, and from there, spreading to the suprabasal layers was observed. Strikingly, in resting stage hair follicles, only the hair germ was infected, whereas the quiescent bulge stem cells (SCs) were resistant to infection. However, during the growth phase, infected cells were also detected in the activated bulge SCs. We demonstrated that cell proliferation was not a precondition for HSV-1 invasion, but SC activation was required as shown by infection of aberrantly activated bulge SCs in integrin-linked kinase (ILK)-deficient hair follicles. These results suggest that the status of the bulge SCs determines whether HSV-1 can reach its receptors, whereas the receptors on basal keratinocytes are accessible irrespective of their proliferation status.

Condyloma acuminatum (CA) is a condition caused by the highly contagious human papillomavirus (HPV), characterized by warts that undergo abnormal cell proliferation. One of the important regulators of cell proliferation is microRNAs (miRNAs). In this study, we aimed to investigate the expression profile of miR-99b in HPV positive CA samples and normal skin. We found significantly lower miR-99b levels in CA samples than in normal skin. Therefore, we investigated the role of miR-99b in regulating the proliferation of primary cultured human epidermal keratinocytes, and found that forced expression of miR-99b inhibited proliferation and induced G1-phase arrest. Based on conserved sequences in 3'UTR for miR-99b binding, we identified the insulin-like growth factor-1 receptor (IGF-1R) gene as a direct target for miR-99b. Further, we confirmed the binding site for miR-99b in the IGF-1R 3'UTR by mutation using a luciferase reporter assay that showed decrease in luciferase activity in the presence of miR-99b in the construct with the wild-type 3'UTR, but not in the construct with the mutant 3'UTR. Moreover, miR-99b over-expression could down-regulate IGF-1R expression, and could repress the PI3K-AKT signaling pathway. Lastly, over-expression of IGF-1R reversed the inhibitory effect of miR-99b on keratinocyte proliferation. Taken together, our results suggest that IGF-1R levels may be modulated by miR-99b in CA: downregulation of miR-99b with concomitant upregulation of its target gene IGF-1R may over-induce the PI3K-AKT signaling pathway, leading to deregulated cell proliferation in CA., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Unlabelled: The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) can both mediate the entry of herpes simplex virus 1 (HSV-1). We have recently shown how these receptors contribute to infection of skin by investigating HSV-1 entry into murine epidermis. Ex vivo infection studies reveal nectin-1 as the primary receptor in epidermis, whereas HVEM has a more limited role. Although the epidermis represents the outermost layer of skin, the contribution of nectin-1 and HVEM in the underlying dermis is still open. Here, we analyzed the role of each receptor during HSV-1 entry in murine dermal fibroblasts that were deficient in expression of either nectin-1 or HVEM or both receptors. Because infection was not prevented by the absence of either nectin-1 or HVEM, we conclude that they can act as alternative receptors. Although HVEM was found to be highly expressed on fibroblasts, entry was delayed in nectin-1-deficient cells, suggesting that nectin-1 acts as the more efficient receptor. In the absence of both receptors, entry was strongly delayed leading to a much reduced viral spread and virus production. These results suggest an unidentified cellular component that acts as alternate but inefficient receptor for HSV-1 on dermal fibroblasts. Characterization of the cellular entry mechanism suggests that HSV-1 can enter dermal fibroblasts both by direct fusion with the plasma membrane and via endocytic vesicles and that this is not dependent on the presence or absence of nectin-1. Entry was also shown to require dynamin and cholesterol, suggesting comparable entry pathways in keratinocytes and dermal fibroblasts., Importance: Herpes simplex virus (HSV) is a human pathogen which infects its host via mucosal surfaces or abraded skin. To understand how HSV-1 overcomes the protective barrier of mucosa or skin and reaches its receptors in tissue, it is essential to know which receptors contribute to the entry into individual skin cells. Previously, we have explored the contribution of nectin-1 and herpesvirus entry mediator (HVEM) as receptors for HSV-1 entry into murine epidermis, where keratinocytes form the major cell type. Since the underlying dermis consists primarily of fibroblasts, we have now extended our study of HSV-1 entry to dermal fibroblasts isolated from nectin-1- or HVEM-deficient mice or from mice deficient in both receptors. Our results demonstrate a role for both nectin-1 and HVEM as receptors and suggest a further receptor which appears much less efficient., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5'-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5'-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Dengue virus (DENV) causes the most prevalent arthropod-borne viral disease in humans. Although Aedes mosquitoes transmit DENV when probing for blood in the skin, no information exists on DENV infection and immune response in the dermis, where the blood vessels are found. DENV suppresses the interferon response, replicates, and causes disease in humans but not wild-type mice. Here, we used mice lacking the interferon-α/β receptor (Ifnar(-/-)), which had normal cell populations in the skin and were susceptible to intradermal DENV infection, to investigate the dynamics of early DENV infection of immune cells in the skin. CD103(+) classical dendritic cells (cDCs), Ly6C(-) CD11b(+) cDCs, and macrophages in the steady-state dermis were initial targets of DENV infection 12-24 hours post-inoculation but then decreased in frequency. We demonstrated recruitment of adoptively-transferred Ly6C(high) monocytes from wild-type and Ifnar(-/-) origin to the DENV-infected dermis and differentiation to Ly6C(+) CD11b(+) monocyte-derived DCs (moDCs), which became DENV-infected after 48 hours, and were then the major targets for virus replication. Ly6C(high) monocytes that entered the DENV-infected dermis expressed chemokine receptor CCR2, likely mediating recruitment. Further, we show that ∼ 100-fold more hematopoietic cells in the dermis were DENV-infected compared to Langerhans cells in the epidermis. Overall, these results identify the dermis as the main site of early DENV replication and show that DENV infection in the skin occurs in two waves: initial infection of resident cDCs and macrophages, followed by infection of monocytes and moDCs that are recruited to the dermis. Our study reveals a novel viral strategy of exploiting monocyte recruitment to increase the number of targets for infection at the site of invasion in the skin and highlights the skin as a potential site for therapeutic action or intradermal vaccination.

Herpes simplex virus-1 (HSV-1) is a human pathogen that utilizes several strategies to circumvent the host immune response. An immune evasion mechanism employed by HSV-1 is retention of interleukin-1β (IL-1β) in the intracellular space, which blocks the pro-inflammatory activity of IL-1β. Here we report that HSV-1-infected keratinocytes actively release the also pro-inflammatory IL-1α, preserving the ability of infected cells to signal danger to the surrounding tissue. The extracellular release of IL-1α is independent of inflammatory caspases. In vivo recruitment of leukocytes to early HSV-1 microinfection sites within the epidermis is dependent upon IL-1 signalling. Following cutaneous HSV-1 infection, mice unable to signal via extracellular IL-1α exhibit an increased mortality rate associated with viral dissemination. We conclude that IL-1α acts as an alarmin essential for leukocyte recruitment and protective immunity against HSV-1. This function may have evolved to counteract an immune evasion mechanism deployed by HSV-1.

Cyprinid herpesvirus 3 (CyHV-3) causes a lethal disease in common and koi carp (Cyprinus carpio). The present study investigated the ability of CyHV-3 to infect common carp during the early stages of its development (from embryos to fingerlings) after inoculation by immersion in water containing the virus. Fish were inoculated at different times after hatching with a pathogenic recombinant CyHV-3 strain expressing luciferase. The sensitivity and permissivity of carp to CyHV-3 were investigated using in vivo bioluminescence imaging. The susceptibility of carp to CyHV-3 disease was investigated by measuring the survival rate. Carp were sensitive and permissive to CyHV-3 infection and susceptible to CyHV-3 disease at all stages of development, but the sensitivity of the two early developmental stages (embryo and larval stages) was limited compared to later stages. The lower sensitivity observed for the early developmental stages was due to stronger inhibition of viral entry into the host by epidermal mucus. In addition, independent of the developmental stage at which inoculation was performed, the localization of light emission suggested that the skin is the portal of CyHV-3 entry. Taken together, the results of the present study demonstrate that carp are sensitive and permissive to CyHV-3 at all stages of development and confirm that the skin is the major portal of entry after inoculation by immersion in infectious water. The results also stress the role of epidermal mucus as an innate immune barrier against pathogens even and especially at the early stages of development.

The controversy over whether vaccine-targeted HPV types will be replaced by other oncogenic, non-vaccine-targeted types remains unresolved. This is in part because little is known about the ecology of HPV types. Patient data has been interpreted to suggest independence or facilitative interactions between types and therefore replacement is believed to be unlikely. With a novel mathematical model, we investigated which HPV type interactions and their immune responses gave qualitatively similar patterns frequently observed in patients. To assess the possibility of type replacement, vaccination was added to see if non-vaccine-targeted types increased their 'niche'. Our model predicts that independence and facilitation are not necessary for the coexistence of types inside hosts, especially given the patchy nature of HPV infection. In fact, independence and facilitation inadequately represented co-infected patients. We found that some form of competition is likely in natural co-infections. Hence, non-vaccine-targeted types that are not cross-reactive with the vaccine could spread to more patches and can increase their viral load in vaccinated hosts. The degree to which this happens will depend on replication and patch colonization rates. Our results suggest that independence between types could be a fallacy, and so without conclusively untangling HPV within-host ecology, type replacement remains theoretically viable. More ecological thinking is needed in future studies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease (LCD). In this study, we investigated the mechanisms of lymphocystis cell (LCC) formation from the viewpoint of gene expression changes in the infected fish. LCC occurrence and virus titers in the experimentally infected Japanese flounder, Paralichthys olivaceus were monitored by visual confirmation and real-time PCR, respectively. The gene expression changes in the fish fin were investigated by microarray experiments. LCCs firstly appeared in the fish at 21 days post infection (dpi). LCD incidence increased with time and reached 92.9% at 62 dpi. LCDV genome was firstly detected from dorsal fins at 14 dpi, and the relative amount of the genome gradually-increased until 56 dpi. Since the occurrence of LCC was approximately synchronized with increasing of the virus genome, virus replication might play important roles for LCC formation. The microarray detected a few gene expression changes until 28 dpi. However, the number of expression changed genes dramatically increased between 28 and 42 dpi in which LCCs formation was active. From the microarray data analyses, apoptosis and cell division related genes were down-regulated, whereas cell fusion and collagen related genes were up-regulated at 42 dpi. Together with the observation of morphological changes of LCCs in previous reports, it is suggested that the following steps are involved in LCC formation: the virus infected cells were (1) inhibited apoptotic death and (2) cell division before enlargement, (3) hypertrophied by cell fusion, and (4) surrounded by a hyaline capsule associated with the alteration of collagen fibers., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We analyzed local reactions of immune homeostasis in the human skin, in particular, effector immune cells CD68 responsible for antigen presentation, during human papillomavirus infection. Under conditions of long-term papillomavirus infection, CD68 markers were identifi ed only in the connective tissue of the skin (derma) and were completely absent in the epidermis, where they were found during physiological and reparative regeneration after thermal injury. We concluded that hypertrophy of the epidermis and connective tissue of the dermal papillary layer in human papillomavirus infection is related to the absence of CD68 immune cells in the epithelial plate and their accumulation in the connective tissue adjacent to the basement membrane of the epidermis. The possibility of epithelium contamination with the virus depends on local immune homeostasis. Therefore, induction of proper CD68 distribution in appropriate structures can contribute to normalization of epithelial-connective tissue interactions.

Despite extensive ex vivo investigation, the spatiotemporal organization of immune cells interacting with virus-infected cells in tissues remains uncertain. To address this, we used intravital multiphoton microscopy to visualize immune cell interactions with virus-infected cells following epicutaneous vaccinia virus (VV) infection of mice. VV infects keratinocytes in epidermal foci and numerous migratory dermal inflammatory monocytes that outlie the foci. We observed Ly6G(+) innate immune cells infiltrating and controlling foci, while CD8(+) T cells remained on the periphery killing infected monocytes. Most antigen-specific CD8(+) T cells in the skin did not interact with virus-infected cells. Blocking the generation of reactive nitrogen species relocated CD8(+) T cells into foci, modestly reducing viral titers. Depletion of Ly6G(+) and CD8(+) cells dramatically increased viral titers, consistent with their synergistic but spatially segregated viral clearance activities. These findings highlight previously unappreciated differences in the anatomic specialization of antiviral immune cell subsets., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Herpes simplex virus 2 infection is characterized by cycles of viral quiescence and reactivation. CD8(+) T cells persist at the site of viral reactivation, at the genital dermal-epidermal junction contiguous to neuronal endings of sensory neurons, for several months after herpes lesion resolution. To evaluate whether these resident CD8(+) T cells frequently encounter HSV antigen even during times of asymptomatic viral infection, we analyzed the transcriptional output of CD8(+) T cells captured by laser microdissection from human genital skin biopsy specimens during the clinically quiescent period of 8 weeks after lesion resolution. These CD8(+) T cells expressed a characteristic set of genes distinct from those of three separate control cell populations, and network and pathway analyses revealed that these T cells significantly upregulated antiviral genes such as GZMB, PRF1, INFG, IL-32, and LTA, carbohydrate and lipid metabolism-related genes such as GLUT-1, and chemotaxis and recruitment genes such as CCL5 and CCR1, suggesting a possible feedback mechanism for the recruitment of CD8(+) T cells to the site of infection. Many of these transcripts are known to have half-lives of <48 h, suggesting that cognate antigen is released frequently into the mucosa and that resident CD8(+) T cells act as functional effectors in controlling viral spread.

Chronic papillomatous dermatitis (CPD) is a stoma site complication due to chronic irritant contact dermatitis. Papillomatosis can also arise in the setting of human papillomavirus (HPV) infection or chronic lymphedema (elephantiasis). Herein, we report the case of a 57-year-old female who presented with a papillomatous growth surrounding a loop ileostomy suspected to be recurrent ovarian serous carcinoma. Excisional biopsy demonstrated nevus sebaceous (NS)-like organoid hyperplasia with koilocytes overlying a dermal scar that exhibited lymphangiectases. Polymerase chain reaction and sequencing for HPV DNA detected HPV 16. In situ hybridization for high-risk HPV DNA showed punctate nuclear pattern in the keratinocytes populating the NS-like hyperplasia indicating integrated HPV 16 DNA. No recurrence has been observed 11 months postexcision. Reports of CPD have documented a spectrum of reactive epidermal hyperplasias including pseudoepitheliomatous, verrucous, papillomatous, syringofibroadenomatous, and rudimentary follicular hyperplasias. HPV DNA has been detected in 3 of 4 CDP cases tested to date and in authentic NS. We postulate that localized lymphedema secondary to scarring coupled with chronic epidermal irritation and inflammation allowed for latent HPV infection to manifest as CPD with NS-like cutaneous hyperplasia.

Herpes simplex virus (HSV) is a member of the herpes virus family that commonly affects the skin. Typical histopathologic findings are usually limited to the epidermis and include intraepidermal vesicles or ulceration and epidermal necrosis. More specific findings in herpes virus infection include enlarged and pale keratinocytes, with steel-gray nuclei and margination of chromatin at the edge of the nucleus and ballooning degeneration. Although histopathologic changes may occasionally involve the hair follicles or sebaceous glands, it is very rare to find HSV involving the eccrine glands. We present a case of a 13-month-old child with a large body burn diagnosed with HSV (in the absence of the epidermis) by the presence of syringitis with herpetic features in the absence of the epidermis to aid in diagnosis.

The E6 proteins from high-risk alpha human papillomavirus (HPV) types (e.g., HPV16) are characterized by the presence of a PDZ-binding motif through which they interact with a number of cellular PDZ domain-containing substrates and cooperate in their degradation. The ability of these E6 proteins to bind to PDZ domain proteins correlates with the oncogenic potential of the virus. The E6 proteins of oncogenic HPV from the genus Betapapillomavirus (betaPV, e.g., HPV8) do not encode a PDZ-binding motif. We found that the PDZ domain protein syntenin-2 is transcriptionally downregulated in primary human epidermal keratinocytes (PHEK) by HPV8 E6. The mRNA levels of the known HPV16 E6 PDZ protein targets Dlg, Scribble, Magi-1, Magi-3, PSD95, and Mupp1 were not changed by HPV8 E6. Decreased protein levels of syntenin-2 were observed in cell extracts from PHEK expressing HPV5, -8, -16, -20, and -38 E6 but not in HPV1 and -4 E6-positive keratinocytes. Surprisingly, HPV16 E6 also repressed transcription of syntenin-2 but with a much lower efficiency than HPV8 E6. In healthy human skin, syntenin-2 expression is localized in suprabasal epidermal layers. In organotypic skin cultures, the differentiation-dependent expression of syntenin-2 was absent in HPV8 E6- and E6E7-expressing cells. In basal cell carcinomas of the skin, syntenin-2 was not detectable, whereas in squamous cell carcinomas, expression was located in differentiated areas. Short hairpin RNA-mediated knockdown of syntenin-2 led to an inhibition of differentiation and an increase in the proliferation capacity in PHEK. These results identified syntenin-2 as the first PDZ domain protein controlled by HPV8 and HPV16 at the mRNA level.

Background: Although several billion corneocytes are shed from human skin daily, metabolic studies from 50 years ago led to the conclusion that corneocyte desquamation had no measurable impact on systemic protein or iron status in humans., Objective: To measure iron content of internal organs after introducing local genetic changes in epidermis that alter iron metabolism in skin., Methods: Iron was measured in tissues and blood from groups of animals 7 weeks after weaning in three different mouse models expressing a transgene in epidermis: a hyperproliferation model in which the HPV16 E7 gene causes a 3-fold increase in epidermal turnover; an epidermal iron sink model in which overexpression of the transferrin receptor causes a 3-4 fold increase of iron in epidermis; a systemic hemochromatosis knockout model that has been crossed with the epidermal iron sink model., Results: In the hemochromatosis model with the iron sink transgene in epidermis, there was a statistically significant reduction in non-heme iron in serum and in the liver and kidney. In all models there was a statistically significant reduction in non-heme iron in the kidney., Conclusion: Local changes in iron metabolism in epidermis can have a measurable impact on systemic iron metabolism. By implication, disruptions in epidermal homeostasis might affect systemic levels of trace nutrients, and circulating toxins might be remediated by sequestering them in epidermis., (Copyright © 2012 Japanese Society for Investigative Dermatology. All rights reserved.)

The contribution of the Wnt signaling pathway to human papilloma virus (HPV)-induced carcinogenesis is poorly understood. In high-grade dysplastic lesions that are caused by high-risk HPVs (HR-HPV), β-catenin is often located in the cell nucleus, which suggests that Wnt pathway may be involved in the development of HPV-related carcinomas. Most of the oncogenic potential of HR-HPVs resides on the PDZ-binding domain of E6 protein. We hypothesized that the PDZ-binding domain of the HPV16-E6 oncoprotein induces the nuclear accumulation of β-catenin due to its capacity to degrade PDZ-containing cellular targets. To test this hypothesis, we evaluated the staining pattern of β-catenin in the skin epidermis of transgenic mice expressing the full-length E6 oncoprotein (K14E6 mice) and measured LacZ gene expression in K14E6 mice that were crossed with a strain expressing LacZ that was knocked into the Axin2 locus (Axin2(+/LacZ) mice). Here, we show that the E6 oncoprotein enhances the nuclear accumulation of β-catenin, the accumulation of cellular β-catenin-responsive genes, and the expression of LacZ. None of these effects were observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6ΔPDZ mice) or in combination with Axin2(+/LacZ). Conversely, cotransfection with either E6 or E6ΔPDZ similarly enhanced canonical Wnt signaling in short-term in vitro assays that used a luciferase Wnt/β-catenin/TCF-dependent promoter. We propose that the activation of canonical Wnt signaling could be induced by the HPV16-E6 oncoprotein; however, the participation of the E6 PDZ-binding domain seems to be important in in vivo models only., (©2011 AACR.)

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies.

Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein β (C/EBPβ) as a critical regulator of CCL20 gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBPβ converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBPβ as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBPβ in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBPβ-inducible and constitutive CCL20 gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity.

Geminiviruses replicate their circular single-stranded DNA genome in nuclei of infected plant cells. Their replication initiator proteins (Reps) possess interaction domains for homo- and hetero-oligomerization as shown previously by in vitro studies and yeast two hybrid assays. Here, homo-oligomerization and cellular localization of the Abutilon mosaic virus (AbMV) Rep was analysed with bimolecular fluorescence complementation (BiFC) in epidermal tissues of Nicotiana benthamiana. BiFC revealed that Rep oligomers accumulated within the nucleoplasm, but were excluded from nucleoli as indicated by a nucleoli/cajal body marker. A similar subcellular distribution was observed for Rep fused to full-length cyan fluorescent protein. To examine whether tagged Reps were functionally active, N. benthamiana plants transgenic for a dimeric AbMV DNA B were inoculated with the BiFC expression constructs and nucleic acids were analysed by rolling circle amplification/restriction fragment length polymorphism as well as Southern blot hybridization. The results confirmed that the modified AbMV Rep was able to transreplicate DNA B., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a mortal and highly contagious disease in common and koi carp. The skin is the major portal of entry of CyHV-3 in carp after immersion in water containing the virus. In the present study, we used in vivo bioluminescence imaging to investigate the effect of skin mucus removal and skin epidermis lesion on CyHV-3 entry. Physical treatments inducing removal of the mucus up to complete erosion of the epidermis were applied on a defined area of carp skin just before inoculation by immersion in infectious water. CyHV-3 entry in carp was drastically enhanced on the area of the skin where the mucus was removed with or without associated epidermal lesion. To investigate whether skin mucus inhibits CyHV-3 binding to epidermal cells, tail fins with an intact mucus layer or without mucus were inoculated ex vivo. While electron microscopy examination revealed numerous viral particles bound on the fins inoculated after mucus removal, no particle could be detected after infection of mucus-covered fins. Finally, anti-CyHV-3 neutralising activity of mucus extract was tested in vitro. Incubation of CyHV-3 with mucus extract reduced its infectivity in a dose dependent manner. The present study demonstrates that skin mucus removal and epidermal lesions enhance CyHV-3 entry in carp. It highlights the role of fish skin mucus as an innate immune protection against viral epidermal entry.

Cutaneous beta human papillomavirus (HPV) types appear to be involved in the development of non-melanoma skin cancer (NMSC); however, it is not entirely clear whether they play a direct role. We have previously shown that E6 and E7 oncoproteins from the beta HPV type 38 display transforming activities in several experimental models. To evaluate the possible contribution of HPV38 in a proliferative tissue compartment during carcinogenesis, we generated a new transgenic mouse model (Tg) where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter. Viral oncogene expression led to increased cellular proliferation in the epidermis of the Tg animals in comparison to the wild-type littermates. Although no spontaneous formation of tumours was observed during the lifespan of the K14 HPV38 E6/E7-Tg mice, they were highly susceptible to 7,12-dimethylbenz(a)anthracene (DMBA)/12-0-tetradecanoylphorbol-13-acetate (TPA) two-stage chemical carcinogenesis. In addition, when animals were exposed to ultraviolet light (UV) irradiation, we observed that accumulation of p21(WAF1) and cell-cycle arrest were significantly alleviated in the skin of Tg mice as compared to wild-type controls. Most importantly, chronic UV irradiation of Tg mice induced the development of actinic keratosis-like lesions, which are considered in humans as precursors of squamous cell carcinomas (SCC), and subsequently of SCC in a significant proportion of the animals. In contrast, wild-type animals subjected to identical treatments did not develop any type of skin lesions. Thus, the oncoproteins E6 and E7 from beta HPV38 significantly contribute to SCC development in the skin rendering keratinocytes more susceptible to UV-induced carcinogenesis.

In equids, bovine papillomaviruses of type 1 (BPV-1) and less frequently type 2 induce common, locally aggressive skin tumours termed sarcoids. Whereas BPV infection in cattle usually involves the epidermis and is productive in this skin layer, infection in equids is currently thought to be abortive, with virus solely residing as multiple episomes in dermal fibroblasts. Based on recent observations that do not agree with this assumption, we hypothesised that BPV also infects equid epidermis and is active in this skin layer. To test this hypothesis, we conducted a proof-of-principle study on eight distinct sarcoids. Presence of viral DNA was addressed by qualitative and quantitative BPV-1 PCR from microdissected sarcoid epidermis, and by subsequent amplicon sequencing. Viral activity was assessed by screening sarcoid epidermis for BPV-1 protein expression using immunohistochemistry (IHC) or immunofluorescence (IF). Virus-free equine skin served as negative control throughout the assays. BPV-1 DNA was demonstrated in all sarcoid epidermis samples, with viral DNA loads ranging between 2 and 195 copies/cell. Identical BPV-1 E5 genes were identified in epidermis and dermis of each of two sarcoids, yet different E5 variants were found in individual lesions. IHC/IF revealed the presence of E5 and E7 protein in sarcoid epidermis, and L1 capsomers in the squamous layer of one lesion. These findings indicate that BPV infection also involves the epidermis, where it may occasionally be productive., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A two-week-old budgerigar (Melopsittacus undulatus) of an outdoor aviary died suddenly and was submitted for determination the cause of illness and death. Macroscopically, the sparsely feathered animal was in a poor body condition. Histopathological examination revealed in various mesenchymal and epithelial tissues, numerous up to 15 microm in diameter large intranuclear, amphophilic to basophilic inclusion bodies with a clearing of the centre. Additionally, a feather dysplasia and retention hyperkeratosis of feather follicles was found. Ultrastructurally, viral particles of approximately 35 nm in diameter were detected in the feather follicle epithelium. A PCR for Avian Polyomavirus on fresh skin samples was negative whereas on formalin-fixed kidney samples with a high amount of viral inclusion bodies yielded a positive result. In addition, viral inclusion body diseases, like Avian Poxvirus, Psittacine Beak and Feather disease virus, Avian Adenovirus, Psittacine Herpesvirus and papillomavirus of psittacines are summarized and compared in the present article.