Ex Vivo Infection of Human Skin Models with Herpes Simplex Virus 1: Accessibility of the Receptor Nectin-1 during Formation or Impairment of Epidermal Barriers Is Restricted by Tight Junctions.

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.

Herpes Simplex Virus 1 Can Bypass Impaired Epidermal Barriers upon Ex Vivo Infection of Skin from Atopic Dermatitis Patients.

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.

Ex Vivo Infection of Human Skin with Herpes Simplex Virus 1 Reveals Mechanical Wounds as Insufficient Entry Portals via the Skin Surface.

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.

Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell, Epi-cDC2s, via different entry pathways.

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.

Measles skin rash: Infection of lymphoid and myeloid cells in the dermis precedes viral dissemination to the epidermis.

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.

In vivo and ex vivo dermoscopy of lesions from implantation of human papillomavirus in tattoos: report of two cases

Abstract The number of individuals with tattoos has been increasing worldwide, alongside with reports of complications varying from reactions to the injected pigments to infections caused by agents inoculated in the pigmentation process. The diagnosis of such unwanted events can be obtained through complementary non-invasive methods, preserving the maximum of the tattoo design. The authors present two cases of patients with warts on tattooing, and correlate their clinical aspects to in vivo and ex vivo dermoscopy, and to the findings in the histopathological examination, aiming to determine patterns that aid the diagnosis of these lesions without performing biopsy.

In vivo and ex vivo dermoscopy of lesions from implantation of human papillomavirus in tattoos: report of two cases.

The number of individuals with tattoos has been increasing worldwide, alongside with reports of complications varying from reactions to the injected pigments to infections caused by agents inoculated in the pigmentation process. The diagnosis of such unwanted events can be obtained through complementary non-invasive methods, preserving the maximum of the tattoo design. The authors present two cases of patients with warts on tattooing, and correlate their clinical aspects to in vivo and ex vivo dermoscopy, and to the findings in the histopathological examination, aiming to determine patterns that aid the diagnosis of these lesions without performing biopsy.

Invasion of Herpes Simplex Virus 1 into Murine Dermis: Role of Nectin-1 and Herpesvirus Entry Mediator as Cellular Receptors during Aging.

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.

Single-cell RNA sequencing reveals cell type-specific HPV expression in hyperplastic skin lesions.

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.

First outbreak of myxomatosis in Iberian hares (Lepus granatensis).

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.

Development, characterization and virus susceptibility of a continuous cell line from the caudal fin of marbled eel (Anguilla marmorata).

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.

The Rho regulator Myosin IXb enables nonlymphoid tissue seeding of protective CD8+ T cells.

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.

Loss of ATP2A2 Allows Herpes Simplex Virus 1 Infection of a Human Epidermis Model by Disrupting Innate Immunity and Barrier Function.

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-ß 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.

Entry of Herpes Simplex Virus 1 into Epidermis and Dermal Fibroblasts Is Independent of the Scavenger Receptor MARCO.

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.

Novel Poxvirus in Proliferative Lesions of Wild Rodents in East Central Texas, USA.

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.

Interleukin-33 is expressed in the lesional epidermis in herpes virus infection but not in verruca vulgaris.

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.

Human papillomavirus oncoproteins induce a reorganization of epithelial-associated γδ T cells promoting tumor formation.

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+ γδlowCCR2+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.

Squamous epitheliotropism of Enterovirus A71 in human epidermis and oral mucosa.

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 Enters Human Keratinocytes by a Nectin-1-Dependent, Rapid Plasma Membrane Fusion Pathway That Functions at Low Temperature.

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.