Visualization of Molluscum Contagiosum Virus in FFPE Skin Sections Using NanoSuit-CLEM: Ultrastructural Evidence of Viral Spread via Skin Barrier Disruption

Abstract

Background: Molluscum contagiosum (MC) is a common viral skin infection caused by members of the Poxviridae family. It primarily affects children, sexually active adults, and immunocompromised individuals. Although MC spreads through direct contact and auto-inoculation, the precise mechanisms by which the virus penetrates the skin barrier remain poorly understood.

Methods: We applied NanoSuit-correlative light and electron microscopy (NanoSuit-CLEM) to formalin-fixed paraffin-embedded (FFPE) skin sections to visualize MC virus particles in situ with high resolution. Melan-A immunohistochemistry using 3,3'-diaminobenzidine (DAB) with osmium staining was performed to identify Henderson-Patterson bodies.

Results: Ultrastructural analysis revealed that MC virus particles were densely localized in the stratum corneum but did not invade deeper epithelial layers in intact skin. However, in areas of epidermal disruption, such as detached or damaged stratum corneum, the virus was observed penetrating into lower layers. While Melan-A immunostaining successfully detected Henderson-Patterson bodies, it failed to identify mature MC virus particles. In contrast, NanoSuit-CLEM combined with Mayer's hematoxylin and lead staining enabled detailed visualization of mature viral particles and their distribution within the stratum corneum.

Conclusions: These findings provide direct ultrastructural evidence that MC virus entry occurs through compromised skin, underscoring the crucial role of the stratum corneum in barrier function. This study highlights the importance of preventing mechanical skin injury, such as scratching or shaving, to limit MC transmission. NanoSuit-CLEM offers a powerful new tool for studying viral pathogenesis in archival tissue samples.

Keywords: Molluscum contagiosum; NanoSuit‐CLEM; skin; viral particles.

Figure 1

CLEM image of Molluscum contagiosum (MC). (A) Cup‐shaped lesion with inverted hyperplastic squamous epithelium lobules extending into the underlying dermis. H&E image. Bar represents 1 mm. (B) Magnified image of (A). Bar represents 0.4 mm. (C) Magnified image of white square in (B). Arrow indicates the eosinophilic Henderson–Patterson bodies (molluscum bodies). Bar represents 120 μm. (D) CLEM image from field FE‐SEM corresponding to (C). Bar represents 120 μm. (E) Magnified image of white square in (D). Arrows indicate MC particles. Bar represents 6.7 μm. (F) Magnified image of black square in (B). Bar represents 120 μm. (G) CLEM image from field FE‐SEM corresponding to (F). Bar represents 120 μm. (H) Magnified image of white square in (G). Arrows indicate MC particles. Bar represents 6.7 μm.

Figure 2

Immunohistochemical analysis of Melan‐A in molluscum contagiosum (MC) bodies. (A–C) The MC specimen is immune‐stained with Melan‐A antibody. Black square indicates the entire infected area, and the white square within the black square is the area in which each figure is magnified. (A) Lower part of the MC‐infected area. Melan‐A is positive for Henderson–Patterson bodies. (B) Middle part of the MC‐infected area. (C) Upper part of the MC infected area. White bar represents 100 μm. (D–F) Corresponding CLEM SEM images of A–C, respectively. DAB‐positive, with Melan‐A stained areas depicted as white areas in the SEM‐BSE images. White bar represents 100 μm. (G–I) Magnified images of D–F (white square), respectively. (G and H) A mosaic and mixed pattern of Melan‐A positive/negative MC viral particles are observed. Melan‐A expression weakens MC viral particles in the higher layers of the skin. White bar represents 10 µm. (I) Melan‐A is scarcely expressed in the upper part of the MC‐infected area. White bar represents 10 µm. (J) Magnified image from the lower part of the lesion. White bar represents 5 µm. (K) Magnified image of the middle part of the MC‐infected lesion. White bar represents 5 µm.

Figure 3

Identification and distribution of MC virus particles in FFPE sections using NanoSuit‐CLEM observation. (A) Overview of the analyzed lesion, divided into four parts (I–IV) for examining the virus particle distribution. Portions B and C (white squares) are selected from part I, representing different regions within the lesion. White bar represents 40 μm. (B) Magnified view of Portion B, adjacent to the basal membrane (BM; stratum basale). A few virus‐like particles are visible in the cytoplasm; however, none cross or are located outside the BM. (C) Magnified view of Portion C, located in the bottom layer of the stratum spinosum. A greater number of MC viral particles are observed in the cytoplasm compared to the BM. White bar represents 5 μm. (D) Part II represents the stratum spinosum, containing multiple MB with eosinophilic cytoplasmic inclusions. White bar represents 40 μm. (E) The lower layer of the stratum spinosum shows numerous virus particles within MB. White bar represents 5 μm. (F) The upper layer of the stratum spinosum displays a higher density and greater number of virus particles in MB. Insets in (E and F) show magnified views of the white squares. (G–L) Parts III and IV represent the stratum corneum, where the lesion center disintegrates, forming a central crater. (G) H&E‐stained section of part III. The white bar represents 40 μm. (H) Virus particles in the central region appear densely packed and indistinguishable, forming a rock‐like flat surface. The white bar represents 5 μm. (I) In another region of the stratum corneum, virus particles remain distinguishable but are densely packed. White bar represents 5 μm. (J) H&E‐stained section in part IV.　White bar represents 40 μm. (K, L) As virus aggregates move upward through the stratum corneum, the solid mass gradually loosens and virus particles become increasingly distinguishable. White bar represents 5 μm.

Figure 4

Analysis of MC virus invasiveness to the epithelium using the NanoSuit‐CLEM method. (A–C) Case 1: Multiple MC virus particles attached to the stratum corneum surface. Most virus particles exhibit a dome‐shaped deformation, indicating firm attachment to the surface. (D–F) Case 2: Similar findings with dome‐shaped MC virus particles attached to the surface. (G–I) Case 3: Some virus particles retain a spherical shape, suggesting weaker attachment. A few particles are observed beneath the epithelial layer (arrows), indicating potential viral infiltration. (J–L) Case 4: Further evidence of dome‐shaped virus particles attached to the corneum surface, showing minimal penetration into deeper layers. (A, D, G, J) H&E staining section. Black bar represents 600 μm. (B, E, H, K) FE‐SEM image (BSE mode). Magnification of black squares of A, D, G, J. Black bar represents 50 μm. (C, F, I, L) FE‐SEM image (BSE mode). Magnification of black squares in B, E, H, K. Black bar represents 5 μm. (M–R) FE‐SEM images following Mayer's hematoxylin and lead staining. (M) Virus aggregation in the Melan‐A‐negative upper layers depicted as white‐stained areas in BSE mode. White bar represents 5 μm. (N, O) Most viral particles remain on the corneum surface. (N) White bar represents 10 μm. (O) White bar represents 2 μm. (P) A few virus particles are observed beneath the corneum (arrow). White bar represents 5 μm. (Q) Virus particles appear lodged in a groove, with some infiltration into deeper areas (arrow). White bar represents 2 μm. (R) Viral infiltration is observed in damaged areas of the corneum (dashed line, arrow). White bar represents 2 μm.