Microscopic observations of SARS-CoV-2 like particles in different oral samples

Abstract

The emerging coronavirus pneumonia epidemic caused by the SARS-CoV-2 infection has spread rapidly around the world. The main routes of transmission of SARS-CoV-2 are currently recognised as aerosol/droplet inhalation. However, the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly known. The current data indicates the presence of viral RNA in oral samples, suggesting the implication of saliva in SARS-CoV-2 transmission, however, no direct observation of SARS-CoV-2 particles in different oral samples has been reported. In this study, we investigated whether particles of SARS-CoV-2 were present in oral samples collected from three symptomatic COVID-19 patients. Using scanning electron microscopy (SEM), the correlative strategy of light microscopy and electron microscopy and immunofluorescence staining, we showed the presence of SARS-like particles in RT-qPCR SARS-CoV-2-positive saliva, dental plaque and gingival crevicular fluid (GCF) samples. In the saliva samples, we demonstrated the presence of epithelial oral cells with morphogenetic features of SARS-CoV-2 infected cells. Inside those cells, vacuoles filled with nascent particles were observed, suggesting the potential infection and replication of SARS-CoV-2 in oral tissues. Our results corroborate previous studies and confirm that the oral cavity may be a potential niche for SARS-CoV-2 infection and a potential source of transmission.

Keywords: COVID-19; dental plaque; saliva; scanning electron microscopy; virology.

FIGURE 1

Scanning electron microscopy images of the ultra‐thin section of dental plaque sample. (A,B) SEM images of dental plaque sampled from Participant 1. (B) High magnification of the boxed region in (A) showing electron dense circular structures with 80–140 nm diameters resembled SARS‐CoV‐2‐like particles (arrows) within vesicles at the cell periphery. (C,D) SEM images of dental plaque sampled from Participant 2. (D) High magnification of the boxed regions in (D) showed SARS‐CoV‐2‐like particles (arrows) with 75–140 nm diameters (arrows) (scale bar A: 5 μm; scale bar B‐D: 1 μm).

FIGURE 2

Scanning electron microscopy images of the ultra‐thin section of gingival crevicular fluid sample. (A,B) SEM images of (GCF) sampled from Participant 1. (B) High magnification of the boxed region in (A) showed SARS‐CoV‐2‐like particles (arrows) within vesicles at the cell periphery. (C,D) SEM images of (GCF) sampled from Participant 2. (D) High magnification of the boxed region in (D) showed SARS‐CoV‐2 like particles (arrows) with 75–140 nm diameters (arrows) (scale bar A: 5 μm; scale bar B, C and D: 1 μm) (scale bar A: 2 μm; scale bar B and D: 1 μm; scale bar C: 3 μm).

FIGURE 3

Scanning electron microscopy of saliva sample. (A,B) Different magnification views of epithelial oral cell observed in saliva sample of (Participant 1). (A) Low magnification of the epithelial oral cell. Boxed regions represent an extensive peri‐nuclear membrane whorls network with vacuoles and (N) nucleus. (B) High magnification view of the boxed region in (A) showed SARS‐CoV‐2‐like particles inside and outside the closed sac resembling morphogenetic matrix vesicae (arrowhead). (C,D) Scanning electron microscopy of epithelial oral cells observed in the saliva sample of Participant 2. (C) Low magnification of the epithelial oral cell. Boxed region showed a peri‐nuclear membrane whorls network with vacuoles resembling morphogenetic matrix vesicae, (N) nucleus. (D) High magnification view of the boxed region in (C) shows SARS‐CoV‐2‐like particles inside open or closed sac with different sizes (arrowhead). (E,F) Scanning electron microscopy of the saliva sample of Participant 3. (E) Low magnification of oral cell cytoplasm. (F) High magnification of the boxed region in (E) shows an extensive network membrane with a closed sac containing SARS‐CoV‐2‐like particles (scale bar A, C and E: 5 μm; scale bar B: 1 μm; scale bar D: 500 nm; scale bar F: 2 μm).

FIGURE 4

Correlative light fluorescence and electron microscopy in an ultra‐thin section of dental plaque sample (Participant 1). Confocal laser scanning microscopy images of 100 nm‐thick ultra‐thin section (Z maximal projection) of dental plaque sample (A,B) DNA stained with Hoechst 33342 (blue) and the SARS‐CoV‐2 particles labelled with anti‐SARS‐CoV‐2 spike protein (orange red). Scanning electron microscopy images (C–E) of the ultra‐thin section shown in (A,B). The boxed region of interest in (A–C) is shown at a higher magnification in (D). Boxed region in (D) is zoomed in (E). Hypo‐electron dense circular structures surrounded by a hyper‐dense crown‐like shapes with 75–140 nm diameters (arrows) are present in the boxed region positive for anti‐SARS‐CoV‐2 fluorescence (scale bar A, B and C: 10 μm; scale bar D: 4 μm; scale bar E: 1 μm).

FIGURE 5

Anti‐SARS‐CoV‐2 immunofluorescence staining on saliva sample from Participant 1 (A‐C), Participant 2 (A1‐C1) and Participant 3 (A2‐C2). (A, A1, A2) DNA was stained using Hoechst 3342 (blue). (B, B1, B2) SARS‐CoV‐2 particles were stained using anti‐SARS‐CoV‐2 antibody (orange‐red), respectively. (C, C1, C2) Colocalization of Hoechst 3342 and anti‐SARS‐CoV‐2 antibody (scale bar: 10 μm).