Histopathology of the Tongue in a Hamster Model of COVID-19

Abstract

Objective: With altered sense of taste being a common symptom of coronavirus disease 2019 (COVID-19), our objective was to investigate the presence and distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within the tongue over the course of infection.

Methods: Golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 and tongues were collected at 2, 3, 5, 8, 17, 21, 35, and 42 days post-infection (dpi) for analysis. In order to test for gross changes in the tongue, the papillae of the tongue were counted. Paraffin-embedded thin sections of the tongues were labeled for the presence of SARS-CoV-2 antigen.

Results: There was no difference in fungiform or filiform papillae density throughout the course of infection. SARS-CoV-2 antigen was observed in the circumvallate papillae taste buds (3-35 dpi) and autonomic ganglia (5-35 dpi), as well as in the serous and mucous salivary glands of the posterior tongue (2-42 dpi).

Conclusion: The presence and distribution of SARS-CoV-2 suggest that the virus could cause taste disturbance by infecting the circumvallate taste buds. This effect could be exacerbated by a diminished secretion of saliva caused by infection of the serous salivary glands and the autonomic ganglia which innervate them.

Keywords: SARS-CoV-2; dysgeusia; hamster.

Figure 1. Anatomy of the Hamster Tongue.

Distribution of tongue papillae and related histology. A midline circumvallate (CV) papilla resides in the posterior region (white oval), foliate on the sides (white curved lines), fungiform on the anterior (white circles), and filiform filling the remaining space. Taste buds (arrowheads) reside on the sides of the CV papillae and the tips of the fungiform papillae but are not found on the filiform papillae. Salivary glands are located beneath the troughs of CV papillae and help dissolve tastants. SC;stratum cornuem, SS; stratum spinosum, SB; stratum basale, LP; lamina propria, TB; taste buds

Figure 2. Papillae Counting.

A) Magnified 8-bit image of the anterior tongue using Image J v1.53a. The region of interest (ROI) was demarcated (grey line) using Image J freehand tool, the fungiform papillae were counted using the Cell Counter plugin for Image J (dark blue dots), and the area (mm²) calculated using the measurement tool in Image J with the ruler behind the tongue for reference. Papillae were excluded from the count (light blue dot) if they were found to not be a complete circle, not completely within the ROI, or not approximately 3:1 in size when compared with surrounding filiform papillae. Scale bar indicates 500μm. B) Magnified 8-bit inverted image of the anterior tongue using Image J v1.53a. Red dots indicate computer generated counting of the papillae using the Image-based Tool for Counting Nuclei (ITCN) plugin for Image J. Scale bar indicates 100μm. C) Fungiform papillae densities were plotted against days post infection (dpi). Error bars indicate mean and standard deviation. No significant differences were found between any of the infected groups (dpi#) or control (ctrl) when tested with ANOVA set to p < 0.05. For all groups, n = triplicates of 4. D) Filiform papillae densities were plotted against days post infection (dpi). Error bars indicate mean and standard deviation. No significant differences were found between any of the infected groups (dpi#) or control (ctrl) when tested with ANOVA set to p < 0.05. For all groups, n = triplicates of 4.

Figure 3. SARS-CoV-2 antigen labeling in the tongue.

Representative images of immunohistochemical labeling (1:200 antibody dilution) of structures of interest with or without infection with SARS-CoV-2. The infected samples at 5 dpi are shown in the lower panels (G, H, I, J, K, L), with arrowheads pointing to positive brown-colored labeling of the SARS-CoV-2 antigen, compared with negative labeling in the mock infected control samples in the upper panels (A, B, C, D, E). The circumvallate papilla taste buds (G) and ganglion cells (L) show positive labeling, while fungiform papilla taste bud (H), serous salivary gland cells (J), mucous salivary gland cells (K) show weakly positive labeling. The filiform papilla cells (I) were negative for labeling. P, papillae. SG, salivary gland. Scale bars indicate 100 μm.