Coronavirus infection in chemosensory cells

Abstract

Clinical manifestations of human coronavirus (HCoV)-related diseases are mostly related to the respiratory system, although secondary complications such as headache, anosmia, ageusia, and myalgia have been reported. HCoV infection and replication in chemosensory cells associated with ageusia and anosmia is poorly understood. Here, we characterized HCoV-OC43 and SARS-CoV-2 infection in two types of chemosensory cells, olfactory and taste cells, with their unique molecular and histological characteristics. We first assessed HCoV-OC43 infection in in vitro cultured human olfactory epithelial cells (hOECs) and fungiform taste papilla (HBO) cells. Interestingly, while both cell types were susceptible to HCoV-OC43 infection, viral replication rates were significantly reduced in HBO cells compared to hOECs. More interestingly, while culture media from hOECs was able to produce secondary infection in Vero cells, there was very limited secondary infection from HBO cells, suggesting that HBO cells may not be able to release infectious virus. On the other hand, unlike HCoV-OC43, SARS-CoV-2 showed comparable levels of viral infection rates in both hOECs and HBO cells. Furthermore, our RT-qPCR-based gene array studies revealed that several key genes involved in taste and olfactory functions were significantly altered by SARS-CoV-2 infection. These results may suggest a possible mechanism associated with chemosensory symptoms, such as anosmia and ageusia in patients infected with SARS-CoV-2.

Keywords: Chemosensory; Coronavirus; HCoV-OC43; Olfactory cells; SARS-CoV-2; Taste cells.

Fig. 1

Immunostaining of HBO cells and hOECs. A, B Both HBO cells and hOECs showed presence of HBO cell-specific (PLC-b2, T1R2, T2R38, and gustducin, A) and hOEC-specific biomarkers (Adcy3, OMP, CNGA2, and b-tubulin III, B). Images were acquired with a Leica TCS-SP2 confocal laser scanning microscope. Transmission images of corresponding fields are shown on the left. Nuclei of cells were stained as blue with DAPI. For controls, immunostaining with antibody specific immunoglobulin demonstrated the absence of nonspecific immunoreactivity (data not shown). Scale bars = 25 μm. C mRNA level of ACE2 in HBO cells and hOECs was processed by RT-qPCR. Data are shown as delta Ct (ΔCt) values of ACE2 gene versus GAPDH gene

Fig. 2

HCoV-OC43 infection in VERO cells, primary human taste (HBO) cells, and olfactory cells (hOECs). A–C VERO, HBO, and hOECs were infected with HCoV-OC43 at different MOI (1, 10, 25, and 50 MOI); RNA and media were collected. A HCoV-OC43-infected VERO cell culture media were processed for RT-qPCR. B HCoV-OC43-infected HBO cell culture media were processed for RT-qPCR. C HCoV-OC43-infected hOEC culture media were processed for RT-qPCR. D–H Vero cells, HBO cells, and hOECs were infected with HCoV-OC43 at 25 MOI; culture media were collected daily, and cellular RNA was extracted at 7 dpi. D HCoV-OC43-infected VERO cell culture media were processed for RT-qPCR. E OC43-infected HBO cell culture media were processed for RT-qPCR. F OC43-infected hOEC culture media were processed for RT-qPCR. G HCoV-OC43-infected Vero cell, HBO cell, and hOEC cellular RNAs were processed for RT-qPCR and shown as bar graph. H Quantification of number of plaques forming units/mL. Data are shown as mean EM of two independent replicates

Fig. 3

SARS-CoV-2 infection in primary human taste (HBO) cells, olfactory cells (hOECs), and Vero cells. HBO cells, hOECs, and Vero cells were infected with SARS-CoV-2, and cellular RNA along with culture media was collected. A SARS-CoV-2-infected hOEC culture media were processed for RT-qPCR. B SARS-CoV-2-infected HBO cell culture media were processed for RT-qPCR. C SARS-CoV-2-infected VERO cell culture media were processed for RT-qPCR. D SARS-CoV-2-infected hOEC cellular RNA was processed for RT-qPCR. E SARS-CoV-2-infected HBO cellular RNA was processed for RT-qPCR. F SARS-CoV-2-infected VERO cellular RNA was processed for RT-qPCR

Fig. 4

Relative quantification of taste and olfactory receptors mRNA expression measured by real-time [quantitative] PCR (RT-PCR) in SARS-CoV-2 infected vs. uninfected HBO cells (A) and hOECs (B). Normalization factors were calculated as the geometric mean of the expression levels of the GAPDH reference gene. Values are expressed as group mean; fold change 2^−∆∆Ct vs. GAPDH gene, stars indicate a significant difference (p < 0.05) between infected (n = 3) and uninfected (n = 3) independent samples. SARS-CoV-2 infection of taste and olfactory cells resulted to changes of expression level of candidate receptors