The present study showed that SARS-CoV-2 infection via the oral route can spread to the nasal cavity and OB in Syrian hamsters. SARS-CoV-2 was detected in the nasal cavity 7 days after oral SARS-CoV-2 inoculation, but the virus infection site was mainly on the lateral side of the nasal cavity. The numbers of ORN-associated cells were reduced in all lineages. SARS-CoV-2 was identified in the olfactory nerve bundles in the pathway from the OM to the OB.
Although the infection conditions in the nasal cavity may be different between the transnasal and transoral infection models of SARS-CoV-2, the virus oral inoculation model may show an infection situation more similar to the actual living environment than the virus nasal administration model. The findings that SARS-CoV-2 entering the host animals via the oral route not only infected the lower respiratory tract, but also the nasal tissues and induced degeneration of the OE, is an important message regarding infection countermeasures in daily life. By inhaling droplets from conversations during eating and drinking, or by eating something touched with SARS-CoV-2-contaminated hands, the virus is likely to adhere to the oral cavity and enter the body, resulting in olfactory dysfunction in some people. Therefore, individuals should thoroughly wash their hands before eating, be careful of splashing droplets during conversation while eating, and avoid sharing meals with others to prevent infection.
Regarding the localization of SARS-CoV-2 infection sites in the nasal cavity and differences in the OE damage depending on the site, a previous report showed that it is more likely for SARS-CoV-2 to be found in the lateral region where there is a greater degree of tissue damage 20, which is consistent with the present results. In addition, this is also analogous to the fact that the OE in the dorsolateral region is susceptible to damage via allergic inflammation 37. Conversely, it has been reported that the NQO1-positive OE is more likely to be damaged by long-term exercise and caloric restriction 38. Thus, it is suggested that the susceptibility of the OM to damage depends on the type of damage and the location of the OE.
Many studies have examined which OE cells are susceptible to infection by SARS-CoV-2, and it is now considered that the virus primarily infects the olfactory cilia and supporting cells but can also infect some basal ORN progenitors 9,19,21,22. However, SARS-CoV-2 can also affect olfactory neurons, albeit slightly 21. In the present validation, the numbers of all ORN-related cells, including ORN progenitors, immature ORNs, and mature ORNs, were reduced in each area of the OE compared to the control group, although differences were observed in each area of the OE. Although the present study was limited to the analysis of day 7 after oral SARS-CoV-2 inoculation, and the temporal changes before and after the infection could not be confirmed, we discuss the following.
In the DS and VS areas where the virus could not be identified, the epithelial layer of the SARS-CoV-2 group was thinner than that of the control group. The numbers of basal cells, immature ORNs, and mature ORNs were reduced, and only the cells in the superficial layer of the OE were dividing, suggesting that the OE was damaged after virus infection and that it may have been in the regeneration stage at the time of examination. As GAP43+ immature ORNs could not be identified and Ki67+ cells were observed in the superficial layer, rather than in the basal layer, we speculate that the time point we verified in this study was the stage when the damaged sustentacular cells had first started to regenerate and that the basal cells (SOX2+ ORN progenitors) and their differentiation process to GAP43+ immature ORNs are suppressed by SARS-CoV-2 infection. Considering that inflammatory cell infiltration is not as abundant in the OM, it is presumed that the OE damage was induced by direct cell damage caused by the virus rather than by the effects of inflammatory cell infiltration. In the previously reported transnasal infection model of SARS-CoV-2, SARS-CoV-2 mainly infected the sustentacular cells, not the ORNs, and most of their cilia were lost early after infection 19,22. It was also reported that SARS-CoV-2 could only be identified in the nasal septum of intranasal infected hamsters up to 3 days after infection 20, which is consistent with our results. In the nasal septum of this animal model, a large proportion of the OE was damaged within a few days after infection, followed by epithelial regeneration over time 20. Although the temporal changes after infection in the oral SARS-CoV-2 inoculation model may differ from those in the transnasal infection model, we speculate that the DS and VS areas, where no SARS-CoV-2 was identified in this study, will regenerate their epithelial tissue.
In the DLT and LT areas where the virus was present, while sustentacular cells and basal ORN progenitors could be seen to some extent, immature ORNs could hardly be seen, and mature ORNs could only be sparsely seen. As several cells negative for SOX2, GAP43, and OMP were found in the middle layer of the OE, it is possible that SARS-CoV-2 impaired the function of mature ORNs and prevented normal ORN protein expression. Taken together, the possible effects of SARS-CoV-2 infection on the ORNs are as follows: (1) sustentacular cells cannot support the ORNs because of viral infection, resulting in difficulty in maintaining the epithelial morphology and reduced function of mature ORNs 9,19,21,22; (2) SARS-CoV-2 directly infects the ONRs and reduces their function, although the percentage may be small 21; and (3) SARS-CoV-2 may inhibit the maturation and differentiation of immature ORNs into mature ORNs.
The olfactory route is considered a potential means for SARS-CoV-2 to invade the central nervous system 39. In this study, we found SARS-CoV-2 in the olfactory nerve bundle around the cribriform plate, indicating that the nasal cavity is an entry route for the virus to invade the central nervous system. In human studies, SARS-CoV-2 has been detected in the OB of autopsied cases 40,41. Imaging studies have also shown hyperintensity of the OB on T2 fluid-attenuated inversion recovery magnetic resonance images 15,42 and a pattern of hypometabolism involving the olfactory system (orbitofrontal cortex and olfactory and rectus gyri) on positron emission tomography of brain 43. A study on rhesus monkeys reported that SARS-CoV-2 primarily invades the central nervous system via the OB 39. In human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2, viral RNA was detected throughout the OB, including in the olfactory nerve layer, 7 days after infection 44. Approximately 10% of patients continued to experience olfactory dysfunction more than 6 months after SARS-CoV-2 infection 45,46, suggesting that the virus affected the central olfactory system, and the results of this study support the possibility of central olfactory dysfunction due to SARS-CoV-2 infection.
In conclusion, this study suggests that transoral SARS-CoV-2 infection may spread to the nasal cavity and then to the central nervous system through the olfactory route. To prevent SARS-CoV-2 infection in daily life, individuals should take adequate infection prevention measures and avoid performing activities associated with transoral infection.