An important factor accelerating the spread of COVID-19 is the high infectivity of this virus. It is related to extraordinarily ability of spike glycoprotein to bind to host receptor with much higher affinity as compared to related SARS-CoV virus. Lower thermostability of SARS-CoV-2 spike protein has also been suggested as a factor contributing to its high infectivity. (1) Although SARS-CoV-2 testing is currently very efficient, public health care systems do not have the capacity to test all the citizens. Thus, the identification of infected but asymptomatic people should be one of the priorities and this should be done as early as possible. Currently, assays that are based on real-time RT-PCR technique are recommended for early detection of the virus. Theoretically, procedures based on RT-PCR are able to detect even a small number of viral RNA particles in biological samples. However, in practice, due to several technical factors, there must be much more viral load in the biological material collected to achieve a reliable diagnosis. Typically nasal and pharyngeal swabs as well as sputum are used as the starting biological material for SARS-CoV-2 testing. It is assumed that this strategy is very efficient in diagnosing infected individuals 5–7 days after onset of symptoms. Unfortunately, it is less efficient in detecting SARS-CoV-2 within 1–4 days after symptoms and in asymptomatic individuals. Therefore, other types of biological samples should be identified to detect SARS-CoV-2 more efficiently about the time of infection.

Because the nasal cavity is the main gate for SARS-CoV-2 entrance, epithelial cells located within this area can be considered as appropriate clinical sample for early virus detection. The nasal cavity contains three main types of mucosa: squamous, respiratory, and olfactory epithelium ( Figure 1 ). Importantly, all these cells are easily accessible for collection by medical staff. According to some gene expression data deposited in databases such as GEO and MGI, respiratory epithelial cells (RECs) express both of the SARS-CoV-2 human proteins required for host cell entry, namely, ACE2 and TRMPSS2 transmembrane proteases (1,2) 1 Table ). On the other hand, recent single cell RNaseq studies in humans showed only TMPRSS2 expression in RECs without detecting ACE2. (3) Other RNaseq studies showed rather low levels of ACE2 in RECs. (4) However, according to the mouse atlas, in embryonic RECs, ACE2 expression was clearly shown by in situ hybridization but TMPRSS2 expression was not examined by this approach. Taken together, current data suggest that RECs present in the nasal cavity express rather low levels of ACE2 and TMPRSS2 proteins as compared to epithelial cells located at lower parts of the human respiratory pathway. However, it should be emphasized that the expression data in RECs are clearly incomplete and require further detailed examination. Before drawing a final conclusions about SARS-CoV-2 affinity to these cells, different aspects such as age-dependence and possible effects of pathological conditions on ACE2/TMPRSS2 expression should also be addressed, preferably at cell-type resolution.

3. The Olfactory Epithelium As a Site of SARS-CoV-2 Replication, Accumulation, and Brain Entrance ARTICLE SECTIONS Jump To

Another suitable source of biological samples for early SARS-CoV-2 detection is the olfactory epithelium (OE), which is easily accessible within the nasal cavity ( Figure 1 ). Recent reports indicate that total anosmia or partial loss of the sense of smell are early markers of SARS-CoV-2 infection. This phenomenon may be caused by different and yet unidentified factors, e.g., “cytokine storm” initiated in some patients or direct damage of the olfactory receptor neurons (ORNs) located in the olfactory epithelium ( Figure 2 ). The latter possibility is particularly likely due to the fact that cells located in the OE express both protein receptors required for effiecient SARS-CoV-2 infection in humans. Several data sets deposited in gene expression databases show relatively high expression levels of ACE2 and TMPRSS2 in human and murine olfactory mucosa ( Table 1 ). In mammals, OE is a continuously regenerating multilayer structure containing both neuronal and non-neuronal cells ( Figure 2 ). The key question is whether ACE2 and TRMPSS2 expression in the OE is neuronal or non-neuronal or whether it occurs in both cell types. Neuronal expression of host receptors will likely facilitate SARS-CoV-2 brain infection through the uptake into ciliated dendrites/soma and subsequent anterograde axonal transport along the olfactory nerve. Non-neuronal expression of ACE2/TRMPSS2 may possibly establish nasal cavity OE as a virus reservoir. Three major RNaseq transcriptome studies conducted in human and murine OE consistently suggest non-neuronal expression of ACE2. (4−6) Hence, ACE2 expression is not clearly detected in mature ORNs, which are the only OE neurons connected to the brain. Expression of TMPRS2 seems to be higher compared to that of ACE2 and takes place likely in both neuronal and non-neuronal OE cells. (5,6) One state-of-the-art RNaseq study showed intriguingly mosaic TMPRSS2 expression which occurs only in subpopulation of mature ORNs, even though the majority of other genes were more evenly expressed in these neurons. (6) It suggests that some olfactory neurons in the OE may be more vulnerable for viral infection than other morphologically similar ORNs. Moreover, expression of murine ACE2 and TMPRSS2 evaluated by microarrays has a tendency to increase with age ( Table 2 ). If it is true in humans, then in elderly people the OE may be more sensitive to SARS-CoV-2 accumulation. However, it should be remembered that although ACE2 is a mandatory factor for viral entry into the cell, TMPRSS2 can probably be replaced by other proteases from this family such as TMPRSS4, TMPRSS11A, 11D, and 11E1. Of these proteases, only TMPRSS4 is also present in the OE, likely in immature neurons and in non-neuronal cells. (5,6)

Figure 2 Figure 2. Basic organization of the olfactory epithelium (OE). Olfactory neurons continuously regenerate through human life and therefore are at different stages of differentiation. Some non-neuronal cells are shown, e.g., progenitors, sustentacular cells, and olfactory ensheathing cells.

Table 2. ACE2 and TMPRSS2 Expression Scores in Mouse Olfactory Epithelium According to the Bgee Database a age of mice ACE2 TMPRSS2 6 weeks old 49.4 78.7 6 months old 61.4 89.5

medulla oblongata where cardiorespiratory controlling nuclei are located. It is known from a previous SARS-CoV pandemic that, that even though lungs were the major site of infection, the brain was also involved in some patients. In addition, it was shown in transgenic mice expressing human ACE2 that SARS-CoV infected the brain through ORNs. (7) Genetically modified mice express only human ACE2 and not human TMPRSS2. This may additionally suggest that murine ORNs express endogenous TMPRSS2, because both proteins are required for efficient infection. Intriguingly, there was an approximately 60 h delay from the time of nasal infection until SAR-CoV virus detection in the olfactory bulb. During that time the virus likely replicated and accumulated in different OE cells, because its subsequent transport to further parts of the brain required a relatively short time of an additional 12–20 h. (7) The results from transgenic hACE2 mice indicate that SARS-CoV probably uses transneuronal/transsynaptic routes employing axonal transport in the brain and this can also be true for SARS-CoV-2. It is known for other viruses, e.g., rabies virus, that they can hijack existing vesicular axonal transport machineries to spread within the brain. There is very recent evidence that SARS-CoV-2 enters early and late endosomal compartments in non-neuronal cells; thus, it may possibly be directed to the vesicular axonal pathway in neurons. (1) However, it should be remembered that the hACE2 mouse is an overexpressor model with expression of human ACE2 controlled by human keratin K18 promoter. For this reason, alternative and more physiological knock-in models for SARS-CoV-2 studies in the nervous system would be desirable. Alternatively to the olfactory axonal route, SARS-CoV-2 may pass from non-neuronal OE cells directly to cerebrospinal fluid surrounding olfactory nerve bundles, located near the cribriform plate. Once in cerebrospinal fluid, the virus could reach most of the brain areas includingwhere cardiorespiratory controlling nuclei are located. (8)