In a major scientific study published in the journal Cell, scientists at the UNC School of Medicine and the UNC Gillings School of Global Public Health have characterized the specific ways in which SARS-CoV-2 – the coronavirus that causes COVID-19 – infects the nasal cavity to a great degree – replicating specific cell types – and infects and replicates progressively less well in cells lower down the respiratory tract, including the lungs.
The findings suggest the virus tends to become firmly established first in the nasal cavity, but in some cases the virus is aspirated into the lungs, where it may cause more serious disease, including potentially fatal pneumonia.
“If the nose is the dominant initial site from which lung infections are seeded, then the widespread use of masks to protect the nasal passages, as well as any therapeutic strategies that reduce virus in the nose, such as nasal irrigation or antiviral nasal sprays, could be beneficial,” said study co-senior author Richard Boucher, MD, the James C. Moeser Eminent Distinguished Professor of Medicine and Director of the Marsico Lung Institute at the UNC School of Medicine.
The other co-senior author of the study was Ralph Baric, Ph.D., the William R. Kenan Distinguished Professor of Epidemiology at the UNC Gillings School of Public Health.
“This is a landmark study that reveals new and unexpected insights into the mechanisms that regulate disease progression and severity following SARS-CoV-2 infection,” said Baric, who also holds a microbiology faculty appointment at the UNC School of Medicine.
“In addition, we describe a new reverse genetic platform for SARS-CoV-2 allowing us to produce key indicator viruses that will support national vaccine efforts designed to control the spread and severity of this terrible disease.”
SARS-CoV-2 initially caused outbreaks in late 2019 in China and spread around the world, infecting nearly 6 million people and killing more than 350,000. The United States accounts for almost a third of those infections and deaths.
The UNC-Chapel Hill team in their study sought to understand better a number of things about the virus, including which cells in the airway it infects, and how it gets into the lungs in the patients who develop pneumonia.
In one set of laboratory experiments, the researchers used different isolates of SARS-CoV-2 to see how efficiently they could infect cultured cells from different parts of the human airway.
They found a striking pattern of continuous variation, or gradient, from a relatively high infectivity of SARS-CoV-2 in cells lining the nasal passages, to less infectivity in cells lining the throat and bronchia, to relatively low infectivity in lung cells.
The scientists also found that ACE2 – the cell surface receptor that the virus uses to get into cells – was more abundant on nasal-lining cells and less abundant on the surface of lower airway cells. This difference could explain, at least in part, why upper airway nasal-lining cells were more susceptible to infection.
Other experiments focused on TMPRSS2 and furin, two protein-cleaving enzymes found on many human cells. It’s thought that SARS-CoV-2 uses those two enzymes to re-shape key virus proteins and enter human cells.
The experiments confirmed that when these human enzymes are more abundant, this particular coronavirus has an increased ability to infect cells and make copies of itself.
The researchers found that the virus can infect airway-lining cells called epithelial cells, and to a limited extent the all-important “pneumocyte” lung cells that help transfer inhaled oxygen into the bloodstream. But SARS-CoV-2 infects almost no other airway cells.
Intriguingly, the virus did not infect airway-lining cells called club cells, despite the fact that these cells express both ACE2 and TMPRSS2. Moreover, the same types of airway epithelial cells from different human donors, especially lower-airway epithelial cells, tended to vary significantly in their susceptibility to infection.
Such findings suggest that there are undiscovered factors in airway cells that help determine the course of infection in individuals – a course known to vary widely from mild or no symptoms all the way to respiratory failure and death.
The team mapped the sites of coronavirus infection in the lungs of several people who had died from COVID-19, and found that these sites exhibited a sort of patchiness and other characteristics consistent with the hypothesis that these sites had originated from infection higher in the airway.
The hypothesis that aspiration of oral contents into the lung is a significant contributor to COVID-19 pneumonia is consistent with the observations that people at higher risk for severe lung disease – the elderly, obese, and diabetic – are more prone to aspiration, especially at night.
The team also found that previously described individual antibodies capable of neutralizing the original SARS coronavirus of 2002 and the MERS coronavirus, which has been spreading slowly in the Middle East since 2012, did not neutralize SARS-CoV-2.
However, blood serum from two of five SARS 2002 patients showed a low level but significant capability to neutralize SARS-CoV-2 infectivity in cultured cells. These data suggests that people who have been exposed to other coronaviruses may carry some other types of antibodies in their blood that provide at least partial protection against SARS-CoV-2.
“These results, using some novel and innovative methodology, open new directions for future studies on SARS-C0V-2 that may guide therapeutic development and practices for reducing transmission and severity of COVID-19,” said James Kiley, Director of the Division of Lung Diseases at the National Heart, Lung, and Blood Institute, part of the National Institutes of Health.
Boucher, Baric, and colleagues note that their study, apart from its specific findings about SARS-CoV-2 infection in the airway, involved the development of key laboratory tools—including a version of SARS-CoV-2 re-engineered to carry a fluorescent beacon—that should be useful in future investigations of the virus.
SARS-CoV-2 Affinity to the Respiratory Epithelium in the Nasal Cavity Is Likely Moderate
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) (Table 1). 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.
1. Fast, Sensitive, and Reliable Tests Are Critical to Slow down a Pandemic
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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.
2. SARS-CoV-2 Affinity to the Respiratory Epithelium in the Nasal Cavity Is Likely Moderate
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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) (Table 1). 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.
Table 1. ACE2 and TMPRSS2 Expression in Human and Mouse Nasal Cavity Epitheliaa
| nasal cavity | hACE2 | hTMPRSS2 | mACE2 | mTMPRSS2 | database |
|---|---|---|---|---|---|
| respiratory epithelium | + | + | + | ND | Bgee, GEO |
| olfactory epithelium | + | ND | + | + | Bgee, GEO |
| olfactory receptor neurons | ND | ND | − or low | + | Bgee, GEO |
Data based on Affymetrix and RNAseq. hACE2, human ACE2; hTMPRSS2, human TMPRSS2; mACE2, mouse ACE2; mTMPRSS2, mouse TMPRSS2. +, positive expression; ND, no data available. Note that olfactory receptor neurons are major part of OE; however, OE also contains several types of non-neuronal cells.
The Olfactory Epithelium As a Site of SARS-CoV-2 Replication, Accumulation, and Brain Entrance
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)
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 |
(a) www.bgee.org, Affymetrix microarrays, score range 0–10).
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 including medulla oblongata where cardiorespiratory controlling nuclei are located.(8)
Brain infection in COVID-19 patients is currently being seriously considered because of many reports of neurological impairments such as stroke, epilepsy ,and encephalitis. The ACE2 expression in glia and in neurons in the brain is low but also well documented. But the specific sites where SARS-CoV-2 enters the brain are not clearly identified.(8)
Mature olfactory neurons present in the OE are probably one such place. However, SARS-CoV-2 virus must first invade high ACE2-expressing yet unidentified non-neuronal OE cells and then pass to low-ACE2-expressing mature ORNs to be finally transported along olfactory axons to the brain.
A good candidate for such cells is specialized glia known as olfactory ensheathing cells (OECs). OECs were previously shown to enhance human herpesvirus-6 replication and accumulation in the OE before virus infected the brain.(9) Many studies have already shown that this type of glia cells can supply axons with macromolecules by way of exosomes and this could be a mechanism of ACE2-independent virus transfer from OEC to ORN axons.
Olfactory Neurons in OE May Mediate Antiviral Responses
It is known that the nervous system can shape responses of the innate immunity system. ORNs which are located with direct contact with the external environment are ideally suited for that role.
Recetly it was shown in fish that ORNs initiate ultrarapid immune responses after binding rhabdovirus surface glycoprotein.(10) The virus binding results in neuronal activation and proinflammatory effects in OE but inhibits inflammation in the brain.
As a consequence, some neurons undergo apoptosis, which may inhibit the reception of olfactory stimuli for some time. This data reveals the possible universal role of ORNs as first line viral sensors and initiators of antiviral protective immune responses.
Based on the above conclusion, there is an exciting possibility that SARS-CoV-2 binding to ORNs initiates that kind of rapid immune response. Induction of the innate immune system through ORNs does not necessarily have to be mediated by ACE2 and TMPRSS2, but it may require additional yet unidentified host protein(s) with the ability to transmit intracellular signaling.
From this point of view, infected people who show signs of olfactory dysfunctions may actually represents those individuals with faster and/or stronger immune response and better body mobilization against the SARS-CoV-2 infection.
Therefore, it will be interesting to examine groups of patients with and without olfactory dysfunction and correlate it with the severity of their symptoms and percentage of recovery. Intriguingly, older patients which are known to be much more sensitive to SARS-CoV-2 infection are also those who have their sense of smell compromised simply because of their age. Reduced numbers of ORNs in older people can potentially slow down their early immune response and, consequently, lead to more severe COVID-19
More information: Yixuan J. Hou et al, SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract, Cell (2020). DOI: 10.1016/j.cell.2020.05.042
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