How might a virus like this get into the nervous system, and why is the olfactory/gustatory system so specifically targeted?
In an article published on the NIH Director’s Blog, Francis Collins describes recent NIH research suggesting that COVID-19’s many neurological symptoms could be explained by the body’s widespread inflammation and associated blood vessel injury, rather than by infection of the brain tissue itself.
Although this could well be the case, this doesn’t tell us much about why smell is such a frequent casualty. Furthermore, most of the patients autopsied were elderly and had significant comorbidities, many apparently dying “with COVID-19” rather than from it. In fact, whether any of them actually lost any sense of smell was undocumented.
The director does point out that in new research from the Journal of Experimental Medicine, the authors did find some immunohistochemical evidence of SARS-CoV-2 in the brain. In particular, antibodies against the spike protein have suggested that SARS-CoV-2, or at least parts of it, can infect neurons of the cerebral cortex.
The authors also found that when they infected human brain organoids and humanized mice overexpressing a human form of ACE2 receptor, they could detect viral mRNA via qPCR within neural cells. For the mice, the virus was introduced either via an intranasal route or via direct injection into the cerebral ventricles.
Using electron microscopy, they could also identify viral particles budding from the host endoplasmic reticulum, and also document significant cell death.
Other researchers have found that ACE2 is expressed in support cells, stem cells, and perivascular cells rather than in neurons. In particular, so-called sustentacular cells of the olfactory epithelial, and pericytes within the olfactory bulb itself were found to have abundant ACE2. The authors then conclude infection of non-neural cells is the likely cause of anosmia and other disturbances in odor perception.
Similarly, in other research using a cell culture system that models the blood-brain barrier, researchers found that the SARS spike protein readily crosses this barrier and infects endothelial cells that line the cerebral vasculature. More generalized neuroinvasion was found in vivo as a result of diffuse inflammation and ischemia secondary to vascular damage.
At this point, it is not known if the virus could simply bud from cell to cell to cross the BBB, or if whole white blood cells are squeezing through to ferry them in.
If this is the whole story, then why do we only lose our cerebral capacity for smell or taste and not everything else? The answer is that this is probably not the whole story. More likely, we are dealing with a very peculiar monster with a hitherto unseen, laser-like precision in its assault on our senses.
While we have not witnessed a virus with this particular proclivity before, virologists are quite familiar with a host of viruses with other particular specificities. For example, both the rabies virus and the polio virus gain access to somatic motor neurons within the spinal cord by first breaching neuromuscular junctions (NMJ) within muscle tissue.
In the case of rabies, nicotinic acetylcholine receptors and neural cell adhesion molecules (NCAMs) have been identified as the primary NMJ receptors for the virus. For the poliovirus, which can replicate in motor neurons, an Ig superfamily member known as CD155 has also been identified as an axonal receptor.
Transneuron spread of these kinds of viruses to higher regions occurs through different types of retrograde and anterograde directed motor proteins, with direct synapse-to-synapse transmission. Other evidence suggests that HSV-1, vesicular stomatitis virus (VSV), Borna disease virus (BDV), influenza A virus, parainfluenza viruses, rabies, and even prions can all enter CNS through an anterograde olfactory route.
Some viruses, like Epstein–Barr virus (EBV) or human herpesvirus 6 (HHV‐6), are believed to hyperstimulate immune cells in the brain, causing them to attack unique proteins found only in myelinating cells, ultimately leading to MS-like degeneration. For example, myelin-specific CD4+ and CD8+ T-cells have recently been found in both peripheral and central nervous systems in some types of viral encephalitis.
In the case of the JC virus, B-cells in immunocompromised patients can infiltrate the CNS and transfer the virus to oligodendrocytes and astrocytes, leading to a fatal inflammatory disease called progressive multifocal leukoencephalopathy (PML). In light of our emerging understanding of these specific viral tropisms in nervous systems, what clues might now be available to figure out what SARS is doing?
One approach would be to try to use our existing knowledge about parallel pathways of smell and taste, and simply ask patients which smells and tastes were lost, which were recovered, and in what order. Aside from the obvious sweet, sour, salty and bitter perceptions, we know that the remainder of our taste stream is mostly just retronasal olfaction.
Whereas this system is optimized to detect tastants internal to the mouth upon exhalation, our regular smell sense is optimized for detecting external molecules that are inhaled across the olfactory epithelium. These primary sensory pathways are complimented by a separate chemesthesis channel which carries chemically initiated senses on the touch channel. Mouthfeel perceptions, like spicy capsaicinoids or cool peppermints, are transduced via TRPV4 receptors and transmitted on the trigeminal nerve.
The key here is to look at the places where these sensations overlap. For example, if the first things a recovering patient is able to detect are say, coffee and cinnamon, then the question is where and how, exactly?
In other words, do they first smell it via inhalation through their nose, or via their mouth exhalation, and can they taste it when they are not breathing at all?
Is it appealing scents, or disagreeable scents that might tend to return first?
Can one smell and taste peanut butter, a skunk, a burning house?
By pooling answers and looking for commonalities, it might be possible to piece together which pathways and higher brain centers are most affected.
In the ongoing search for clues, the next step might be to ask if there are any drugs that might help restore smell or taste, and how these drugs typically operate. In some cases, a combination therapy of steroids like dexamethasone and theophylline seemed to help recover function.
Both seem to be needed because at least one patient who stopped taking the theophylline arm of their treatment lost their sense of smell again. It was only recovered when the theophylline was resumed. For cases of total anosmia that also involve severe olfactory distortion and even hallucination, low doses of the antipsychotic haloperidol confer significant relief. Other breaking research now suggests that anosmia might be effectively remedied with a steroid fluticasone nasal spray, while dysgeusia overcome with triamcinolone oral paste.
Although some kinds of neurons have been shown to express low levels of the ACE2 receptors for the SARS-Cov-2 spike protein, possible co-expression of other important proteases or other receptor cofactors, like TMPRSS2 or Neuropilin-1, remains to be fully investigated. Comparing the mechanisms of entry of other related coronoviruses, or even unrelated viruses that similarly infect the brain via an olfactory route, could provide more clues.
A case in point is the OC43 coronavirus, which is one of the viruses that causes the common cold. OC43 can gain access to the brain via the N-acetyl-9-O-acetylneuraminic acid and then hijack axonal transport to reach higher olfactor areas such as the piriform cortex.
The most recent version of OC43, known as genotype D, was discovered in 2004 and is believed to have originated from a recombination event between related genotypes B and C.
As in the case of OC43, some researchers also believe that a novel furin cleavage site, a defining feature of the SARS-CoV-2 virus, might have originated from a recombination event with another coronavirus. However, recombination between more distantly related classes of coronaviruses that would contain a furin site may not be as likely, if even possible at all, as recombination within classes.
Reduction of smell and taste is now recognized as one of the cardinal symptoms of COVID-19. The deficit appears to be most often transient, with a regaining of smell and taste after several days to weeks, but the anosmia differs from other virus-associated deficits in its sudden onset and its rapid recovery.
Multiple reviews have covered this topic—so why is the current review needed? We have come to realize that to understand COVID-19, it is necessary to consider multiple dimensions, from the cellular-molecular level to psychophysical and clinical features, as well as genetics and epidemiology.
Relating different aspects of the disease in a holistic approach has been lacking in previous reviews; we will show that taking into account and integrating multiple disciplines provides a more complete insight and synthesis.
Anosmia and hypogeusia were not initially recognized to be linked to COVID-19; they were mentioned to affect only about 5% of COVID-19 patients in one of the first studies from China (Mao and others 2020), but a much higher prevalence was reported in subsequent studies from Europe, the Middle East, and North America (Agyeman and others 2020; Hannum and others 2020; Passarelli and others 2020; Printza and Constantinidis 2020; Sedaghat and others 2020; Tong and others 2020; von Bartheld and others 2020).
Why is the reduction in smell and taste one of the first symptoms of COVID-19, and why were these deficits recognized to be a cardinal symptom of COVID-19 only when the pandemic had moved beyond East Asia? We discuss possible explanations for early symptoms and for the population differences and their implications.
Key to understanding such differences in infectivity of SARS-CoV-2 may lie in the frequency of variants in the virus entry proteins, ACE2 and TMPRSS2, which may depend on cell type and population, with implications for infectivity, virus spread, and therefore managing the COVID-19 pandemic.
It has been a major mystery how the virus affects the senses of smell and taste. Significant progress has now been made to begin to elucidate the cellular and molecular mechanisms of coronavirus-induced anosmia. Recent work has provided new insights into the cell types in the olfactory epithelium that express the relevant virus entry proteins (Bilinska and others 2020) and that accumulate the virus after infection (Bryche and others 2020).
Much less is known about the underlying mechanisms that may explain taste reduction in COVID-19. ACE2 is expressed in epithelial cells of the tongue (Sato and others 2020; Vaira and others 2020c; Xu and others 2020; Cooper and others 2020), but probably not in taste buds (Wang and others 2020e), yet ACE2 inhibitor drugs are known to induce taste (and smell) disorders (Irvin and Viau 1986; Naik and others 2010; Bertlich and others 2020).
In the olfactory epithelium, the evidence suggests a distinct cascade of cellular events that can explain the transient anosmia in COVID-19. Whether and how the SARS-CoV-2 virus may utilize a route from the nose to infect the brain has been and still is a question of major interest and concern. We review a series of relevant studies that provide deeper insights on this topic, and we propose new hypotheses of how SARS-CoV-2 may gain access to the brain without relying on transport within olfactory neurons.
In this context, we explain the importance of developing and investigating new transgenic mouse models for future research in this field. We also review the prospects for making use of the anosmia seen in COVID-19 as an early, rapid, and surprisingly effective diagnostic screening tool.
What Is the Prevalence of Smell and Taste Dysfunction in COVID-19 Patients?
The literature on the prevalence of chemosensory dysfunctions in COVID-19 initially appeared to be confusing: wide ranges of prevalence were reported by different studies. In the first two months of the COVID-19 pandemic, clinicians considered such deficits to be a rare occurrence (Chen and others 2020a; Guan and others 2020; Mao and others 2020; Wang and others 2020d; reviewed by da Costa and others 2020). The first report that recognized smell and taste reduction to be a much more prevalent symptom came out of Germany (Streeck 2020), and subsequent studies have confirmed that a high prevalence of approximately 60% is the norm, especially outside of East Asia (von Bartheld and others 2020).
Several reviews have summarized findings of the early studies on this topic (Agyeman and others 2020; Hannum and others 2020; Passarelli and others 2020; Printza and Constantinidis 2020; Sedaghat and others 2020; Tong and others 2020), which were followed by a more comprehensive review and meta-analysis (von Bartheld and others 2020). Worldwide, the prevalence of olfactory deficits in COVID-19 patients was calculated to be 44.1%, the prevalence of taste deficits was 43.3%, and the prevalence for any chemosensory deficits was 49.0% (Table 1).
The prevalence of anosmia/ageusia in COVID-19 is generally thought to be an underestimate (Vaira and others 2020b; Tong and others 2020; von Bartheld and others 2020), because most studies rely on the patient telling the researcher about their subjective impressions, although some studies report that the results of subjective and objective measures are roughly equivalent (Parma and others 2020).
Several researchers have noticed a possible difference in the prevalence of chemosensory deficits between populations in East Asia and in Western countries (Dell’Era and others 2020; Lovato and others 2020; Lechien and others 2020a; Meng and others 2020; Qiu and others 2020).
Intriguingly, the most recent and comprehensive systematic review, of 30,264 patients, demonstrated a significant, nearly 3-fold higher prevalence in olfaction and/or taste impairment in populations in Western countries than in populations in East Asia, and the difference appears to be independent of age or disease severity (von Bartheld and others 2020, Fig. 1A and andB).B).
There are no data yet specifically on populations in Africa, South America, or South Asia in this respect. The nasal epithelium has been shown to have a larger viral load than the epithelium in the lower respiratory tract (Hou and others 2020; Meinhardt and others 2020; Rockx and others 2020; Wang and others 2020c; Zou and others 2020). Therefore, population differences in viral load could have far-reaching implications for infectivity and virus spreading, and ultimately for successful management of the pandemic.
Prevalence of Dysfunctions in Smell, Taste, and Any Chemosensory Perception in COVID-19 Patients According to Our Recent Review and Meta-Analysis (von Bartheld and others 2020).
|Sensory Deficit||Population||Cohort Number||Prevalence (%)|
|Smell and/or taste|
Anosmia in COVID-19: What Is the Underlying Mechanism?
The anosmia induced by SARS-CoV-2 has several unique features. Its high prevalence (in Western countries) is remarkable, as is its sudden onset, its rather short duration, and in most cases, a rapid recovery, as well as the fact that the anosmia (and taste dysfunction) can be the only symptoms in a significant fraction of patients, and that the anosmia often presents without nasal congestion or rhinorrhea.
The chemosensory deficits are typically transient and last from several days to about 2 weeks (most resolve or significantly improve within 7–10 days, Lechien and others 2020a; Lee and others 2020; Printza and Constantinidis 2020; von Bartheld and others 2020).
One study reported that smell is lost slightly earlier (peak on day 3) than taste (peak on days 5–7, as illustrated in Fig. 2; Vaira and others 2020a). The timing and duration of the sensory deficits are so unique in COVID-19, they may give important clues about the potential underlying mechanism, as discussed below.
The rather distinct clinical features of the anosmia differ dramatically from the SARS pandemic in which only one single case of anosmia was reported (Hwang 2006), versuss literally millions of cases in COVID-19 (von Bartheld and others 2020). This is surprising, because the two viruses have considerable genomic identity of 79% to 82% (Wu and others 2020a). So what is it at the cellular and molecular level in COVID-19 that enables such a potent effect on the senses of smell and taste?
Four different principal scenarios have been considered to explain the smell dysfunction in COVID-19 patients, as illustrated in Figure 3A-E: (1) nasal obstruction/congestion and rhinorrhea, (2) loss of olfactory receptor neurons, (3) brain infiltration affecting olfactory centers, and (4) damage of support cells in the olfactory epithelium. We will evaluate for each scenario to what extent the proposed mechanism is consistent with, or supported by, the available data.
- Many viral infections cause nasal obstruction, congestion and rhinorrhea, thereby impeding odorant access to the sensory epithelium and preventing the binding of the odorants to olfactory receptors (Doty and Mishra 2001; Hummel and others 2017). This possibility of physical obstruction (conductive olfactory loss) was initially considered a likely explanation of the anosmia in COVID-19 (Eliezer and others 2020; Gane and others 2020; Qiu and others 2020), but has now been all but ruled out by several studies, primarily because a large fraction (nearly 60%, von Bartheld and others 2020) of patients with anosmia do not have nasal congestion, obstruction or rhinorrhea (Kaye and others 2020; Lechien and others 2020b; Printza and Constantinidis 2020; Tong and others 2020; Vaira and others 2020b; von Bartheld and others 2020; Xydakis and others 2020), and because these patients lack any significant mucosal swelling of the nasal cleft or sinuses on radiographic imaging (Naeini and others 2020).
- Does the virus infect olfactory receptor neurons, leading to their death? Such a sensorineural olfactory loss has been considered a plausible explanation of the anosmia (Baig and others 2020; Meinhardt and others 2020; Sia and others 2020; Ueha and others 2020; Wang and others 2020b). However, at closer look, there are three major inconsistencies with this scenario: the time course of cellular regeneration versus clinical recovery, the lack of expression of viral entry proteins, and the absence of the virus within olfactory neurons. When olfactory receptor neurons die, their replacement requires 8 to 10 days (Brann and Firestein 2014; Schwob 2002; Schwob and others 1995), plus about 5 days for cilia maturation (Liang 2020), but the time course of smell recovery in COVID-19 often is less than one week (Dell’Era and others 2020; Kaye and others 2020; Lee and others 2020; Printza and Constantinidis 2020; Sayin and Yazici 2020; Sedaghat and others 2020; Vaira and others 2020a; von Bartheld and others 2020). Thus, functional recovery after anosmia often is faster than the time it takes for neuron replacement, cilia maturation, and the growth of the new axons from the olfactory epithelium through the cribriform plate to form synapses in the olfactory bulb (Bryche and others 2020; Liang 2020; Schwob 2002; Soler and others 2020; Fig. 4). Regarding expression of the virus entry proteins, Butowt and Bilinska (2020) were the first who predicted, based on in-silico data, that mature olfactory receptor neurons do not express ACE2, and therefore are not likely to be infected by SARS-CoV-2. They were also the first to localize the virus entry proteins to distinct cell types in the olfactory epithelium (Bilinska et al. 2020; Table 2). There is now an emerging consensus that mature olfactory neurons do not express the virus entry proteins, ACE2 and TMPRSS2, at least not at significant levels, and not in the large majority of the mature olfactory neurons in mouse and human (Baxter and others 2020; Bilinska and others 2020; Brann and others 2020; Chen and others 2020b; Fodoulian and others 2020; Gupta and others 2020; Klingenstein and others 2020; Ziegler and others 2020—see Table 2). A recent study that localized the SARS-CoV-2 virus in the hamster olfactory epithelium confirmed this notion by showing that sustentacular cells contained the virus, but not olfactory neurons (Bryche and others 2020). This means that the olfactory neurons are not the initial and primary target of the virus. Taken together, these facts seem to rule out that many cases of anosmia in COVID-19 can be explained by direct virus-induced damage and death of olfactory receptor neurons, although death of the olfactory neurons is likely involved in prolonged cases of anosmia.
Chronology and Specifics of the Evidence for Expression of SARS-CoV-2 Entry Proteins ACE2 and TMPRSS2 in Identified Cell Types of the Olfactory Epithelium.
|Reference||Date of Publication||Species||Technique||ORN||SuC||ORN||SuC|
|Bilinska and others 2020||May 7, 2020||Mouse||ISH||+/−||+ (most)|
|Ziegler and others 2020||May 28, 2020||Mouse||RNAseq||−||+/− (<1%)||−||+|
|Brann and others 2020||July 28, 2020||Mouse||RNAseq||−||+/− (<3%)||−||+ (~50%)|
|Preprints—not yet peer reviewed|
|Gupta and others 2020||April 1, 2020||Human||RNAseq||−||+/− (<1%)|
|Fodoulian and others 2020||April 2, 2020||Human||RNAseq||−||+||−||+|
|Chen and others 2020b||May 9, 2020||Human||ICC||−||++ (most)|
|Baxter and others 2020||May 15, 2020||Mouse||RNAseq||−||+/−|
|Klingenstein and others 2020||July 15, 2020||Human||ICC||−||++ (most)||−||++ (most)|
- Does the virus infiltrate the brain, possibly from the nose, and affect olfactory centers (olfactory bulb and cortex), thereby reducing smell sensations? This scenario has been considered by several investigators (Aragão and others 2020; Baig and others 2020; Briguglio and others 2020; DosSantos and others 2020; Gilani and others 2020; Karimi-Galougahi and others 2020; Lechien and others 2020a; Li and others 2020b; Meinhardt and others 2020; Politi and others 2020; Sia and others 2020). The sudden loss of smell (and taste), followed by a rapid recovery, is a strong argument against this possibility, as is the fact that the olfactory neurons, which constitute one direct route to the brain by anterograde axonal transport, do not express the obligatory entry proteins for the virus (as detailed above). No study to date has shown that the olfactory receptor neurons or olfactory bulb neurons accumulate the virus acutely in normal (non-genetically modified) animals, at least not within the first 2 weeks after infection (Bryche and others 2020). Accordingly, the third scenario is highly unlikely to explain the often rapid and transient anosmia in COVID-19. There is currently no evidence that the SARS-CoV-2 virus itself can reach the brain through the olfactory route in the acute phase of anosmia; alterations of brain tissues by magnetic resonance imaging were not a consistent finding and may have been caused by virus-induced inflammation or by vascular/systemic routes (Aragão and others 2020; Cooper and others 2020; Politi and others 2020; Sedaghat and others 2020; Wang and others 2020b). The data from genetically modified mouse models, as discussed below, are inconsistent regarding brain infiltration (Bao and others 2020; Sun and others 2020a; Sun and others 2020b).
- Could the virus produce damage to the support cells in the olfactory epithelium and thereby diminish rapidly, but transiently, the sense of smell? This mechanism is supported by the abundant expression of the two entry proteins, ACE2 and TMPRSS2, in sustentacular cells in the olfactory epithelium (Figs. 4 and and5;5; Bilinska and others 2020; Brann and others 2020; Chen and others 2020b; Klingenstein and others 2020—Table 2), and by the presence of the virus primarily, if not exclusively, in the sustentacular cells (Bryche and others 2020; Meinhardt and others 2020). The initial reports of ACE2 expression in sustentacular cells based on RNAseq reported that only between 1% and 3% of these cells expressed ACE2 (Brann and others 2020; Ziegler and others 2020), while immunocytochemistry indicated that the large majority, if not all sustentacular cells contain ACE2 protein (Table 2). The most likely explanation for this discrepancy is that RNAseq is an inadequate technique for quantification and estimation of the extent of protein expression (Brann and others 2020). Interestingly, death of sustentacular cells does not seem to necessarily cause death of olfactory receptor neurons; the study by Bryche and others (2020) has shown that the neurons’ cilia (the dendritic extensions of the olfactory receptor neurons that bind the odorant molecules) can transiently retract or lose protein expression, implying temporary neuronal dysfunction despite persistence of olfactory nerve axons. Death and regeneration of sustentacular cells occurs much faster than death and regeneration of olfactory neurons (Bryche and others 2020; Jia and others 2010; Schwob 2002), which have to mature their dendrites and grow new axons through the cribriform plate into the olfactory bulb (Fig. 4). Therefore, rapid replenishment of sustentacular cells is consistent with the rapid recovery of the sense of smell that is clinically observed in most cases (Fig. 2). Is damage or inactivation of sustentacular cells in the olfactory epithelium sufficient to cause functional deficits of smell sensation and is it consistent with the time course and the peculiarities of the impairment reported by COVID-19 patients? The answer to these questions requires some background knowledge about the multitude of functions that sustentacular cells may perform in the olfactory epithelium.
Does the Coronavirus Cause Anosmia by Selectively Damaging Support Cells in the Olfactory Epithelium?
Sustentacular cells have been proposed to be involved in peripheral processing of odorants in multiple ways. They appear to endocytose (clear) the odorant-binding proteins after signal transduction at the neurons’ cilia to allow the next round of odorant receptor binding, thereby increasing sensitivity (Heydel and others 2013; Strotmann and Breer 2011). Sustentacular cells express multiple CYP450-family monooxygenases, which hydroxylate and help to remove toxic volatiles (Heydel and others 2013).
Sustentacular cells may supply neuronal cilia with some of the glucose required to meet the high energy demands of the olfactory transduction cascade (Cooper and others 2020; Villar and others 2017). Sustentacular cells also maintain the structural integrity of the olfactory epithelium (Bryche and others 2020; Jia and others 2010). Hence, these support cells are closely associated, both metabolically and functionally, with olfactory neurons and with odorant signal transduction (Fig. 4).
Recently, the SARS-CoV-2 virus was localized after nasal infection, and the time course of its effects on the olfactory system was determined (Bryche and others 2020). The virus localized exclusively to sustentacular cells and caused a massive degeneration of the olfactory epithelium and a widespread loss of the sustentacular cells, along with the olfactory cilia.
The rapid loss of the sustentacular cells was reminiscent to that seen when the olfactory epithelium was treated with nickel sulfate in neurotoxic concentrations—most of the olfactory axons remained intact, implying that many olfactory receptor neurons survived (Bryche and others 2020; Jia and others 2010). The cilia began to recover within 7 to 10 days after infection (Bryche and others 2020). This suggests that the odorants would fail to bind to their cognate odorant receptors until cilia are structurally and functionally restored (Liang 2020). Sustentacular cells appear to be essential for the maintenance and normal function of the cilia extending from the knobs (Fig. 4).
Accordingly, the coronavirus-induced anosmia or hyposmia may be a direct effect of the virus on the function of sustentacular cells, by reducing the odorant clearing function, or they may be indirect, by causing secondary metabolic or other dysfunction of the olfactory receptor neurons, since the sustentacular cells also serve to protect these neurons. Sustentacular cells regenerate after damage with a faster rate than olfactory receptor neurons (Schwob 2002; Schwob and others 1995; Fig. 4), which may explain why the COVID-19 anosmia is usually short lasting (Fig. 2).
It is tempting to speculate that a similar function of support cells exists in the taste buds, since the taste defects occur with a very similar time course as the olfactory defects (Lee and others 2020; Vaira and others 2020a; Fig. 2), but there are no studies yet available to support this hypothesis, and a recent study using RNAseq did not find significant ACE2 or TMPRSS2 expression in mouse taste buds (Wang and others 2020e). As an alternative or supplement to the virus-induced destruction of sustentacular cells, there may be consequences of immune cell infiltration from the basal lamina into the olfactory epithelium.
Immune cell infiltration by macrophages and lymphocytes has been shown for mammalian and human olfactory epithelium infected by SARS-CoV-2 (Bryche and others 2020; Meinhardt and others 2020), and this appears to be accompanied by a significant increase in the levels of the proinflammatory cytokine, tumor necrosis factor alpha (Torabi and others 2020). It has been suggested that inflammation-mediated loss of odorant receptor expression may contribute to the anosmia in COVID-19 (Rodriguez and others 2020; Torabi and others 2020; Yan and others 2020). The potential roles of inflammation in olfactory dysfunction was recently reviewed (Oliviero and others 2020; Rodriguez and others 2020).
It is curious that three recent studies reported contradictory findings about virus accumulation. Two studies claimed that the virus was present in some olfactory receptor neurons, in human and hamster (Meinhardt and others 2020; Sia and others 2020), while another study reported that, in hamster, the virus was present exclusively in sustentacular cells (Bryche and others 2020). How can this be reconciled?
The former studies did not identify cell types in the olfactory epithelium, they only visualized the virus, and their interpretation of virus being located in olfactory neurons is questionable: in the Meinhardt study, the authors apparently mis-identified obliquely sectioned sustentacular cells for olfactory neuron processes in their figure 4A (“knobs” are much too large), as also noted by Cooper and others (2020). Accordingly, the data presented by Meinhardt and others (2020) and Sia and others (2020) may be consistent with those of Bryche and others (2020), who employed double labeling with cell-type specific markers to unambiguously identify the virus-containing cell types in the olfactory epithelium.
Why do some COVID-19 patients have longer-lasting anosmia? While the large majority regain their sense of smell within 1 to 3 weeks, there are reports of some patients remaining anosmic or hyposmic for months or more. The most likely explanation is that in those cases, a larger area of the sensory epithelium was affected, possibly with a more profound destruction of the epithelium that included death of a larger number of olfactory receptor neurons. The extent of epithelial destruction varied in both the human and animal studies (Bryche and others 2020; Meinhardt and others 2020).
Taken together, it is most likely that the anosmia and hyposmia observed in COVID-19 patients is caused by viral entry, infection, and death of sustentacular cells, which does not necessarily lead to infection, damage, death, and the need for regeneration of olfactory receptor neurons. Therefore, the scenario (4), specific elimination of the function of sustentacular cells, is the most likely mechanism for the transient smell dysfunction in COVID-19. What would be needed for definitive proof of this hypothesis? Histological examination (biopsies) of human olfactory epithelium during progressive stages of COVID-19 infection, ideally by comparing biopsies from cases with anosmia and biopsies from cases without anosmia.
Is There a Route for SARS-CoV-2 from the Nose to the Brain?
The SARS-CoV-2 virus has been shown to be present in the brain parenchyma and cerebrospinal fluid in humans (Meinhardt and others 2020; Moriguchi and others 2020; Paniz-Mondolfi and others 2020; Wu and others 2020b) and in some of the animal models (Jiang and others 2020; Sia and others 2020; Sun and others 2020b, Table 3), but it is still unclear how the virus manages to get there. The possible routes include three main pathways: Neuronal, by moving along cranial nerves (nervus terminalis, olfactory, trigeminal, facial, glossopharyngeal, vagal); vascular/systemic, mediated via endothelial cells or leukocytes that cross the blood-brain barrier; and gaining access to cerebrospinal fluid-containing spaces; or a combination of some of these three pathways (Briguglio and others 2020; Dubé and others 2018; Li and others 2020b; Plakhov and others 1995; Zou and others 2020; Zubair and others 2020).
We will focus in this section on the potential routes through the cribriform plate. Many investigators have discussed the possibility that SARS-CoV viruses infect the brain through an olfactory route (Baig and others 2020; Butowt and Bilinska 2020; Gilani and others 2020; Li and others 2020b; McCray and others 2007; Meinhardt and others 2020; Netland and others 2008; Sia and others 2020; Ueha and others 2020; Zhou and others 2020; Zubair and others 2020).
Less often, it is remembered that a second cranial nerve enters the brain through the cribriform plate: the nervus terminalis. Intuitively, the olfactory and terminal nerves are a plausible pathway to the brain, because these neurons are the only cranial nerve neurons that have a peripheral dendritic process with direct access to the virus in the nasal cavity, and a central axon that reaches the brain, without any synaptic transfer through a pseudounipolar ganglion cell as in the other sensory systems. The four possible routes from the nose to the brain, through the cribriform plate, are illustrated in Figure 6A-D.
Genetically Modified Mouse Models Expressing Human ACE2.
|Short Name (Box 1)||Level of Expression||Promoter||Site of Integration||Technology||SARS-CoV in Brain||Reference|
|“Tseng mouse”||High overexpression||Artificial CAG||Random||Expression cassette|
|Not evaluated||Tseng and others 2007|
|High overexpression||Cytokeratin K18||Random||Expression cassette|
|Yes||McCray and others 2007;|
Netland and others 2008
|“Qin mouse”||Mild overexpression||Exogenous murine ACE2||Random||Expression cassette|
|No||Yang and others 2007;|
Bao and others 2020
|“Baric mouse”||High overexpression||FOXJ1||Random||Expression cassette|
|Yes (but only in deceased mice)||Menachery and others 2016;|
Jiang and others 2020
|“Sun mouse”||Physiological||Endogenous murine ACE2||Knock-in (ACE2 locus)||CRISPR/Cas9 elements microinjection||Yes||Sun and others 2020b|
|“Zhao mouse”||Transient overexpression||Viral CMV||No integration to host genome||Recombinant adenoviral vector transduction||No||Sun and others 2020a|
The Olfactory Nerve
It is now well established that most of the olfactory receptor neurons do not express the virus entry proteins, ACE2 and TMPRSS2 (Baxter and others 2020; Bilinska and others 2020; Brann and others 2020; Chen and others 2020b; Fodoulian and others 2020; Gupta and others 2020; Klingenstein and others 2020; Table 2), and consistent with the absence of the entry proteins, there is no convincing evidence that olfactory receptor neurons accumulate SARS-CoV-2, neither in animal models (Bryche and others 2020; Sia and others 2020) nor in humans (Meinhardt and others 2020).
But just because the olfactory receptor neurons do not express the two entry proteins for the virus, or only at very low levels (TMPRSS2), is it safe to assume that SARS-CoV viruses cannot utilize the olfactory route to the brain? Unfortunately, the answer is no. There is circumstantial evidence that SARS viruses can move beyond sustentacular cells and can reach the brain. One can consider several potential mechanisms that may allow such a transfer to happen. We know that the virus can and does readily enter sustentacular cells (Bryche and others 2020).
A key question is: how can the virus possibly transfer from sustentacular cells to either the olfactory neurons or to other cells or structures that allow it to gain access to the cerebrospinal fluid? Is close vicinity between cell types sufficient to transfer SARS-CoV-2 to a neighboring cell that is not synaptically connected (Fig. 4)? If this indeed happens, the virus may utilize an organelle exchange system (exosome pathway) between support cells and neurons, as has been shown to exist between donor and host cells in other systems (Sadeghipour and Mathias 2017).
Another mechanism has been proposed by DosSantos and others (2020), using the information that some stem cells in the olfactory epithelium express low levels of ACE2 (Krolewski and others 2013; Brann and others 2020; Durante and others 2020; Fodoulian and others 2020). It is—at least theoretically—possible that the virus may move from sustentacular cells to stem cells, which generate immature olfactory receptor neurons, and when these turn into mature olfactory receptor neurons (with axons extending into the olfactory bulb), they may transfer the virus directly to the olfactory bulb and beyond (Fig. 6A).
The Nervus Terminalis (Cranial Nerve “0”)
All mammals have a collection of neurons with cell bodies dispersed along the course of the olfactory nerve and olfactory bulb that are thought to have chemosensory and/or autonomic/endocrine functions, including regulation of mucous secretion in the nasal mucosa. They connect the nasal epithelium with brain centers caudal to the olfactory bulb—the medial forebrain (septum), preoptic area, and hypothalamus (Larsell 1950). These neurons are relatively sparse in humans (30–1500 cells); they are much larger in number in some marine mammals (10,000–20,000, Larsell 1950; Oelschläger and others 1987). Whether these cells express ACE2 or TMPRSS2 is currently not known, but ACE2 expression may be hypothesized, based on the presumed function of regulating blood flow in marine mammals (Oelschläger and others 1987). In mouse, the nervus terminalis cells are known to innervate not only blood vessels (including fenestrated capillaries)–thus resembling circumventricular organs—but some of the cells are also in direct contact with the subarachnoid space (Jennes 1987). These properties make the nervus terminalis a nearly ideal conduit for SARS-CoV-2 transmission to caudal brain centers, to the cerebrospinal fluid, and into the vascular system (Fig. 6B), especially if virus entry proteins are expressed. The targets of the nervus terminalis—including the hypothalamus—may also transfer the virus in the brain parenchyma via ACE2-expressing neurons (Nampoothiri and others 2020; Pal and Banerjee 2020). SARS-CoV-1 has been shown in humans and in an animal model to accumulate in the hypothalamus (Gu and others 2005; Netland and others 2008).
Cerebrospinal Fluid–Containing Spaces
Cerebrospinal fluid (CSF) drains through the cribriform plate into lymphatic vessels and this space is in immediate vicinity of, and between, the olfactory nerve fibers (Norwood and others 2019). Although the flow is primarily in the direction from the brain toward the nasal cavity, it is conceivable—and there is precedent—that some compounds can travel in the opposite direction (Lochhead and Thorne 2012). Substances that cross the nasal epithelium and reach the lamina propria may either absorb into the vasculature, or they may enter spaces between the perineural sheaths surrounding the olfactory nerve and thereby gain access to the CSF and the brain (Lochhead and Thorne 2012), as illustrated in Figure 6C.
The nasal passages are highly vascular. Substances that enter the blood vessels may cross the blood-brain barrier in circumventricular organs, or they may bypass the blood-brain barrier via direct nose-to-brain pathways to enter the brain as described above. The faster transport from the nasal epithelium to the olfactory bulb and brainstem is thought to be mediated by extracellular bulk flow within perivascular spaces of cerebral blood vessels rather than via intracellular transport along cranial nerves (Lochhead and Thorne 2012). Circumventricular organs, as illustrated in Figure 6D, may take up the virus from the vasculature through ACE2-expressing tanycytes (Nampoothiri and others 2020).
Insights From Animal Models
It is known from animal models that different viruses use different pathways and combinations of pathways to invade the brain from the periphery (Dubé and others 2018; Perlman and others 1990; Plakhov and others 1995). Some viruses transfer from neuron to neuron, using anterograde and retrograde axonal transport, which takes approximately one day for a directly connected neuron (Dubé and others 2018; Netland and others 2008), while other viruses can enter spaces containing cerebrospinal fluid, for example, in openings of the cribriform plate, from where they rapidly distribute throughout the ventricular spaces in the brain and infect neurons, including some that have no direct connections with the olfactory system (Netland and others 2008; Plakhov and others 1995).
When human ACE2 was overexpressed in a mouse model using the cytokeratin K18 promoter (see Box 1), the virus responsible for SARS, SARS-CoV-1, rapidly infected the brain after intranasal inoculation (McCray and others 2007; Netland and others 2008), and the mice died within less than a week from brain infection, apparently due to death of virus-infected neurons in the brainstem. Since cytokeratin K18 happens to be expressed in sustentacular cells, but not in olfactory receptor neurons (Schwob and others 1995), the SARS virus appears to have taken a route that originated with the support cells in this mouse model, enabled by the abundant (because overexpressed) ACE2 protein in these cells. Interestingly, the pathway of SARS-CoV-1 from the nose to the brainstem could not be explained solely by olfactory neuron-to-neuron transport, because in some cases, the olfactory bulb was spared (McCray and others 2007), and because transport was too fast, and rapidly reached neurons not connected to the olfactory system (Netland and others 2008).
Taken the above into account, we expand the hypothesis proposed by Li and others (2020b): Since sustentacular cells extend the entire thickness of the olfactory epithelium, SARS-CoV-2 can gain access to the lamina propria, and may be extruded into the cerebrospinal fluid-containing spaces within the cribriform plate, and then spread rapidly throughout the ventricular system, infecting initially cell types that are close to the ventricular ependyma (such as dorsal raphe in the brainstem, neurons in the hypothalamus and basal ganglia), but rarely reach brain parts that are remote from the ventricular system (e.g., the cerebellum). This hypothesis is consistent with the findings of both, human studies (Meinhardt and others 2020; Nampoothiri and others 2020) as well as animal models examining the neurotropism of viruses, including coronaviruses (Perlman and others 1990; Netland and others 2008).
Synthesis from Animal and Human Studies
A recent study has reported how frequently brain regions contained SARS-CoV-2 virus in COVID-19 patients (Meinhardt and others 2020). Although this study did not provide the timeline after infection, only the endpoint, it is interesting to compare with the brain nuclei that contained SARS-CoV-1 virus in the animal model (McCray and others 2007; Netland and others 2008). Of particular interest is that in humans, even though the olfactory mucosa had the highest viral load in nearly all cases that were examined (Meinhardt and others 2020), the medulla oblongata was more often positive for the virus than the olfactory bulb, which speaks against a neuron-to-neuron transfer of the virus along the olfactory nerve, and is more consistent with a spread through the cerebrospinal fluid compartment, as indicated also by the animal model (McCray and others 2007; Netland and others 2008).
The study on this mouse model has also provided some information on the timing and sequence in which the virus appears in the brain after intranasal infection (Netland and others 2008). Interestingly, the arrival of the virus in the olfactory bulb did not precede other sites, as one would expect if it was transferred from olfactory receptor neurons to mitral cells and then to second- and third-order targets of the olfactory bulb, but rather appeared simultaneously in the olfactory bulb, raphe neurons in the medulla and in neurons of the hypothalamus and basal ganglia (Netland and others 2008), suggesting that the neuron-to-neuron transport was not the only route in the brain.
Similarly, in humans with COVID-19 and high viral loads in the olfactory epithelium, more cases with significant amounts of detectable virus actually involved the medulla than the olfactory bulb, and they were equal between the olfactory bulb and trigeminal ganglion (Meinhardt and others 2020). Taken together with the mouse time course studies, the currently available data suggest that there probably is a transfer of the virus from the olfactory epithelium through the cribriform plate to the brain, but in addition to being anterogradely transported along axons and transferred to second-order neurons in the olfactory bulb, the virus also appears to utilize another route, likely cerebrospinal fluid spaces which penetrate the cribriform plate along with the olfactory nerve fibers, and by entering channels formed by olfactory ensheathing cells (Butowt and Bilinska 2020; Li and others 2020b; Norwood and others 2019; van Riel and others 2015), or by using nervus terminalis cells as conduits (Fig. 6B).
Accessing the cerebrospinal fluid would allow the virus to rapidly distribute throughout the ventricular system, reaching first nuclei that have periventriclar locations, such as raphe and hypothalamus—which is where Netland and others (2008) describe heavy accumulation coincident with infection of the olfactory bulb. Animal models with physiological levels of human ACE2 expression will be valuable tools to determine the precise pathway of SARS-CoV-2 to brain infection (Box 1).
Is there ACE2-independent virus entry and transfer? For the interpretation of data obtained in the study mentioned above, it must also be considered that the novel coronavirus may utilize an ACE2-independent route to transfer from sustentacular cells to olfactory receptor neurons. SARS-CoV-1 and other coronaviruses may use additional lower-affinity co-receptors besides the main high-affinity receptor. For example, it was shown that SARS-CoV-1, in addition to ACE2, may use CD209 glycoproteins as alternative host receptors (Jeffers and others 2004). SARS-CoV-2 can utilize CD147 to enter some cell types (Wang and others 2020a). Although olfactory receptor neurons do not express any or very little ACE2, they do express CD147 as shown by multiple microarray and RNAseq studies (Krolewski and others 2013; Nickell and others 2012; Saraiva and others 2015). Therefore, it is possible that some viral particles pass from sustentacular cells to olfactory neurons by using a CD147-dependent mechanism. Alternatively, the virus may utilize an exosome pathway that is known to allow viruses to transfer between cells (Sadeghipour and Mathias 2017). Furthermore, it is possible that SARS-CoV-2 itself upregulates ACE2 in host tissues (Nampoothiri and others 2020; Ziegler and others 2020)—which adds another level of complexity in identifying relevant cell types and potential routes of infection.
Potential Consequences of Viral Brain Infection for Neurodegenerative Diseases
Once a virus has entered the brain, it can persist there for many years, and such long-term presence may lead to inflammation that is thought to play a role in chronic neurological diseases (Desforges and others 2019; Dubé and others 2018). These are additional reasons why it is important to better understand whether and how the novel SARS-CoV-2 virus may utilize a route through the cribriform plate to the brain. COVID-19 patients can present with a variety of neurological symptoms (Mao and others 2020; Wang and others 2020b). The long-term presence of the virus in the brain may lead to inflammation and perhaps initiate or aggravate chronic neurological diseases such as multiple sclerosis and Parkinson’s disease (Desforges and others 2019; Dubé and others 2018; Serrano-Castro and others 2020). The pathway from the nose to the brain must be considered among other potential routes of SARS-CoV-2 from the periphery to the brain (Baig and others 2020; Butowt and Bilinska 2020; DosSantos and others 2020; Li and others 2020b). Given that the olfactory epithelium has such intense expression of the entry proteins for the SARS-CoV-2 virus—the highest expression level in the nasal cavity—and is located on the main route of infection through aerial spread, neurologists need to be vigilant to the possibility of brain infection by SARS-CoV-2 using a route from the nose to the brain.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7488171/#bibr126-1073858420956905
More information: Eric Song et al. Neuroinvasion of SARS-CoV-2 in human and mouse brain, Journal of Experimental Medicine (2021). DOI: 10.1084/jem.20202135
David H. Brann et al. Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-associated anosmia, Science Advances (2020). DOI: 10.1126/sciadv.abc5801