“SARS-CoV-2 can persist within the olfactory epithelium”
SARS-CoV-2 can persist within the olfactory epithelium, not only for several weeks following Covid-19, but also for months after symptoms disappear, says a French study posted on November 18, 2020 on the bioRxiv prepublication site. The data from this work also seem to indicate that the coronavirus could enter the central nervous system by traveling, retrograde, from the olfactory nerve pathways.
Obtained in Covid-19 patients and on the golden Syrian hamster, an animal model of coronavirus infection, these new data provide a mechanism that may explain the persistence or relapse of the total or partial loss of smell (anosmia) in of Covid-19 patients. These results also raise new questions about the possibility that SARS-CoV-2 can reach the central nervous system (brain, brain stem) by the retrograde route, therefore moving up the nervous threads of the olfactory system.
The presence of the virus was confirmed after examination of the cells of the olfactory epithelium. The sample consisted, using a cytological brush and under endoscopic guidance, in carrying out rotational and translational movements at the level of the conchae of the nasal cavities, most often the inferior concha, in order to bring back a sample rich in cells. Remember that the conchae are small bone lamellae located in the lateral wall of the nasal cavity.
Nasal brushing performed in patients with a recent loss of smell, as well as in people with complaints of prolonged or relapsed anosmia, has observed the presence of SARS -CoV-2 in the sample. Even though this same virus was not detected by RT-PCR technique on nasopharyngeal swabs. This shows that the diagnosis of infection of the nasal mucosa by SARS-CoV-2 via nasal brushing is more efficient than that carried out after the now classic nasopharyngeal swab by simple swab, at least in patients with loss of smell.
It all started when the team of Hervé Bourhy, Marc Lecuit and Pierre-Marie Lledo from the “Lyssavirus, epidemiology and neuropathology”, “Biology of infections” and “Perception and memory” laboratories at the Institut Pasteur (Paris) recruited for their study. five patients who came to consult in an ENT department at Lariboisière hospital (Paris) for a loss of smell and a suspicion of Covid-19 in the context of the first wave.
The clinical condition of these patients does not require hospitalization. With the exception of one patient who complained of a progressive loss of smell, the anosmia of the other four was sudden onset. This prominent symptom is accompanied by changes in taste, except for one of them. Depending on the case, other symptoms are present, such as diarrhea, cough, difficulty in breathing, conjunctivitis, fever, fatigue, headache, muscle pain, laryngitis or simple sore throat. In one of the patients, loss of smell was the first symptom to appear. The anosmia was severe in four of the patients, moderate in another. In these four anosmic patients, the loss of taste (ageusia) is also severe. It is moderate for the other patient.
Four patients have a runny nose (rhinorrhea). Two complained of nasal irritation. Another has hyperacusis (hypersensitivity to sounds). No patient has a stuffy nose. Four patients also presented a taste disorder (dysgeusia) with, depending on the case, less acuteness for sweetness, bad taste in the mouth, reduced or exaggerated acuity for bitter, reduced sensation for salty or ‘acid. In two patients, the dysgeusia was such that they could not tell the difference between meat and fish.
Eager to find out whether the loss of smell is linked to infection of the olfactory lining, the researchers decided to remove cells from these five patients by nasal brushing. Only two of them tested positive for SARS-CoV-2 by RT-PCR after nasopharyngeal swabbing. Virologists will then detect viral RNA in the cell sample from the olfactory mucosa, which certifies the presence of an infection with SARS-CoV-2. One patient has a high viral load in the olfactory mucosa (2.25 million copies / microliter) while it is too low to be quantifiable in others.
The researchers then set out to search, by immunofluorescence, for the presence of the virus in the cell sample obtained by nasal brushing in search of viral proteins. The labeling is positive in three patients: it attests to the presence of a SARS-CoV-2 antigen (NP nucleoprotein, present inside the viral particles) in mature sensory neurons. These data show that SARS-CoV-2 infection is associated with inflammation of the olfactory mucosa in patients with a smell disorder.
The scientists then decided to assess the profile of inflammation mediators, namely locally produced cytokines and other inflammatory mediators. It appears that the pro-inflammatory cytokine interleukin 6 (IL-6) is abundantly produced in three patients carrying SARS-CoV-2 antigens.
At this stage, it becomes clear that the SARS-CoV-2 coronavirus has a particular tropism for the olfactory epithelium and that the resulting infection is associated with significant local inflammation.
“SARS-CoV-2 coronavirus has a particular tropism for the olfactory epithelium”
Infection of olfactory sensory neurons and their supporting cells
But what exactly are the target cells of the coronavirus at this level? Researchers will determine that many cell types of the olfactory neuroepithelium (located in the roof of the nasal cavities and involved in the perception of odor molecules) are infected during acute infection with SARS-CoV-2 when the loss occurs. of smell.
Researchers have indeed detected that mature olfactory sensory neurons are not the only cells infected by the coronavirus. Supporting cells for sensory nerve cells (sustentacular cells) and immune cells (myeloid cells) are also. Ditto for still immature sensory neurons of the nasal mucosa. Under the microscope, some of these infected neurons are about to be phagocytosed by cells belonging to the immune system’s first line of defense (innate immunity).
The hamster, a model of SARS-CoV-2 infection
The researchers then decided to turn to a natural animal model of SARS-CoV-2 infection, in this case the golden Syrian hamster (Mesocricetus auratus) which expresses an ACE2 receptor capable of interacting with SARS-CoV -2. It will be recalled that the S protein (Spike, spicule in French) of the SARS-CoV-2 envelope is used by virions to bind to the ACE2 receptor present on the target cells.
These rodents will be inoculated intranasally with SARS-Cov-2 and followed for several days. Infected hamsters will present two to four days after high viral loads in the respiratory tract, especially in the turbinates. The viral load remains detectable for the next 14 days.
Viral RNA in the brains of infected hamsters
The researchers report a surprising result: they detected the presence, two days after the intranasal inoculation of SARS-CoV-2, of SARS-CoV-2 RNA in several regions of the hamster brain, in this case in the olfactory bulb (ovoid structure, located at the base of the brain), but also in regions of the brain far from the nasal cavities, in this case in the cerebral cortex, the brainstem (diencephalon, midbrain, bridge, elongated medulla) and the cerebellum, but without clearly visualizing viral antigens. In other words, the researchers quantified by PCR the genetic material of the virus (RNA) in hamster brains but still detected viral proteins by immunofluorescence.
Anosmia and ageusia in infected hamsters
Hamsters infected with SARS-Cov-2 also exhibit taste disturbances. They no longer show a strong preference for sugary water and take longer to smell hidden foods.
But what about the cellular damage caused by the upper and lower respiratory airways in these hamsters experimentally infected intranasally with SARS-Cov-2? In these rodents, the researchers will more particularly study the highest part of the turbinates, that covered by the olfactory neuroepithelium made up of hair cells, namely the sensory neurons and their support cells (sustentacular).
It appears that in large portions of the olfactory epithelium, these two categories of cells lose their eyelashes two days after inoculation with the coronavirus, as evidenced by the images obtained by electron microscopy. On the fourth day post-inoculation, it is observed that viral particles bud on the surface of these infected cells.
At the same time, within the olfactory mucosa, the researchers detected the presence of viral antigens in the cytoplasm of mature and immature sensory neurons, as well as in immune cells infiltrating the neuroepithelium. The latter, drawn to the site of the viral infection, were themselves infected after having phagocytosed contaminated olfactory cells. In addition, regions of the olfactory neuroepithelium containing infected cells appear architecturally disorganized, unlike adjacent areas free from infection which, in turn, retain their cellular arrangement in several layers (stratified appearance).
These cells of the olfactory mucosa regained their ciliated appearance 14 days after infection, at the same time as the hamsters regained their sense of smell. These results therefore show that anosmia is the consequence of sensory impairment associated with viral infection and not due to simple nasal obstruction or tissue inflammation.
Above all, the researchers found the presence of SARS-CoV-2 in the olfactory nerve threads located near the olfactory neuroepithelium. They thus detected viral antigens in the axons of olfactory sensory neurons that reach the olfactory bulbs.
This work shows that intranasal inoculation of SARS-CoV-2 in hamsters results in infection of olfactory sensory neurons and anosmia, mimicking what is histologically and clinically observed in Covid-19 patients.
Retrograde axonal transport
The SARS-CoV-2 nucleoprotein (nucleocapsid protein) was detected at the junction between the olfactory bulb and the olfactory nerve, which tends to show that the virus travels in a retrograde manner, in other words, it can “go up” along the axons. This internal coronavirus protein has also been detected in immune cells infiltrating the olfactory bulb.
According to the authors, “the observation of the presence of viral antigens all along the route which leads from the sensory olfactory organ [the olfactory neuroepithelium of the turbinates of the nasal cavity] to the olfactory bulb, shows that SARS-CoV -2 enters the central nervous system through the olfactory system ‘in the infected hamster.
Neuroinvasion and neuroinflammation in infected hamsters
SARS-CoV-2, therefore, induces in the olfactory bulbs of infected rodents, an intense inflammatory reaction (over expression of genes involved in inflammation) as well as a strong local antiviral response (production of molecules associated with innate immunity and adaptive, activation of immune cells).
The researchers used a sophisticated molecular biology tool: “single-cell messenger RNA sequencing” or Single-cell RNA-sequencing (ScRNA-seq), which makes it possible to detect which genes are particularly active in each of the cells of the tissue or of the organ studied. Since the expression of a gene involves the DNA being converted into messenger RNA (mRNA), the presence of these mRNA molecules provides information on how genes work. This tool confirmed the activity of genes involved in inflammation within the olfactory bulb as well as the activation of non-neuronal cells (astrocytes, microglial cells).
According to the researchers, this result, coupled with the presence of viral proteins in the olfactory bulbs which are emanations from the brain, supports “the hypothesis that neuroinvasion by SARS-CoV-2 sustains neuroinflammation.” In other words, if there is inflammation, it is a sign that the virus has successfully entered the central nervous system.
And the researchers said that increased production of inflammatory molecules (cytokines and chemokines) persisted until day 14 after infection in these hamsters, even though they had recovered from their anosmia and ageusia. This shows that, in these rodents experimentally infected with SARS-CoV-2, an inflammatory process can occur during symptomatic infection and persist during the asymptomatic post-infectious period.
Prolonged / recurrent anosmia in Covid-19 patients
In view of these data obtained in this animal model, what about a possible persistence of SARS-CoV-2 in Covid-19 patients complaining of persistent neurological symptoms and / or sensory disturbances, even three months after the onset of symptoms? This is the important question that clinicians then turned to.
To do this, the infectious diseases department of Hôtel-Dieu (Paris) recruited, as part of a Covid-long consultation led by Prof. Dominique Salmon, four new Covid-19 patients who presented either a loss of prolonged sense of smell or relapse of their anosmia. These people were included in the study between June 15 and 29, 2020, at a time when the incidence of SARS-CoV-2 was then less than 10 per 100,000 inhabitants in the capital. It is therefore unlikely that these people were re-infected with the coronavirus at that time.
These four new patients, who were not hospitalized, complained of an uninterrupted loss of smell (up to six months after the onset of symptoms) as well as persistent ageusia in three of them. They also had other neurological disorders (itchy nose, left intercostal pain and left arm pain, burning sensations, dizziness, among others). Two of these patients had a bad taste in their mouth. Two other patients had a reduced or exaggerated feeling for bitterness, reduced taste acuity for salty or sour.
Negative on nasal swab but positive on nasal brushing
None of these four patients complaining of prolonged or recurrent anosmia was then found positive on PCR on nasopharyngeal swab. However, in each of them, the examination of the olfactory mucous membrane cells collected after nasal brushing revealed the presence of SARS-CoV-2 RNA. The detection of viral RNA is based on a special PCR technique called RT-qPCR.
According to the researchers, the presence of SARS-CoV-2 RNA and proteins in the olfactory neuroepithelium of the nasal cavity (although the infectivity of the virus could not be assessed by cell culture in the laboratory) could mean that some patients, especially those with mild or non-mild symptoms, could be involved in virus transmission when considered virus-free based on a negative nasal swab PCR test result.
Of the four patients studied, three carried a high viral load in the olfactory mucosa (between 0.1 and 0.4 million copies of viral RNA per microliter). Likewise, the presence in the olfactory epithelium of the SARS-CoV-2 nucleoprotein was detected by immunofluorescence in three out of four patients.
Finally, numerous immune cells were observed in the olfactory mucosa in each of these patients. Elevated levels of interleukin-6 (IL-6) were recorded in these patients with prolonged or persistent anosmia, like the first patients who experienced loss of smell during acute viral infection and this which had been observed in hamsters experimentally infected with SARS-CoV-2.
“the olfactory neuroepithelium of patients with persistent loss of smell remains inflammatory and infected, with persistence of SARS-CoV-2 RNA in each of them,”
Neuroinvasive potential of SARS-CoV-2
This work is reminiscent of the fact that viral RNA or SARS-CoV-2 proteins have already been detected in the brain and/or cerebrospinal fluid, at the autopsy of patients who died of Covid-19, by German researchers in June 2020 in an article posted on bioXriv (preprint) as well as by an American team in August in the New England Journal of Medicine.
The question of the neuroinvasive potential of SARS-CoV-2 is therefore once again raised, even if other physiopathological mechanisms have already been mentioned: transport by the retrograde route via olfactory sensory neurons and/or via the vagus nerve into the central nervous system, damage to the blood-brain barrier at the interface between blood and brain, intracerebral infiltration of infected immune cells (T lymphocytes and/or peripheral macrophages).
This new study therefore revives the debate on retrograde transport of SARS-CoV-2 to the central nervous system from the olfactory nerve pathways in humans. Above all, it shows that a persistent smell disorder for several months in Covid-19 patients could reflect the prolonged presence of the virus in the olfactory neuroepithelium, and therefore a persistent viral infection of this particular micro environment.
No doubt studies of a larger number of Covid-19 patients with persistent anosmia, or even other recurrent neurological symptoms, should follow.
Dias De Melo G, Lazarini F, Levallois S, et al. COVID-19-associated olfactory dysfunction reveals SARS-CoV-2 neuroinvasion and persistence in the olfactory system. bioRxiv. Posted November 18, 2020. doi:10.1101/2020.11.18.388819