Inflammatory Response and Defects on Myelin Integrity in the Olfactory System of K18hACE2 Mice Infected with SARS-CoV-2.

Viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use respiratory epithelial cells as an entry point for infection. Within the nasal cavity, the olfactory epithelium (OE) is particularly sensitive to infections which may lead to olfactory dysfunction. In patients suffering from coronavirus disease 2019, deficits in olfaction have been characterized as a distinctive symptom. Here, we used the K18hACE2 mice to study the spread of SARS-CoV-2 infection and inflammation in the olfactory system (OS) after 7 d of infection. In the OE, we found that SARS-CoV-2 selectively targeted the supporting/sustentacular cells (SCs) and macrophages from the lamina propria. In the brain, SARS-CoV-2 infected some microglial cells in the olfactory bulb (OB), and there was a widespread infection of projection neurons in the OB, piriform cortex (PC), and tubular striatum (TuS). Inflammation, indicated by both elevated numbers and morphologically activated IBA1+ cells (monocyte/macrophage lineages), was preferentially increased in the OE septum, while it was homogeneously distributed throughout the layers of the OB, PC, and TuS. Myelinated OS axonal tracts, the lateral olfactory tract, and the anterior commissure, exhibited decreased levels of 2',3'-cyclic-nucleotide 3'-phosphodiesterase, indicative of myelin defects. Collectively, our work supports the hypothesis that SARS-CoV-2 infected SC and macrophages in the OE and, centrally, microglia and subpopulations of OS neurons. The observed inflammation throughout the OS areas and central myelin defects may account for the long-lasting olfactory deficit.

Visualizing in deceased COVID-19 patients how SARS-CoV-2 attacks the respiratory and olfactory mucosae but spares the olfactory bulb.

Anosmia, the loss of smell, is a common and often the sole symptom of COVID-19. The onset of the sequence of pathobiological events leading to olfactory dysfunction remains obscure. Here, we have developed a postmortem bedside surgical procedure to harvest endoscopically samples of respiratory and olfactory mucosae and whole olfactory bulbs. Our cohort of 85 cases included COVID-19 patients who died a few days after infection with SARS-CoV-2, enabling us to catch the virus while it was still replicating. We found that sustentacular cells are the major target cell type in the olfactory mucosa. We failed to find evidence for infection of olfactory sensory neurons, and the parenchyma of the olfactory bulb is spared as well. Thus, SARS-CoV-2 does not appear to be a neurotropic virus. We postulate that transient insufficient support from sustentacular cells triggers transient olfactory dysfunction in COVID-19. Olfactory sensory neurons would become affected without getting infected.

Olfactory epithelium histopathological findings in long-term coronavirus disease 2019 related anosmia.

BACKGROUND: Olfactory dysfunction represents one of the most frequent symptoms of coronavirus disease 2019, affecting about 70 per cent of patients. However, the pathogenesis of the olfactory dysfunction in coronavirus disease 2019 has not yet been elucidated. CASE REPORT: This report presents the radiological and histopathological findings of a patient who presented with anosmia persisting for more than three months after infection with severe acute respiratory syndrome coronavirus-2. CONCLUSION: The biopsy demonstrated significant disruption of the olfactory epithelium. This shifts the focus away from invasion of the olfactory bulb and encourages further studies of treatments targeted at the surface epithelium.

Neurological Insights of COVID-19 Pandemic.

The novel coronavirus SARS-CoV-2, which was identified after a recent outbreak in Wuhan, China, in December 2019, has kept the whole world in tenterhooks due to its severe life-threatening nature of the infection. The virus is unlike its previous counterparts, SARS-CoV and MERS-CoV, or anything the world has encountered before both in terms of virulence and severity of the infection. If scientific reports relevant to the SARS-CoV-2 virus are noted, it can be seen that the virus owes much of its killer properties to its unique structure that has a stronger binding affinity with the human angiotensin-converting enzyme 2 (hACE2) protein, which the viruses utilize as an entry point to gain accesses to its hosts. Recent reports suggest that it is not just the lung that the virus may be targeting; the human brain may soon emerge as the new abode of the virus. Already instances of patients with COVID-19 have been reported with mild (anosmia and ageusia) to severe (encephalopathy) neurological manifestations, and if that is so, then it gives us more reasons to be frightened of this killer virus. Keeping in mind that the situation does not worsen from here, immediate awareness and more thorough research regarding the neuroinvasive nature of the virus is the immediate need of the hour. Scientists globally also need to up their game to design more specific therapeutic strategies with the available information to counteract the pandemic. In this Viewpoint, we provide a brief outline of the currently known neurological manifestations of COVID-19 and discuss some probable ways to design therapeutic strategies to overcome the present global crisis.

SARS-CoV-2: Olfaction, Brain Infection, and the Urgent Need for Clinical Samples Allowing Earlier Virus Detection.

The novel SARS-CoV-2 virus has very high infectivity, which allows it to spread rapidly around the world. Attempts at slowing the pandemic at this stage depend on the number and quality of diagnostic tests performed. We propose that the olfactory epithelium from the nasal cavity may be a more appropriate tissue for detection of SARS-CoV-2 virus at the earliest stages, prior to onset of symptoms or even in asymptomatic people, as compared to commonly used sputum or nasopharyngeal swabs. Here we emphasize that the nasal cavity olfactory epithelium is the likely site of enhanced binding of SARS-CoV-2. Multiple non-neuronal cell types present in the olfactory epithelium express two host receptors, ACE2 and TMPRSS2 proteases, that facilitate SARS-CoV-2 binding, replication, and accumulation. This may be the underlying mechanism for the recently reported cases of smell dysfunction in patients with COVID-19. Moreover, the possibility of subsequent brain infection should be considered which begins in olfactory neurons. In addition, we discuss the possibility that olfactory receptor neurons may initiate rapid immune responses at early stages of the disease. We emphasize the need to undertake research focused on additional aspects of SARS-CoV-2 actions in the nervous system, especially in the olfactory pathway.

Type I Interferon Signaling to Dendritic Cells Limits Murid Herpesvirus 4 Spread from the Olfactory Epithelium.

Murid herpesvirus 4 (MuHV-4) is a B cell-tropic gammaherpesvirus that can be studied in vivo Despite viral evasion, type I interferons (IFN-I) limit its spread. After MuHV-4 inoculation into footpads, IFN-I protect lymph node subcapsular sinus macrophages (SSM) against productive infection; after peritoneal inoculation, they protect splenic marginal zone macrophages, and they limit MuHV-4 replication in the lungs. While invasive infections can be used to test specific aspects of host colonization, it is also important to understand natural infection. MuHV-4 taken up spontaneously by alert mice enters them via olfactory neurons. We determined how IFN-I act in this context. Blocking IFN-I signaling did not increase neuronal infection but allowed the virus to spread to the adjacent respiratory epithelium. In lymph nodes, a complete IFN-I signaling block increased MuHV-4 lytic infection in SSM and increased the number of dendritic cells (DC) expressing viral green fluorescent protein (GFP) independently of lytic infection. A CD11c+ cell-directed signaling block increased infection of DC only. However, this was sufficient to increase downstream infection, consistent with DC providing the main viral route to B cells. The capacity of IFN-I to limit DC infection indicated that viral IFN-I evasion was only partly effective. Therefore, DC are a possible target for IFN-I-based interventions to reduce host colonization.IMPORTANCE Human gammaherpesviruses infect B cells and cause B cell cancers. Interventions to block virus binding to B cells have not stopped their infection. Therefore, we must identify other control points that are relevant to natural infection. Human infections are difficult to analyze. However, gammaherpesviruses colonize all mammals. A related gammaherpesvirus of mice reaches B cells not directly but via infected dendritic cells. We show that type I interferons, an important general antiviral defense, limit gammaherpesvirus B cell infection by acting on dendritic cells. Therefore, dendritic cell infection is a potential point of interferon-based therapeutic intervention.

Host entry by gamma-herpesviruses--lessons from animal viruses?

The oncogenicity of gamma-herpesviruses (γHVs) motivates efforts to control them and their persistence makes early events key targets for intervention. Human γHVs are often assumed to enter naive hosts orally and infect B cells directly. However, neither assumption is supported by direct evidence, and vaccination with the Epstein-Barr virus (EBV) gp350, to block virion binding to B cells, failed to reduce infection rates. Thus, there is a need to re-evaluate assumptions about γHV host entry. Given the difficulty of analysing early human infections, potentially much can be learned from animal models. Genomic comparisons argue that γHVs colonized mammals long before humans speciation, and so that human γHVs are unlikely to differ dramatically in behaviour from those of other mammals. Murid Herpesvirus-4 (MuHV-4), which like EBV and the Kaposi's Sarcoma-associated Herpesvirus (KSHV) persists in memory B cells, enters new hosts via olfactory neurons and exploits myeloid cells to spread. Integrating these data with existing knowledge of human and veterinary γHVs suggests a new model of host entry, with potentially important implications for infection control.

Air-assisted intranasal instillation enhances adenoviral delivery to the olfactory epithelium and respiratory tract.

Intranasal instillation is used to deliver adenoviral vectors to the olfactory epithelium and respiratory tract. The success of this approach, however, has been tempered by inconsistent infectivity in both the epithelium and lungs. Infection of the epithelium may be hampered in part by the convoluted structure of the cavity, the presence of mucus or poor airflow in the posterior cavity. Delivery of adenovirus to the lungs can be uneven in the various lobes and distal bronchioles may be poorly infected. Current approaches to circumvent these issues rely principally on intubation or intratracheal instillation. Here we describe a technique that significantly improves adenoviral infectivity rates without requiring surgical intervention. We use compressed air to increase circulation of instilled adenovirus, resulting in enhanced infection in both the epithelium and lungs. This procedure is straightforward, simple to perform and requires no specialized equipment. In the epithelium, neurons and sustentacular cells are both labeled. In the lungs, all lobes can be infected, with penetration to the most distal bronchioles. The use of compressed air will likely also be useful for enhancing the distribution of other, desired agents within the epithelium, central nervous system and respiratory tract.

Detection of parainfluenza virus 3 in turbinate epithelial cells of postviral olfactory dysfunction patients.

OBJECTIVES/HYPOTHESIS: Postviral olfactory dysfunction (PVOD) develops after a common cold, but little is known about the viral pathogen inducing olfactory dysfunction. We hypothesized that human parainfluenza virus 3 (PIV3) may cause PVOD. We therefore assayed the nasal cavity mucosae of PVOD patients for the presence or persistence of PIV3. METHODS: We assessed 25 patients (5 men, 20 women), ranging in age from 31 to 85 (mean, 51) years, diagnosed with PVOD and 22 controls (18 men, 4 women) diagnosed with nasal septal deviation between July 2005 and August 2006. Inferior turbinate epithelial cells were collected using a Rhino-probe mucosal curette, and PIV3 was assayed by seminested reverse-transcription polymerase chain reaction. RESULTS: PVOD occurred most frequently between May and July. Hyposmia was observed in 60% of patients and anosmia in 40%. The most common clinical symptoms were rhinorrhea, sore throat, nasal obstruction, fever, myalgia, cough, and hoarseness. Patients usually visited the outpatient clinic within 3 months after the onset of olfactory dysfunction. Twenty-two of 25 (88.0%) epithelial samples from PVOD patients were positive for PIV3 compared with 2 of 22 (9.1%) epithelial samples from controls. CONCLUSIONS: The high detection rate of PIV3 in the turbinate epithelial cells of PVOD patients suggests that PIV3 may be the causative virus of PVOD.

Efficient adenoviral vector-directed expression of a foreign gene to neurons and sustentacular cells in the mouse olfactory neuroepithelium.

Replication deficient recombinant adenoviral vectors are efficient gene transfer agents for postmitotic cells, including neurons and glial cells. In this paper we have examined the effectiveness of adenoviral vector-mediated gene transfer to the olfactory epithelium of adult mice. We show that Ad-LacZ, a prototype first generation adenoviral vector containing an expression cassette for the reporter gene LacZ, directs transgene expression to mature and immature olfactory neurons and to sustentacular cells. The technique to apply the vector to the nasal cavity and the amount of viral vector per mouse are important variables that determine the success of viral vector-mediated gene transfer to the mouse olfactory neuroepithelium. Slow infusion of the viral vector solution in fully anaesthetized mice yields the best result in terms of the number of epithelial cells transduced. Infection of the olfactory neuroepithelium with a moderate amount of viral vector (10(9) plaque-forming units (PFU)) results in transgene expression in many cells throughout the epithelium for 8-12 days, followed by a decline in transduced cells at 25 days postinstillation of the virus This decrement in transgene expression is consistent with the natural turnover process that occurs in the epithelium throughout adulthood. At high viral loads (1.3 x 10(10) PFU) extinction of transgene expression occurs as early as 8 days postinjection and is accompanied by epithelial degeneration indicating that the vector dose used should be carefully chosen. Taken together, the current observations demonstrate that adenoviral vectors are effective tools to genetically modify the adult mouse olfactory neuroepithelium in vivo.

Morphologic changes in the nasal cavity associated with sialodacryoadenitis virus infection in the Wistar rat.

A sequential study of lesions of the nasal cavity associated with sialodacryoadenitis virus (SDAV) infection was made in the laboratory rat. Wistar rats were intranasally inoculated with approximately 10(3) TCID50 of the coronavirus SDAV. Transverse sections of four regions of the nasal cavity from inoculated and control animals were examined by light microscopy and immunohistochemistry at 2, 4, 6, 8, 10, and 14 days postinoculation (PI). Lesions were observed in the following regions of the upper respiratory tract: respiratory epithelium, transitional epithelium, olfactory epithelium, nasolacrimal duct, vomeronasal organ, and the submucosal glands of the nasal passages. In general, in structures lined by ciliated epithelial cells, there was focal to segmental necrosis with exfoliation of affected cells and polymorphonuclear cell infiltration during the acute stages, progressing to squamous metaplasia during the reparative stages. Repair in these regions was essentially complete by 14 days PI. In the olfactory epithelium and the vomeronasal organ, there was interstitial edema with necrosis and exfoliation of epithelial cells and minimal to moderate inflammatory cell response during the acute stages. Residual reparative lesions were still evident in the olfactory epithelium, the columnar epithelium and neuroepithelium of the vomeronasal organ, and the nasolacrimal duct at 14 days PI. Viral antigen was demonstrated by immunohistochemistry in all regions during the acute stages of the disease, with the exception of the vomeronasal organ. In view of these findings, infections of the respiratory tract with viruses such as SDAV could have significant effects on functions such as olfaction and chemoreception for > or = 2 weeks postexposure in this species.

Role of gp63 and gIII of Aujeszky's disease virus in the invasion of the olfactory nervous pathway in neonatal pigs.

The purpose of this study was to examine in which way envelope glycoproteins gp63 and gIII of Aujeszky's disease virus (ADV) are involved in neuropathogenesis in pigs. The Ka strain of ADV and its single deleted mutants were examined with respect to invasion and spread in the olfactory nervous pathway after intranasal inoculation in neonatal pigs. The olfactory mucosa, olfactory bulb and lateral olfactory tract representing the 1st, 2nd and 3rd neuronal level of the olfactory pathway respectively, were examined for virus by isolation and for antigen by immunocytochemical localization. The Ka undeleted strain and its gIII deleted mutant invaded and spread to all the neuronal levels in a similar way. The gp63 deleted mutant invaded and spread in the olfactory mucosa similarly to the Ka parental strain but its replication and degree of spread were diminished in all the levels of the central nervous system (CNS). It is, therefore, concluded that gp63 is involved in the neurotropism of ADV, while gIII is not.