Neuropathogenesis of SARS-CoV-2 Infection

Introduction

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for causing the novel coronavirus disease 2019 (COVID-19) that became a global concern after it emerged from Wuhan, China in December 2019. Its high community transmission propelled its spread across several countries, gripping the world in a major health crisis that was eventually declared a pandemic by the World Health Organization (WHO). The ongoing pandemic of COVID-19 has presented new challenges to scientists. Currently, there is a lack of information about the different aspects of SARS-CoV-2 to enable researchers worldwide to understand the virus as they race to find an effective vaccine. With each passing day, new features are being unraveled about its behavior, and new information gathered as the virus evolves across different populations. While there is no specific treatment available, those employed are based on prior experience from other viral infections and depend on the severity of the case being presented. Clinical and epidemiological findings have revealed that some patients are asymptomatic, and others have mild symptoms such as fever, fatigue, sore throat, and dry cough, and recover on minimal medical intervention. In contrast, many others show rapid deterioration and develop complications that include gastrointestinal, cardiovascular, dermatologic, and neurological disturbances, along with respiratory, renal, and liver dysfunction, which can lead to sepsis, encephalitis, and death.

There is a soaring mortality rate associated with SARS-CoV-2 infection, and the number of infected cases is still rising across different parts of the world. Based on the hospital admission data, epidemiologists have reported that elderly patients and people of any age with underlying comorbidities are highly susceptible to the complications associated with COVID-19. These comorbid conditions include hypertension, cardiovascular disease, diabetes, chronic respiratory disease, liver disease, hemoglobin disorders, cancer, renal disease, neurological diseases, and obesity and may affect disease outcomes. Indeed there is concern that patients on immunosuppressive therapies for inflammatory conditions such as multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, and chronic inflammatory demyelinating polyneuropathy (CIDP) may have a higher risk of infection or develop a severe COVID-19. However, data is lacking and so far there is no supporting evidence. Overall, these comorbid conditions lead to a weakened immune state, which decreases the ability to fight the viral infection. Given the robust immune response incurred by the virus, it is currently unclear if treatment with specific immunomodulators that are used to treat chronic autoimmune disorders predisposes patients to severe SARS-CoV-2 infection, or protects the host by dampening immune cell activation.

A peculiar feature of COVID-19 is the heterogeneous spectrum of clinical manifestations that affect different organ systems. It appears that SARS-CoV-2 primarily starts as an infection in the upper respiratory tract, which then moves to the lower respiratory tract from where it can also invade the nervous system. Hospitalization data strongly suggests that the neurological symptoms of the SARS-CoV-2 cannot be overlooked, yet there is a huge debate regarding the neurological impact of the virus. In this chapter, we provide an overview of studies that assessed the neuroinvasive potential of COVID-19 and discuss possible neuropathogenic mechanisms used by SARS-CoV-2 to invade the nervous system. As we grapple with the effects of this novel virus and the unique challenges of diagnosing and managing COVID-19, it is critical that all of the effects of this novel virus on the nervous system are considered so that these can be better managed and the outcomes of individuals infected with the virus can be improved.

The Neuroinvasive Potential of Coronaviruses (CoV)

To understand the neuroinvasiveness of coronaviruses (CoV), it is essential to consider their structural features. The electron microscopic structure of CoV appears as an enveloped particle having a spherical, oval, or pleomorphic crown-like shape with a diameter ranging between 80 and 120 nm. These structural features are encoded by the genome of CoV, which is comprised of a single-stranded nonsegmented positive-sense RNA of about 27–32 kb. The entire genome is bound by the nucleocapsid (N) protein to form a helical symmetric nucleocapsid. The surface is coated with large projections of membrane glycoproteins, termed spike (S) proteins, and some CoV even have shorter projections of the hemagglutinin-esterase (HE) protein. The S protein is the primary facilitator for entry into the host cell by mediating binding to target receptors present on the cellular surface. The outer viral envelope contains the membrane (M) and envelope (E) proteins. The M protein is a type III transmembrane glycoprotein that helps maintain the CoV conformation and antigenicity, whereas the E protein acts as an ion-channeling viroporin that is involved in envelope formation, budding, and pathogenesis, and interacts with other CoV proteins and host proteins. The N protein interacts with the M protein to stabilize the assembly of envelope complexes during the formation of new viral particles. Together, each of these four key structural proteins (N, S, E, M) confer unique properties to CoV for invading the host and spreading the infection to different tissues.

SARS-CoV-2 is the newest addition to the beta-CoV genus as the seventh member of the family of human CoV (HCoV), which consists of respiratory viruses including HCoV-229E (named after a student specimen coded 229E), HCoV-OC43 (organ culture 43), HCoV-NL63 (NetherLand 63), HCoV-HKU1 (Hong Kong University 1), SARS-CoV (severe acute respiratory syndrome), and MERS-CoV (Middle East respiratory syndrome). The genomic data of SARS-CoV-2 shows sequence similarity of 79%–80% to SARS-CoV, 50% to MERS-CoV, and the highest similarity of 96% to a bat family of CoV. SARS-CoV, MERS-CoV, and SARS-CoV-2 cause a severe respiratory illness that hints toward their lethal nature, making them highly pathogenic. At the same time, other members of the family have been associated with a mild or moderate form of respiratory disease. To understand the neuropathogenesis of novel SARS-CoV-2 infection, it is important to familiarize ourselves with the impact of other members of the CoV family on the nervous system and what is known from past experience with SARS and MERS, the relevant aspects of which are included here.

Several reports have shown neurovirulence of CoV in pediatric and adult cases with no evidence for a direct link. In a study by Li et al. cytokine profiles were determined in the central nervous system (CNS) and respiratory tract of hospitalized pediatric cases with CNS illness and CoV infection. Cytokine profiling in the serum showed significantly higher granulocyte colony-stimulating factor (G-CSF) in individuals infected with CoV that had complications in the CNS or respiratory tract, compared to healthy subjects, although levels were markedly higher in those with CNS complications. Further, the levels of cytokines such as interleukins (IL-6, IL-8), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly higher in the cerebrospinal fluid (CSF) of patients with CNS infection relative to levels in the serum. It is noteworthy that this report was the first to have found a high incidence of CoV infection in pediatric cases with CNS illness, showing characteristic cytokine profiles in the CSF, which suggests the host immune response, plays a role in the progression of the infection and its associated complications.

Similarly, multiple reports suggest an association of CoV infection with chronic CNS diseases, including MS, encephalitis, acute flaccid paralysis, Guillain-Barré syndrome (GBS), acute disseminated encephalomyelitis (ADEM), hemorrhage, seizures, and stroke. The initial reports on SARS patients have shown evidence of SARS-CoV infection in neurons in the brain, as detected by the presence of viral particles and genetic material using electron microscopy, immunohistochemistry, and polymerase chain reaction (PCR). Interestingly, autopsy reports of SARS patients have also shown cerebral edema and meningeal vasodilation, as well as virus-infiltrated monocytes and lymphocytes in the vessel wall, viral particles in the brain, and changes in neurons with demyelinating nerve fibers. Clinical findings have found that SARS-CoV can even cause neurological complications such as encephalitis, axonopathic polyneuropathy, myopathy or rhabdomyolysis, aortic ischemic stroke, and olfactory neuropathy. The neuroinvasive potential of MERS-CoV was shown in a retrospective study in which 25.7% of infected patients developed confusion, and 8.6% had seizures. Another study found a delayed appearance of neurological features after the presentation of respiratory symptoms in 20% of patients with MERS, including disturbances in consciousness, neuropathy, ischemic stroke, paralysis, and GBS. Inoculation of SARS-CoV and MERS-CoV in transgenic mice through the intranasal route has revealed the ability of these viruses to infect brain areas such as the thalamus and brainstem through olfactory nerves. The mice induced with low doses of MERS-CoV showed that the viral particles were confined to the brain region only, not lungs, strongly hinting that neuroinvasion-induced mortality in the infected mice.

Despite the neurological impact, the exact route of infection used by SARS-CoV and MERS-CoV for neuroinvasion in humans is unknown. Studies suggest that the hematogenous or lymphatic route of infection does not play a significant role in driving initial CNS infection due to the absence of virus in nonneuronal cells in the infected areas of the brain. Some studies suggest that CoV may invade the CNS through a synaptic route by infecting the peripheral nerve terminals. Although the exact mechanisms by which CoV affects the nervous system remain unclear, the above findings demonstrate that infection with SARS-CoV and MERS-CoV is associated with neurological manifestations and complications, which support recent findings of brain invasion in patients infected with SARS-CoV-2.

The Neuroinvasive Potential of SARS-CoV-2

There is limited knowledge about the emergence of SARS-CoV-2; however, it is believed that it evolved from other CoV such as SARS-CoV and/or MERS-CoV through a series of mutagenic events. As such, it behaves similarly to these viruses in terms of its pathogenicity, structure, and route of infection. Given the evidence that SARS-CoV and MERS-CoV are neurotropic viruses, this suggests the same may be true of SARS-CoV-2. Epidemiological findings show that the median duration of disease progression from the onset of illness was 5 days to dyspnea, 7 days to hospital admission, and 8 days to the intensive care unit. Notably, this latency period is likely long enough for viral particles to invade the nervous system and damage the neurons.

The effect of SARS-CoV-2 on the nervous system can be gauged at three levels: symptoms arising due to viral infection, collateral effect on the nervous system from the immune response to the virus, and complications in patients with neurological comorbidities. It has now become clear that in addition to having a major impact on the respiratory and cardiovascular systems, it also affects the nervous system. Altogether, the clinical and experimental studies report commonly observed manifestations and postinfection complications that clearly reflect neurovirulence in COVID-19, including headache, stroke, nausea, dizziness, vomiting, disturbed olfaction, seizures, disturbed sensorium, altered consciousness, ataxia, encephalopathy/encephalitis, hypogeusia, hypoplasia, neuralgia, myelitis, and skeletal muscle symptoms. Findings from some of the major studies highlighting the neurological impact of SARS-CoV-2 infection are summarized in Table 2.1 and will be further detailed throughout this volume.

Additional neurologic complications have been reported in studies of infected patients with SARS-CoV-2 that have used neuroimaging with computerized tomography (CT) and magnetic resonance imaging (MRI) along with electroencephalogram (EEG) data. These have reported hemorrhagic necrotizing encephalopathy, epileptogenicity, and encephalomalacia, which could be the outcome of the severe cytokine storm observed in some COVID-19 cases. There have also been reports of a temporary loss of speech, confusion, lethargy, and signs of disorientation, with brain scans showing injury to the thalamus accompanied by hemorrhage and necrotizing encephalopathy. Even though there are only a few reports confirming that the neurovirulence in COVID-19 is primarily linked to encephalitis and meningitis, its lethal impact cannot be underlooked.

As will be discussed in more detail in Chapter 10 , there is also mounting evidence of changes in olfaction and taste in SARS-CoV-2-infected patients, and in some cases, patients have experienced a bitter taste while fighting off the infection. This sets the basis for anosmia and dysgeusia observed in some infected patients, possibly due to reduced sensitivity of neurosensory reflexes by SARS-CoV-2. This is partially supported by data from experimental animals infected with SARS-CoV or MERS-CoV, where olfactory nerves were shown as a route of brain invasion.

Several reports indicate the presence of virus particles in various parts of the nervous system, such as the brain and CSF, which could incur damage. To this end, a putative route of infection of the brain by SARS-CoV-2 has been proposed, and it is suggested that isolating viral particles from the endothelium of cerebral microcirculation, CSF, glial cells, and neuronal tissue would serve as confirmatory evidence for the neurotrophic potential of SARS-CoV-2. Pathological findings from postmortem analysis of COVID-19 patients have shown neurodegeneration and edema in the brain tissue. The first report of viral encephalitis that appeared to be the result of direct SARS-CoV-2 invasion of the brain was reported from China’s Beijing Ditan Hospital, where the presence of the virus was revealed in the CSF of a patient by PCR-based genome sequencing. Similarly, imaging data on brain autopsies of 19 cases have shown brain lesions in eight patients, which were related to hemorrhagic and posterior reversible encephalopathy syndrome. Taken together, these observations point to a viral-mediated blood-brain barrier (BBB) disruption, enhancing its permeability and thus contributing to the localized edema and inflammatory response seen within the parenchyma. Further, it has not yet been fully discerned if respiratory failure in COVID-19 is linked to the neurovirulence potential of SARS-CoV-2. Thus, continued investigation to determine the causes of these neurological symptoms is required in order to fully understand the overall effects of SARS-CoV-2.

Intriguingly, dysregulated metabolism at a neuropeptide and neurotransmitter level has also been linked to disease severity in COVID-19 patients. Proteomic and metabolic alterations have been reported in the sera of severe cases of COVID-19 that were linked to serotonin, kynurenine, tryptophan, and polyamine pathways. These findings have also highlighted the role of platelets in SARS-CoV-2 infection, which was in consensus with clinical studies showing complications in coagulopathy and delirium, with a possible link to altered neurotransmitters like serotonin. Similarly, aberrations in the biosynthesis of the neurotransmitter dopamine have also been linked with certain pathophysiological processes of SARS-CoV-2. Considering the neurovirulence of SARS-CoV-2 infection, it is unclear if metabolic alterations occur in the peripheral blood circulation, and if altered metabolites cross the BBB and impact the nervous system. A recent study has performed metabolite profiling in the CSF and in blood samples from severe SARS-CoV-2 infection cases and delirium-prone patients, which provides clues into the altered brain metabolism observed in these patients. This demonstrated a significant difference in the concentrations of metabolites such as acylcarnitines and polyamines, particularly phenethylamine (PEA) between the CSF and blood in delirium-prone patients compared to healthy controls. The observed difference was linked to altered activity of the enzyme monoamine oxidase B (MAOB) that is involved in platelet regulation and coagulation and in the metabolism of neurotransmitters, with known association with delirium, anosmia, neuropsychiatric disorders, and neurodegenerative diseases. Notably, the computational structural analysis showed significant similarity between the angiotensin-converting enzyme 2 (ACE2), which mediates the entry of SARS-CoV-2 into host cells, and MAOB, with 95% similarity observed in the SARS-CoV-2 spike protein binding region of ACE2. This suggests that SARS-CoV-2 interacts with MAOB through its spike protein and alters its activity at the molecular level, setting up the ground for neurological complications in COVID-19.

To find promising drug targets for treating SARS-CoV-2, it is crucial to understand the mechanisms of neuroinvasion by the virus and the impact it has on the nervous system. It will also be interesting to explore the interactions between the BBB and SARS-CoV-2 proteins in gaining access to the nervous system. Thus far, there has been no evidence of direct meningeal invasion of SARS-CoV-2, making it unclear if reported neurological features, such as headache or encephalopathy, are a consequence of the systemic inflammatory response or are secondary to CNS investment by the virus. It is also possible that respiratory muscle injury and/or reduced sensorium may be responsible for the observed respiratory failure in COVID-19 patients. In light of the possible involvement of the neuromuscular system in COVID-19 patients, neurophysiological assessment should be included in patients that present with the disease. Ultimately, understanding the neuroinvasive propensity of SARS-CoV-2 may provide clues for the treatment and prevention of COVID-19 transmissions.