Mechanisms of Entry Into the Central Nervous System by Neuroinvasive Pathogens

Background: The literature on neurological manifestations, cerebrospinal fluid analyses, and autopsies in patients with COVID-19 continues to grow. The proposed mechanisms for neurological disease in patients with COVID-19 include indirect processes such as inflammation, microvascular injury, and hypoxic-ischemic damage. An alternate hypothesis suggests direct viral entry of SARS-CoV-2 into the brain and cerebrospinal fluid, given varying reports regarding isolation of viral components from these anatomical sites. Evidence acquisition: PubMed, Google Scholar databases, and neuroanatomical textbooks were manually searched and reviewed. Results: We provide clinical concepts regarding the mechanisms of viral pathogen invasion in the central nervous system (CNS); advances in our mechanistic understanding of CNS invasion in well-known neurotropic pathogens can aid in understanding how viruses evolve strategies to enter brain parenchyma. We also present the structural components of CNS compartments that influence viral entry, focusing on hematogenous and transneuronal spread, and discuss this evidence as it relates to our understanding of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Conclusions: Although there is a paucity of data supporting direct viral entry of SARS-CoV-2 in humans, increasing our knowledge of the structural components of CNS compartments that block viral entry and pathways exploited by pathogens is fundamental to preparing clinicians and researchers for what to expect when a novel emerging virus with neurological symptoms establishes infection in the CNS, and how to design therapeutics to mitigate such an infection.

State-of-the-Art Review Section Editors: Fiona Costello, MD, FRCP(C) Sashank Prasad, MD Mechanisms of Entry Into the Central Nervous System by Neuroinvasive Pathogens Navid Valizadeh, MBBCh, Emily A. Rudmann, BS, Isaac H. Solomon, MD, PhD, Shibani S. Mukerji, MD, PhD Background: The literature on neurological manifestations, cerebrospinal fluid analyses, and autopsies in patients with COVID-19 continues to grow. The proposed mechanisms for neurological disease in patients with COVID-19 include indirect processes such as inflammation, microvascular injury, and hypoxic-ischemic damage. An alternate hypothesis suggests direct viral entry of SARS-CoV-2 into the brain and cerebrospinal fluid, given varying reports regarding isolation of viral components from these anatomical sites. Evidence Acquisition: PubMed, Google Scholar databases, and neuroanatomical textbooks were manually searched and reviewed. Results: We provide clinical concepts regarding the mechanisms of viral pathogen invasion in the central nervous system (CNS); advances in our mechanistic understanding of CNS invasion in well-known neurotropic pathogens can aid in understanding how viruses evolve strategies to enter brain parenchyma. We also present the structural components of CNS compartments that influence viral entry, focusing on hematogenous and transneuronal spread, and discuss this evidence as it relates to our understanding of severe acute respiratory syndrome coronavirus-2 (SARSCoV-2). Conclusions: Although there is a paucity of data supporting direct viral entry of SARS-CoV-2 in humans, increasing our knowledge of the structural components of CNS compartments that block viral entry and pathways exploited by pathogens is fundamental to preparing clinicians and researchers for what to expect when a novel emerging virus with neurological symptoms establishes infection in the Division of Neuroimmunology and Neuro-infectious Disease (NV, ER, SSM), Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts; John C Lincoln Hospital (NV), Phoenix, Arizona; Harvard Medical School (ER, IHS, SSM), Boston, Massachusetts; and Department of Pathology (IHS), Brigham and Women’s Hospital, Boston, Massachusetts. S. S. Mukerji is supported by the National Institute of Mental Health at the National Institutes of Health (Grant number K23MH115812), James S. McDonnell Foundation and the Rappaport Foundation. The authors report no conflicts of interest. N. Valizadeh and E. Rudmann contributed equally to the manuscript. Address correspondence to Shibani S. Mukerji, MD, PhD, Division of Neuroimmunology and Neuro-infectious Disease, Department of Neurology, Massachusetts General Hospital, 55 Fruit Street Wang 855, Boston, MA 02114; E-mail: [e-mail] Valizadeh et al: J Neuro-Ophthalmol 2022; 42: 163-172 CNS, and how to design therapeutics to mitigate such an infection. Journal of Neuro-Ophthalmology 2022;42:163–172 doi: 10.1097/WNO.0000000000001455 © 2022 by North American Neuro-Ophthalmology Society G lobal surveillance has revealed that the prevalence of neurological complications in COVID-19 patients now ranges from 35% to 85% (1,2), with a large fraction of patients reporting persistent symptoms beyond 90 days postacute infection (3). Neurological symptoms in these patients are highly heterogenous, with observed syndromes including acute encephalopathy (1,4–7), headache (1,8), anosmia and ageusia (9,10), fatigue (9,11,12), memory impairment (9,12), seizure (1,13), and acute cerebrovascular disease (1,2,14–17). There have also been case reports of para-infectious and postinfectious complications, such as acute demyelinating encephalomyelitis (ADEM) (18), acute necrotizing encephalopathy (19,20), and Guillain–Barré syndrome (21,22). The study of postacute sequelae of COVID-19 is an area of active research, with ongoing observation and treatment trials globally (e.g., NCT04360538, NCT04895189, NCT04576351, NCT04411147). The mechanisms underlying neurological and neuropsychiatric sequelae have been widely discussed, yet remain speculative. One theory, endorsed by a specialty group within the World Federation of Neurology, proposes direct viral entry of SARS-CoV-2 into the central nervous system (CNS) (23). Alternate theories assuming an indirect mechanism include infection-mediated inflammation, microvascular pathology, and hypoxic-ischemic injury (24). The aim of this review is to discuss major routes of entry into the CNS used by well-characterized neuro-invasive pathogens, including hematogenous and transneuronal spread, and discuss this evidence as it relates to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). 163 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review Hematogenous Mechanisms of Pathogen Entry Into the Central Nervous System Two bulwarks stand against microbial neuroinvasion from the intravascular compartment: the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier (BCSFB). Both are multi-layered structures composed of cells with specialized intercellular junctions (Fig. 1). On the apical surface, an endothelial cell layer is in direct contact with blood. Endothelial cells are closely approximated to one another, joined both via standard intercellular adherence proteins and via tight junctions (TJ) composed of transcellular proteins (e.g., zonula occludens-1 and -2, claudin-5, etc.). Deep to the endothelium lies a pericyte cell layer and the endothelial basement membrane (BM). At the capillary level, the BM is in close contact with a layer of closely interlocked astrocyte foot processes termed the glia limitans (GL). At the post-capillary venule level, a dilated perivascular space intervenes between the endothelial BM and the GL, giving rise to the concept of a ‟2walled castle-moat” system of BBB protection (25). In contrast, the BCSFB is characterized by fenestrations that interrupt the endothelial lining of choroid plexus capillaries, found in the choroid plexus of the lateral, third, and fourth ventricles. These microvessels are encircled by choroid plexus stroma. The basal aspect of the stroma is in contact with epithelial BM, which anchors a monolayer of CSF-facing choroid plexus epithelial cells. Similar to the endothelial cells of the BBB, choroid plexus epithelium is connected via TJ. Despite this intricate defensive architecture, pathogen access to neural tissue has been described by several nonmutually exclusive mechanisms that include trafficking while being harbored within immune cells, direct invasion and transcellular passage through the endothelium, and induction of increased barrier permeability to facilitate paracellular or transcellular traversal. Trafficking of Pathogens Via Immune Cells Into the Central Nervous System Circulating leukocytes migrate from the bloodstream to the CNS as part of routine surveillance in a tightly regulated multistep process. Although phagocytic leukocytes normally contribute to clearance of viral, bacterial, and parasitic pathogens (26), infected leukocytes traversing the BBB can facilitate CNS infection (Fig. 1). This “Trojan horse”-mediated trafficking is the primary proposed mechanism for HIV entry into the CNS via infected T-lymphocytes and monocytes, although alternative mechanisms including paracellular transit are possible (27,28). Circulating HIV-infected immune cell populations upregulate adhesion complexes on brain endothelial cells, which can give rise to a local increase in binding affinity (28–30). Notably, HIV deploys several mechanisms to bias transmigration potential toward infected cells, including viral protein such as gp120-induced or Tat-induced BBB disruption, upregulation of matrix metalloproteinases such as (MMP)-2 and (MMP)-9, upregulation of adhesion molecules 164 in infected lymphocytes or monocytes (30), and elevated fitness in response to chemokines including C–C motif chemokine ligand 2 (CCL2) (29,30). Although our understanding of transmigration as a mechanism for HIV neuropathogenesis is incomplete, it has relevance to other intracellular pathogens which may use a similar mechanism. Antecedent coronaviruses to SARS-CoV-2 have exhibited the capability to infect human monocytes, despite primary tropism for respiratory tissue. For example, HCoV-229E, a causative agent of mild respiratory infection akin to the “common cold,” infects human monocyte cell lines in vitro and stimulates increased chemokine production, provoking migration of other monocytes. Angiotensin converting enzyme-2 (ACE2) is the main identified cell surface receptor for viral entry of several coronaviruses including SARS-CoV-2 (31). The ACE2 receptor is known to be expressed on specific macrophage and dendritic cell subpopulations, and some studies have identified viral components in leukocytes (32). Despite the presence of viral components in immune cells, robust data supporting SARS-CoV-2 exploitation of the migratory capacity of immune cells to cross the BBB in humans are limited. Most autopsy series investigating brain tissue report minimal perivascular immune cell infiltration (reviewed in Ref. 33,34), even among cases with imaging suggestive of vessel wall inflammation (35) or with autopsyreported intramural inflammatory infiltrates (36). Among the minority of human autopsy studies demonstrating inflammatory infiltration, SARS-CoV-2 RNA or protein in parenchymal immune cells has not been detected (37–41). As a counterpoint, among the reports with SARS-CoV-2 viral proteins identified in brain parenchyma by immunohistochemistry (38,41), presence of spike or nucleocapsid protein was not associated with inflammation. These data, in combination with other corroborating human studies, suggest that SARSCoV-2 replication in CNS compartments is unlikely (33). Blood–Brain-Barrier Permeability and Paracellular Entry Paracellular entry is the passive diffusion of molecules from the blood into the CNS through intercellular spaces intrinsic to the endothelium, which are typically restricted by TJ proteins (Fig. 1) (26). Use of this mechanism principally requires disruption or circumvention of BBB anatomy. Animal models consistently show diminished TJ expression and increased BBB permeability when exposed to cytokines and chemokines such as interleukin (IL)-1b, IL-6, tumor necrosis factor alpha (TNFa), and CCL2 (42,43), underscoring that compromised BBB can be a collateral effect of a host inflammatory response/cascade. West Nile virus (WNV) is a prototypical neuroinvasive pathogen that achieves CNS invasion by multiple mechanisms, including enhancement of BBB permeability. Murine models of WNV neuroinvasion show elevated Evan Valizadeh et al: J Neuro-Ophthalmol 2022; 42: 163-172 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review FIG. 1. Pathways of hematogenous entry into the central nervous system (CNS). Top Panel: The blood–brain barrier (BBB) apical surface is comprised of an endothelial cell layer that is in direct contact with pericapillary venule. Endothelial cells are closely approximated to one another, joined by intercellular adherence proteins and tight junctions (TJ). Deep to the endothelium is the pericyte cell layer and endothelial basement membrane (BM). At the capillary level, the BM is in close contact with a layer of closely interlocked astrocyte foot processes termed the glia limitans. Bottom left: the Trojan horse mechanism of neuroinvasion includes infected circulating immune cells that upregulate endothelial cellular adhesion molecules (1) Upregulation of E-selectin, V-CAM1, and selectins facilitates immune cell migration along the vessel wall (2) Increased levels of interleukins and other cytokines alters BBB permeability and promotes immune diapedesis. Infected immune cells traverse the BBB, carrying virus within (3) The infected immune cell enters the CNS, leading to increases in local cytokine and chemokine levels and recruitment of leukocytes (4) Bottom right: Increases in circulating proinflammatory cytokines and chemokines disrupt TJ proteins, reduce transendothelial electrical resistance, and induce increasing porosity in the BBB facilitating paracellular viral entry. In addition, endothelial cells and pericytes that express viral receptors may become directly infected, resulting in transendothelial entry. In some cases, pathogens may enter the CNS through adsorptive endocytosis and emerge on the basolateral aspect of the BBB. Created with BioRender.com. blue dye leakage and CSF IgG with decreased expression of TJ proteins (44,45). Furthermore, MMP-9, which executes barrier breakdown by cleaving TJ proteins and the adjacent extracellular matrix (26), is increased in circulation after WNV infection (26,45,46). “Cytokine storm” is a well-appreciated COVID-19 phenomenon, responsible for significant morbidity in critically ill patients. Supraphysiologic levels of acute phase proteins such as ferritin and c-reactive protein and elevated cytokines such as IL-6, IL-8, and IL-10 are associated with poor prognosis (32,47–49). Transcriptional upregulation of cytokines after infection of primary macrophages by SARS-CoV-2 in vitro (32) and deep immune-profiling of human blood suggest a hyperinflammatory phenotype with a combination of inade- Valizadeh et al: J Neuro-Ophthalmol 2022; 42: 163-172 quate adaptive immune response and hyperactive inflammatory myeloid and neutrophil system may be a prominent feature of COVID-19 critical illness (49). CSF analyses on hospitalized COVID-19 patients suggest that some individuals have an elevated albumin CSF-to-serum ratio, indicating BBB disruption (reviewed in Ref. 50). Correlations between intrathecal proinflammatory cytokines (e.g., IL-1b, IL-6, IL-8, TNFa, IL-12p70) and BBB status are mixed, and the relationship between compromised BBB integrity and neurological findings is not well established (5,50–52). There is limited evidence of SARS-CoV-2 RNA in CSF aside from rare case reports, and in a small minority of patients included in case series (50). Furthermore, when detected, SARS-CoV-2 165 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review RNA is typically close to the limits of assay detection and is not always associated with CSF pleocytosis or markers of increased BBB permeability (53). Although absence of intrathecal viral nucleic acid alone does not eliminate the possibility of neuroinvasion, as evidenced by WNV neuroinvasive disease (54,55), the paucity of CSF cellular inflammation observed in most COVID-19 patients is atypical for a neuroinvasive pathogen (5,51,52). Transendothelial-Mediated Pathogen Entry Into the Central Nervous System Endothelial cell pathology in the setting of SARS-CoV-2 infection has been widely reported on lung autopsies (56,57) and direct infection has been reported in some extrapulmonary tissues (58). The brain endothelium, however, is not frequently permissive to infection (26). Several viruses are capable of replication within brain endothelial cells and release virions into brain tissue (e.g., Nipah virus, Zika virus) (26), which can precipitate devastating morbidities such as endothelial necrosis (26). Recently, ACE2 RNA was identified in endothelial cells and pericytes using single cell RNA sequencing, and ACE2 protein expression was observed in endothelial cells collected from human brain biopsies and autopsies. Neuropilin-1 (NRP1), another cell receptor for SARS-CoV-2, is expressed in adult endothelial cells (38) and choroid plexus epithelial cells in adults and pediatric brain tissue (59,60). In vitro models using cerebral organoids suggest SARS-CoV-2 infection is possible in a manipulated system, and infection is accompanied by decreased expression of TJ proteins (61). Similar findings are observed in human brain microvascular endothelial cell culture models demonstrating that SARS-CoV-2 spike protein can alter BBB dynamics by increasing intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM1) cell surface proteins and gene expression of MMP-3, -9, and -12 (62). There is a paucity of data in human autopsies suggesting brain endothelial infection. Rare reports of detectable SARSCoV-2 RNA in the endothelium at autopsy include a pediatric case series of patients with severe COVID-19 and multisystem inflammatory syndrome in children in which 2 of 5 patients had detectable RNA in brain endothelial cells (37), and a widely cited case report of a patient with Parkinson Disease and fatal COVID-19 (63). These findings have yet to be replicated in larger studies (57). There have been some reports to suggest that although productive infection may not be present, S1 or other spike protein subunits may be harbored in human endothelial cells, and like HIV proteins, may result in inflammation and tissue damage (64). Neuronal Transport Into the Central Nervous System From the Peripheral Nervous System Mechanisms of neuronal entry from infected peripheral nerves into the CNS are often species-specific and strain166 specific, and mediate pathogenesis in 4 major viral families (Herpesviridae, Rhabdoviridae, Flaviviridae, and Picornaviridae) (reviewed in Ref. 26,65). Transolfactory Spread of Pathogens Into the Central Nervous System WNV, in addition to enhancing BBB permeability, can potentially infiltrate the brain via neuronal transit into the olfactory bulb. In mouse models, WNV neuroinvasion reliably occurs within days of inoculation and is observed in the olfactory bulb and spinal cord (66). In hamster models, sciatic nerve WNV inoculation results in viral migration from the periphery into the spinal cord, infecting anterior horn motor neurons. Using compartmentalized neuronal cultures, WNV can spread in both retrograde and anterograde directions (67), and virions found close to axodendritic synapses strongly suggests transsynaptic spread as a pathogenic mechanism of WNV. Coronaviruses with varying structural similarity to SARS-CoV-2 have demonstrated neurotropism and CNS entry capability. Porcine hemagglutinating encephalomyelitis virus (PHEV) is a Betacoronavirus with neurotropic properties. In animal studies, PHEV invades the dorsal root ganglion cell body and spreads to adjacent satellite cells by budding and release from infected neurons (68). After hindlimb inoculation, PHEV is detectable in the ipsilateral dorsal root ganglion, followed by the spinal cord, and finally the contralateral primary motor cortex of the animal (68,69). Neuron-to-neuron transmission in the periphery relies on a constitutive exocytic mechanism, whereas trans-synaptic spread in the CNS uses virion release from coated vesicles into the synaptic cleft (68,69). Although human data are lacking, PHEV provides a template of coronavirus neuron-to-neuron spread from the periphery into the CNS. Human coronavirus OC43 (HCoV-OC43) is also detectable in the olfactory bulb of mice 4 days postnasal inoculation, before other brain regions (70), and is subsequently detectable in the piriform cortex and, eventually, brainstem (71). Although human data on HCoV-OC43 neuroinvasion are sparse, rare reports of viral components (RNA and protein) have been published on fatal encephalitis among immunocompromised infants (72) and an immunocompetent teenager presenting with ADEM (73). SARS-CoV-2 shares close homology with SARS-CoV-1, which has demonstrated capacity to invade the CNS in animal models (reviewed in Ref. 74). In K18-hACE2 mice, which express the humanized ACE2 receptor, intranasal inoculation results in abundant viral antigen in the olfactory bulb 2–3 days postinoculation. Neurons in the olfactory network, including the piriform cortex, are positive for SARS-CoV-1 protein, and with increasing viral inoculum, there is wider dissemination throughout the brain, to regions including basal ganglia, dorsal raphe nuclei, frontal and parietal cortices, thalami, pons, and medulla (75,76). In Valizadeh et al: J Neuro-Ophthalmol 2022; 42: 163-172 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review an autopsy series of 4 patients with fatal SARS-CoV-1 infection, nucleocapside protein and RNA polymerase gene fragments were identified at low levels in the cerebrum in all decedents using immunohistochemistry and in situ hybridization (77). In another autopsy series of 8 patients, SARSCoV-1 RNA was uniformly detected in the hippocampus and cortex (78). Given the high prevalence of anosmia in patients with COVID-19, transolfactory spread into the CNS has been widely suspected. The distribution of cell surface receptors that allow for SARS-CoV-2 viral entry into the human olfactory neuroepithelium sheds some light on the viability of the olfactory nerve (Fig. 2) as a port of entry for the virus to enter the CNS. In the upper airway, ACE2 gene expression was found in nasal epithelial and secretory cells (79,80), such as sustentacular cells, horizontal basal cells, goblet cells, and Bowman glands, but importantly, not in olfactory sensory neurons (OSN). These single cell RNA sequencing data have been corroborated with immunohis- tochemistry of human nasal epithelium (81). Transmembrane protease serine 2 is a primer of the SARS-CoV-1 spike protein (82), and is responsible for cleavage at S1 and S2, facilitating viral fusion with host cell membranes (31). The expression profile in nasal epithelium also resembles that of ACE2, with enriched expression on epithelial and secretory cells (79,80). CD147 is a transmembrane glycoprotein that has a functional role in SARS-CoV-1 infection, and a similar role has been proposed in SARS-CoV-2. Subsequent studies using recombinantly expressed proteins found no support for SARS-CoV-2 spike protein interaction with CD147 (83,84), although it may influence ACE2 levels via an indirect mechanism. At this time, robust evidence of CD147-influenced CNS entry in SARS-CoV-2 infection is lacking. Finally, NRP1 is a transmembrane glycoprotein expressed in endothelial cells, vascular smooth muscle cells, macrophages, dendritic cells, and T-lymphocytes (85). Although early, there is some evidence to support its role as a cofactor in SARS-CoV-2 infection (86,87). NRP1 is FIG. 2. Schematic of the olfactory neuroepithelium. The olfactory neuroepithelium is in the posterosuperior discrete region of the nasal mucosa. Olfactory sensory neuron (OSN) cell bodies are located in the deep and mid-sections of the olfactory neuroepithelium, with dendrites containing odorant receptors extending to the apical surface and embedded within the superficial mucosal layer. OSNs are surrounded by sustentacular support cells. The deep layer of the epithelium contains progenitor cells intermixed with basal and dendrite cells that overlay the basal lamina propria. OSN axons penetrate this basal layer and traverse the lamina cribrosa of the cribriform plate in nerve bundles that project directly to the adjacent olfactory bulbs nestled on the inferior aspect of the medial frontal lobes. From here, second-order neurons pass posteriorly in the olfactory tracts. These project to the ipsilateral anterior olfactory nucleus, the contralateral olfactory bulb (OB) via the anterior commissure, and terminate in the septal nuclei surrounding the paraterminal gyrus. From here, 2 fiber bundles project: The medial striae (which subdivides into the medullary striae, terminating in the habenular nuclei and tegmentum and salivary nuclei; and the olfactory-hypothalamic-tegmental pathway, terminating in the dorsal motor nucleus of the vagus in the medulla) and the lateral striae that convey substantially more efferent fibers to the primary olfactory cortex (e.g., piriform cortex, amygdala, parahippocampal gyrus, olfactory tubercle, anterior olfactory nucleus). Created with BioRender. com. Valizadeh et al: J Neuro-Ophthalmol 2022; 42: 163-172 167 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review expressed in the human olfactory epithelium and brain, including in mature astrocytes, endothelial cells, and microglia. In the human brain, RNA and protein expression are highest in the hippocampal formation and retina, but also appear at low levels in the olfactory epithelium and olfactory bulb. In experiments interrogating olfactory epithelium from autopsies of 6 patients with COVID-19, expression of NRP1 co-localized with OLIG2, a marker associated with immature OSN (86). Most published autopsy data show normal appearance of the olfactory bulb and absent or mild degrees of microgliosis and astrogliosis (33,88). This summation reflects findings in approximately 140 patients, most of whom had severe disease (33). The most commonly reported abnormalities in the olfactory nerve are microgliosis and astrogliosis without robust inflammatory infiltrate (38,40,88). SARS-CoV-2 RNA appears in the olfactory bulb and adjacent frontal lobe in some reports (40,89); however, the significance of this finding remains unclear and the cell types harboring viral RNA are not known. Remarkably, there is no definitive correlation between presence of viral RNA in the olfactory bulb and tissue damage or reported anosmia. There are numerous examples of glial cell activation in the olfactory bulb without detectable virus using multiple molecular techniques (38,88,89) or without prominent histopathological changes. In a minority of autopsy cases, severe inflammation and structural damage to the olfactory bulb were observed, with ultrastructural detection of spherical virallike particles in the gyrus rectus in one case,94 and intracytoplasmic viral-like inclusion bodies in the olfactory bulb in another case (90); replication of these data is required in large studies. Alternative Routes for Transneuronal Central Nervous System Entry Although ocular surfaces are recognized as a potential route of infection for SARS-CoV-1 and SARS-CoV-2 (91), ocular inoculation is understood to cause infection by transport from eye to nasopharynx via the lacrimal duct (92) or via infection of ocular surfaces directly. There is a dearth of pathological investigation of the eye and its relationship to brain findings (34). The vagus nerve is also a conduit to the CNS and has been proposed as a pathway for avian influenza and swine hemagglutinating encephalomyelitis viruses in animal experiments (69,93). Although animal experiments do support roles for transneuronal CNS entry in pathophysiology of other neurotropic viruses via multiple peripheral nerves, including the trigeminal and vagus nerves, human evidence to suggest a role of these routes during SARS-CoV-2 infection is lacking. CONCLUSION Pathogens have evolved alongside host evolution to overcome natural barriers to infection. Advancing our under168 standing of host weaknesses to pathogen cell entry can prepare us for emerging infections and possibly drug development to block invasion. The neuroinvasive potential of SARS-CoV-2 has been a major topic of discussion since the start of the pandemic; at this time, most evidence suggests that direct viral entry into the CNS is a rare phenomenon in humans and would not explain the neurological symptoms experienced by millions of people with COVID-19. Unfortunately, SARS-CoV-2 continues to circulate, mutate, and infect, and may eventually evolve to use any number of mechanisms to cross CNS barriers. Continued research dedicated to delineating mechanisms of pathogen entry into the CNS in this era of emerging neuroinfectious diseases is needed, and persistent surveillance of viral genetic material in neuroanatomical spaces via deep sequencing is essential while we remain in this current pandemic, and for rapid detection of future outbreaks. ACKNOWLEDGMENTS The authors wish to acknowledge the contributions to our understanding of COVID-19 by front-line health care personnel. REFERENCES 1. Chou SHY, Beghi E, Helbok R, Moro E, Sampson J, Altamirano V, Mainali S, Bassetti C, Suarez JI, McNett M. Global incidence of neurological manifestations among patients hospitalized with COVID-19—a report for the GCS-neuroCOVID consortium and the ENERGY consortium. JAMA Netw Open. 2021;4:e2112131. 2. Mao L, Jin H, Wang M, Hu Y, Chen S, He Q, Chang J, Hong C, Zhou Y, Wang D, Miao X, Li Y, Hu B. Neurologic manifestations of hospitalized patients with coronavirus disease 2019 in Wuhan, China. 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