Central nervous system as a target of novel coronavirus infections: Potential routes of entry and pathogenic mechanisms.

Since the COVID-19 pandemic started in December 2019, there have been several reports of patients succumbing to neurological complications. Early reports were suggestive of a possibility, while by early 2020 it was clearly evident that although SARS-CoV-2 primarily attacks the respiratory system, the brain is one of the most affected organs post-recovery. Although it may be premature to comment on the long-term effects of COVID-19 in brain, some reliable predictions can be made based on the data currently available. Further, exploring the CNS connections of SARS-CoV-2 is of keen interest for neuroscience researchers. As soon as the virus enters the nasal region, it is exposed to the olfactory nervous system which is interlinked with the visual system, and hence we explore the mechanism of entry of this virus into CNS, including brain, olfactory and retinal nervous systems. In this review, we have thoroughly reviewed reports about both SARS-CoV-1 and SARS-CoV-2 with respect to their ability to breach the blood-brain and blood-retinal barriers. We have compiled different neurological conditions resulting from COVID-19 and looked into viral infections related to COVID-19 to understand how the virus may gain control of the olfactory and visual systems. Once the dust settles on the pandemic, it would be interesting to explore the extent of viral infection in the CNS. The longterm effects of this virus in the CNS are not yet known, and several scientific research papers evolving in this field will throw light on the same.

Novel polyomavirus associated with brain tumors in free-ranging raccoons, western United States.

Tumors of any type are exceedingly rare in raccoons. High-grade brain tumors, consistently located in the frontal lobes and olfactory tracts, were detected in 10 raccoons during March 2010-May 2012 in California and Oregon, suggesting an emerging, infectious origin. We have identified a candidate etiologic agent, dubbed raccoon polyomavirus, that was present in the tumor tissue of all affected animals but not in tissues from 20 unaffected animals. Southern blot hybridization and rolling circle amplification showed the episomal viral genome in the tumors. The multifunctional nuclear protein large T-antigen was detectable by immunohistochemical analyses in a subset of neoplastic cells. Raccoon polyomavirus may contribute to the development of malignant brain tumors of raccoons.

A new model for Hendra virus encephalitis in the mouse.

Hendra virus (HeV) infection in humans is characterized by an influenza like illness, which may progress to pneumonia or encephalitis and lead to death. The pathogenesis of HeV infection is poorly understood, and the lack of a mouse model has limited the opportunities for pathogenetic research. In this project we reassessed the role of mice as an animal model for HeV infection and found that mice are susceptible to HeV infection after intranasal exposure, with aged mice reliably developing encephalitic disease. We propose an anterograde route of neuroinvasion to the brain, possibly along olfactory nerves. This is supported by evidence for the development of encephalitis in the absence of viremia and the sequential distribution of viral antigen along pathways of olfaction in the brain of intranasally challenged animals. In our studies mice developed transient lower respiratory tract infection without progressing to viremia and systemic vasculitis that is common to other animal models. These studies report a new animal model of HeV encephalitis that will allow more detailed studies of the neuropathogenesis of HeV infection, particularly the mode of viral spread and possible sequestration within the central nervous system; investigation of mechanisms that moderate the development of viremia and systemic disease; and inform the development of improved treatment options for human patients.

The multibasic cleavage site in H5N1 virus is critical for systemic spread along the olfactory and hematogenous routes in ferrets.

The route by which highly pathogenic avian influenza (HPAI) H5N1 virus spreads systemically, including the central nervous system (CNS), is largely unknown in mammals. Especially, the olfactory route, which could be a route of entry into the CNS, has not been studied in detail. Although the multibasic cleavage site (MBCS) in the hemagglutinin (HA) of HPAI H5N1 viruses is a major determinant of systemic spread in poultry, the association between the MBCS and systemic spread in mammals is less clear. Here we determined the virus distribution of HPAI H5N1 virus in ferrets in time and space-including along the olfactory route-and the role of the MBCS in systemic replication. Intranasal inoculation with wild-type H5N1 virus revealed extensive replication in the olfactory mucosa, from which it spread to the olfactory bulb and the rest of the CNS, including the cerebrospinal fluid (CSF). Virus spread to the heart, liver, pancreas, and colon was also detected, indicating hematogenous spread. Ferrets inoculated intranasally with H5N1 virus lacking an MBCS demonstrated respiratory tract infection only. In conclusion, HPAI H5N1 virus can spread systemically via two different routes, olfactory and hematogenous, in ferrets. This systemic spread was dependent on the presence of the MBCS in HA.

Human herpesvirus-6 entry into the central nervous system through the olfactory pathway.

Viruses have been implicated in the development of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and multiple sclerosis. Human herpesvirus-6 (HHV-6) is a neurotropic virus that has been associated with a wide variety of neurologic disorders, including encephalitis, mesial temporal lobe epilepsy, and multiple sclerosis. Currently, the route of HHV-6 entry into the CNS is unknown. Using autopsy specimens, we found that the frequency of HHV-6 DNA in the olfactory bulb/tract region was among the highest in the brain regions examined. Given this finding, we investigated whether HHV-6 may infect the CNS via the olfactory pathway. HHV-6 DNA was detected in a total of 52 of 126 (41.3%) nasal mucous samples, showing the nasal cavity is a reservoir for HHV-6. Furthermore, specialized olfactory-ensheathing glial cells located in the nasal cavity were demonstrated to support HHV-6 replication in vitro. Collectively, these results support HHV-6 utilization of the olfactory pathway as a route of entry into the CNS.

Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors.

To understand how the nervous system processes information, a map of the connections among neurons would be of great benefit. Here we describe the use of vesicular stomatitis virus (VSV) for tracing neuronal connections in vivo. We made VSV vectors that used glycoprotein (G) genes from several other viruses. The G protein from lymphocytic choriomeningitis virus endowed VSV with the ability to spread transsynaptically, specifically in an anterograde direction, whereas the rabies virus glycoprotein gave a specifically retrograde transsynaptic pattern. The use of an avian G protein fusion allowed specific targeting of cells expressing an avian receptor, which allowed a demonstration of monosynaptic anterograde tracing from defined cells. Synaptic connectivity of pairs of virally labeled cells was demonstrated by using slice cultures and electrophysiology. In vivo infections of several areas in the mouse brain led to the predicted patterns of spread for anterograde or retrograde tracers.

Subclinical brain injury caused by H5N1 influenza virus infection.

Although H5N1 influenza A viruses can cause systemic infection, their neurotropism and long-term effects on the central nervous system (CNS) are not fully understood. We assessed H5N1viral invasion of the CNS and its long-term effects in a ferret model. An H5N1 virus caused nonsuppurative encephalitis, which lasted for 3 months without neurologic signs. Further, another H5N1 virus caused nonsuppurative vasculitis with brain hemorrhage. Three-dimensional analysis of viral distribution in the brain identified the olfactory system as a major route for brain invasion. The efficient growth of virus in the upper respiratory tract may thus facilitate viral brain invasion.

Experimental intranasal infection of equine herpesvirus 9 (EHV-9) in suckling hamsters: kinetics of viral transmission and inflammation in the nasal cavity and brain.

Equine herpesvirus 9 (EHV-9), the newest member of the equine herpesvirus family, is a highly neurotropic herpesvirus that induces encephalitis in a variety of animals. To access transmission of EHV-9 in the nasal cavity and brain, a suckling hamster model was developed so that precise sagittal sections of nasal and cranial cavities including the brain could be processed, which proved useful in detecting viral transmission as well as extension of pathological lesions. Suckling hamsters were inoculated intranasally with EHV-9, and were sacrificed at 6, 12, 18, 24, 36, 48, and 60 h post inoculation (PI). Sagittal sections of the entire head, including nasal and cranial cavities including the brain, were made to assess viral kinetics and identify the progress of the neuropathological lesions. At 12 to 24 h PI the virus attached to and propagated in the olfactory epithelium, and infected adjacent epithelial cells. At 48 h PI, immunohistochemistry for EHV-9 viral antigen showed that virus had extended from the site of infection into the olfactory bulb and olfactory nerve. These results indicate that EHV-9 rapidly invades the brain via the olfactory route after experimental intranasal infection.

Pathogenesis of aerosolized Eastern Equine Encephalitis virus infection in guinea pigs.

Mice and guinea pigs were experimentally exposed to aerosols containing regionally-distinct strains (NJ1959 or ArgM) of eastern equine encephalitis virus (EEEV) at two exclusive particle size distributions. Mice were more susceptible to either strain of aerosolized EEEV than were guinea pigs; however, clinical signs indicating encephalitis were more readily observed in the guinea pigs. Lower lethality was observed in both species when EEEV was presented at the larger aerosol distribution (> 6 mum), although the differences in the median lethal dose (LD50) were not significant. Virus isolation and immunohistochemistry indicated that virus invaded the brains of guinea pigs within one day postexposure, regardless of viral strain or particle size distribution. Immunohistochemistry further demonstrated that neuroinvasion occurred through the olfactory system, followed by transneuronal spread to all regions of the brain. Olfactory bipolar neurons and neurons throughout the brain were the key viral targets. The main microscopic lesions in infected guinea pigs were neuronal necrosis, inflammation of the meninges and neuropil of the brain, and vasculitis in the brain. These results indicate that guinea pigs experimentally infected by aerosolized EEEV recapitulate several key features of fatal human infection and thus should serve as a suitable animal model for aerosol exposure to EEEV.

Brain lesions induced by experimental intranasal infection of Japanese encephalitis virus in piglets.

Non-suppurative encephalitis was experimentally induced in three-week-old piglets by a single intranasal inoculation of Japanese encephalitis virus (JEV) isolated from field pigs. The lesions consisted of glial cell aggregates and perivascular cuffing throughout the olfactory tract and pyriform cortex. JEV antigens were detected in the cytoplasm and neuronal processes of small nerve cells in the granule cell layer of the olfactory bulb, in the neuronal processes of the olfactory tract and in the cytoplasm of neurons in the pyriform cortex. The distribution of the antigens corresponded closely with the distribution of brain lesions. These findings suggest that JEV may enter the brain by the olfactory pathway in addition to via haematogenous spread in piglets.

Pathogenesis of equine herpesvirus-1 infection in the mouse model.

Equine herpesvirus-1 (EHV-1) is a major equine pathogen causing respiratory diseases, abortions and severe neurological disorders. The basis of neurological disturbances is, as in other organs, infection of endothelial cells, followed by vasculitis, thrombosis and ischaemic damage of the parenchyma. Here, a murine model was used to explore the mechanism of entry to, and spread within the brain, the cell affinity of the agent and the modulating role of the immune defence, which are all factors governing the pathogenesis of the neurological disease. Because controversial views exist about these mechanisms, we undertook a neuropathological study with intranasally infected adult mice. EHV-1 entered the brain through the olfactory neuroepithelium and along the olfactory nerves, and spread transsynaptically in rostro-caudal direction, using olfactory and limbic neuronal networks. Exclusively neurons were infected. The cellular immune reaction exerted a restraining effect on virus dissemination. Following nasal infection, the olfactory route was the major pathway for virus entry and dissemination, involvement of the trigeminal nerve in virus spread seems much less probable. In the adult mouse brain EHV-1 behaves as a typical neurotropic agent, using, similarly to other herpesviruses, the neuronal networks for dissemination. Vasculitis, the predominant type of lesion in natural infection, and endothelial cell positivity for EHV-1 were detectable only in the lung. Thus, this agent exhibits in the mouse a dual affinity: it is neurotropic in the brain, and endotheliotropic in visceral organs. Consideration of pathogenetic aspects of equine and experimental murine EHV-1 infections also helps a better understanding of human herpetic brain disease.

Olfactory inputs to hypothalamic neurons controlling reproduction and fertility.

In order to gain insight into sensory processing modulating reproductive behavioral and endocrine changes, we have aimed at identifying afferent pathways to neurons synthesizing luteinizing hormone-releasing hormone (LHRH, also known as gonadotropin-releasing hormone [GnRH]), a key neurohormone of reproduction. Injection of conditional pseudorabies virus into the brain of an LHRH::CRE mouse line led to the identification of neuronal networks connected to LHRH neurons. Remarkably, and in contrast to established notions on the nature of LHRH neuronal inputs, our data identify major olfactory projection pathways originating from a discrete population of olfactory sensory neurons but fail to document any synaptic connectivity with the vomeronasal system. Accordingly, chemosensory modulation of LHRH neuronal activity and mating behavior are dramatically impaired in absence of olfactory function, while they appear unaffected in mouse mutants lacking vomeronasal signaling. Further visualization of afferents to LHRH neurons across the brain offers a unique opportunity to uncover complex polysynaptic circuits modulating reproduction and fertility.

Invasion and spread of equine herpesvirus 9 in the olfactory pathway of pigs after intranasal inoculation.

The neuropathogenesis of equine herpesvirus 9 (EHV-9) in pigs was investigated by intranasal inoculation of the virus together with intramuscular administration of dexamethasone (DM). All infected pigs developed characteristic meningo-encephalitis, accompanied by basophilic intranuclear inclusion bodies in the neuronal cells. One non-DM-treated and two DM-treated pigs had prominent malacic lesions in the rhinencephalon. Associated with the encephalitic lesions, there was invariably an increase in the number of nucleated cells in the cerebrospinal fluid (CSF). EHV-9 antigen was first detected in the nasal and olfactory epithelial cells in the nasal cavity, and in the neuroglial cells in the olfactory bulb. Subsequently it was demonstrated in the amygdaloid and caudate nuclei, and putamen. The virus was not isolated from the CSF. These results suggest that, after intranasal inoculation, EHV-9 replicates in the olfactory epithelial cells, spreading to the central nervous system via the olfactory pathway.

Bovine herpesvirus 5 glycoprotein E is important for neuroinvasiveness and neurovirulence in the olfactory pathway of the rabbit.

Glycoprotein E (gE) is important for full virulence potential of the alphaherpesviruses in both natural and laboratory hosts. The gE sequence of the neurovirulent bovine herpesvirus 5 (BHV-5) was determined and compared with that of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gE open reading frames showed that they had 72% identity and 77% similarity. To determine the role of gE in the differential neuropathogenesis of BHV-1 and BHV-5, we have constructed BHV-1 and BHV-5 recombinants: gE-deleted BHV-5 (BHV-5gEDelta), BHV-5 expressing BHV-1 gE (BHV-5gE1), and BHV-1 expressing BHV-5 gE (BHV-1gE5). Neurovirulence properties of these recombinant viruses were analyzed using a rabbit seizure model (S. I. Chowdhury et al., J. Comp. Pathol. 117:295-310, 1997) that distinguished wild-type BHV-1 and -5 based on their differential neuropathogenesis. Intranasal inoculation of BHV-5 gEDelta and BHV-5gE1 produced significantly reduced neurological signs that affected only 10% of the infected rabbits. The recombinant BHV-1gE5 did not invade the central nervous system (CNS). Virus isolation and immunohistochemistry data suggest that these recombinants replicate and spread significantly less efficiently in the brain than BHV-5 gE revertant or wild-type BHV-5, which produced severe neurological signs in 70 to 80% rabbits. Taken together, the results of neurological signs, brain lesions, virus isolation, and immunohistochemistry indicate that BHV-5 gE is important for efficient neural spread and neurovirulence within the CNS and could not be replaced by BHV-1 gE. However, BHV-5 gE is not required for initial viral entry into olfactory pathway.

Neural invasion of two virulent suid herpesvirus 1 strains in neonatal pigs with or without maternal immunity.

The neural invasion of two virulent Suid Herpesvirus 1 (SHV1) strains was examined in neonatal pigs with or without maternal immunity. One-week-old pigs with comparable levels of maternal immunity (SN-titer = 12-48) were intranasally inoculated with 10(7.0) TCID50 of either of the Ka or E21 strains. The invasion of the strains was examined in the nasal mucosa and in three neuronal levels of the trigeminal nervous pathway as well as in three levels of the olfactory nervous pathway by virus titration and immunohistochemistry (IHC). In control pigs without specific antibodies, both strains invaded up to the end level of each neural pathway. In pigs with maternal immunity, the Ka strain invaded only up to the 2nd level of each pathway with titers being significantly lower (p<0.05) than in the negative controls. However, the E21 strain invaded up to the end levels in both neural pathways of immune pigs with virus titers being similar to those observed in non-immune pigs (p>0.05). IHC revealed that maternal antibodies can protect against a fibroblast-mediated spread of the Ka strain in the lamina propria of the nasal mucosa, as well as against a local spread of the Ka and E21 strains from neurons to their satellite cells in the trigeminal ganglion. In conclusion, the nature of virus strain determines the invasion of SHV1 within the nervous system of maternally-immune neonatal pigs.

Comparative neurovirulence and tissue tropism of wild-type and attenuated strains of Venezuelan equine encephalitis virus administered by aerosol in C3H/HeN and BALB/c mice.

To assess the potential for aerosol administration of vaccines for Venezuelan equine encephalitis virus (VEE), we compared the neurovirulence and tissue tropism of the wild-type Trinidad donkey (TrD) strain to those of the attenuated TC83 and V3526 strains of VEE in mice. Six to 8-week-old female C3H/HeN and BALB/c mice were aerosol exposed to one of the three VEE strains. Three mice of each strain were euthanatized at different times and their tissues were processed and stained using hematoxylin and eosin, immunohistochemistry, and in situ hybridization. All three viral strains infected the brains of mice and induced encephalitis. TrD spread caudally from the olfactory bulbs to all regions of the brain, caused widespread necrotizing panencephalitis by day 5, and resulted in 100% mortality (geometric mean = 7 days) in both mouse strains. By comparison, TC83 relatively spared the caudal regions of the brain but still caused 100% mortality in the C3H/HeN mice (geometric mean = 12 days), yet it did not kill any BALB/c mice. V3526 infectivity of the brain was the most limited, mainly affecting the neocortex and diencephalon. This virus was not lethal in either mouse strain. The TrD strain also infected the olfactory neuroepithelium, local lymphoid tissues, teeth, and vomeronasal organs, whereas the affinity of TC83 and V3526 outside the brain was essentially limited to the olfactory neuroepithelium. Attenuated VEE strains administered to mice by aerosol have restricted tissue tropism as compared with wild-type virus; however, even attenuated strains can infect the brain and induce encephalitis.

Spread of measles virus through axonal pathways into limbic structures in the brain of TAP1 -/- mice.

The spread of measles virus into the brain was studied exploiting the olfactory pathway, which represents an important route of neuroinvasion by viruses. The virus was injected into the main olfactory bulb of wild-type mice and mice with disrupted TAP1 gene (TAP refers to the Transporter associated with Antigen Presentation), which codes for products essential for the cell-mediated immune response. Virus invasion was monitored for 4 weeks by immunohistochemistry. The distribution of measles virus was found to be restricted to brain areas connected with the olfactory bulbs. However, in the wild-type mice there was a marked infiltration of lymphocytes in the infected brain structures, and the virus did not pass beyond the piriform cortex. In the TAP1 -/- mice the virus spread more extensively along olfactory projections into the limbic system and monoaminergic brainstem neurons. Infected mice of both types developed seizures, which may have been focally evoked from the piriform cortex. This study provides evidence that measles virus can spread through axonal pathways in the brain. The findings obtained in the gene-manipulated mice point out that a compromised immune state of the host may potentiate targeting of virus to the limbic system through olfactory projections.