Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in humans can cause pneumonia, acute respiratory distress syndrome, acute lung injury, cytokine storm syndrome and death [1
]. Although SARS-CoV-2 infection primarily causes respiratory disease, some patients develop symptoms of neurological disease, such as headache, loss of taste and smell, ataxia, meningitis, cognitive dysfunction, memory loss, seizures and impaired consciousness [3
]. SARS-CoV-2 infection also induces long-term neurological sequelae in at least one-third of human cases. Infection with other coronaviruses, such as mouse hepatitis virus (MHV) in mice and SARS-CoV-1 and Middle East Respiratory Syndrome (MERS) virus in humans, has been shown to cause neurological disease [11
]. However, little is known about the pathophysiology of SARS-CoV-2-associated neurological disease in humans.
Central nervous system (CNS) cells that express the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) include neurons, glial cells and astrocytes [13
]. ACE2 is expressed in multiple human brain areas, including the amygdala, cerebral cortex and brainstem, with the highest expression levels found in the pons and medulla oblongata in the brainstem that contain the medullary respiratory centers of the brain [8
]. Several human autopsy reports have documented the presence of SARS-CoV-2 RNA in brain tissues [16
]. Human iPSC-derived neural progenitor cells (NPCs) have been shown to be permissive to SARS-CoV-2 infection, and both viral proteins and infectious viral particle production were detected in neurospheres and brain organoids infected with SARS-CoV-2 [18
]. Human autopsy reports have shown evidence of lymphocytic panencephalitis, meningitis and brainstem perivascular and interstitial inflammatory changes with neuronal loss in COVID-19 patients [20
]. These data suggest that SARS-CoV-2 can productively infect human CNS cells [21
]. However, the contributions of CNS cell infection and induced neuroinflammation to the pathogenesis of SARS-CoV-2-associated disease are not well understood.
Small animal models provide a means for studying the neurological complications associated with SARS-CoV-2 infection. K18-hACE2-transgenic mice were originally developed for the study of SARS-CoV-1 pathogenesis. hACE2 expression in K18-hACE2 transgenic mice is driven by the human cytokeratin 18 (K18) promoter. It was recently reported that intranasal inoculation with SARS-CoV-2 results in a rapidly fatal disease in K18-hACE2 mice [22
]. These studies were focused on describing the acute lung injury in SARS-CoV-2-infected K18-hACE2 mice that was associated with high levels of inflammatory cytokines and accumulation of immune cells in the lungs [22
]. In these published studies, neither infectious virus nor viral RNA was detected in the olfactory bulbs or brains of the majority of the infected animals, indicating restricted neurotropism of SARS-CoV-2 in K18-hACE2 mice. In the present study, we show that intranasal infection of six-week-old K18-hACE2 mice by SARS-CoV-2 can cause severe neurological disease, with the brain being a major target organ for infection by this route of infection, and neuroinflammation and neuronal death contributing to the infection-associated morbidity and mortality. The data also suggest that SARS-CoV-2 can be trafficked to the brain via the olfactory bulb with subsequent transneuronal spread, as has been reported for other coronaviruses [26
This study demonstrates a critical role of direct infection of CNS cells and of the inflammatory response in mediating SARS-CoV-2-induced lethal disease in K18-hACE2 mice. Intranasal inoculation of the virus results in a lethal disease with high levels of virus replication in the brain. Virus infection of the CNS was accompanied by an inflammatory response as indicated by the production of cytokines/chemokines, infiltration of leukocytes into the perivascular space and parenchyma and CNS cell death. Our data also indicate that following infection by the intranasal route, the virus enters the brain by traversing the cribriform plate and infecting neuronal processes located near the site of intranasal inoculation.
Some animal coronaviruses, such as MHV readily infect neurons and cause lethal encephalitis in mice [11
]. SARS-CoV-1 infection also induces severe neurological disease after intranasal administration in K18-hACE2 mice [27
]. Similarly, in our study, SARS-CoV-2 virus antigen was detected throughout the brain, including the cortex, cerebellum and hippocampus. The onset of severe disease in SARS-CoV-2-infected mice correlated with peak viral levels in the brain, immune cell infiltration and CNS cell death. Peak virus titers in the brains were approximately 1000 times higher than the peak titers in the lungs, suggesting a high replicative potential of SARS-CoV-2 in the brain. The relative upregulation of cytokine and chemokine mRNAs was approximately 10 to 50 times higher in the brain compared to the lungs, strongly suggesting that extensive neuroinflammation contributed to clinical disease in these mice.
It was recently reported that SARS-CoV-2 infection of K18-hACE2 mice causes severe pulmonary disease with high virus levels detected in the lungs of these mice and that mortality was due to the lung infection [22
]. In these studies, viral RNA was undetectable in the brains of the majority of the infected animals, indicating a limited role of brain infection in disease induction. An important distinction between our study and others is that we detected high infectious virus titers in the olfactory system and brains of 100% of the infected K18-hACE2 mice. This phenotype was not consistently observed in the aforementioned K18-hACE2 mouse studies [22
]. Moreover, none of the published studies evaluated the extent of neuroinflammation and neuropathology at the later stages of infection. Our results showed that the inflammatory response was more pronounced in the brain than in the lungs on days 5 and 6 after infection. Although both our study and the previous studies infected mice via the intranasal route, the other studies used older (7-to 9-week-old) K18-hACE2 mice and a lower viral dose (104
PFU), and in one study, only analyzed samples 3 days after infection [22
]. In our study, six-week-old K18-hACE2 mice were infected with 105
PFU. However, unpublished data from our laboratory demonstrated that six-week-old K18-hACE2 mice infected with a lower viral dose (103
PFU) also exhibit a similar phenotype, suggesting that the brain is a major site of infection following infection by the intranasal route, regardless of the virus dose used. Additional studies are needed to clarify the parameters that differentially affect tissue tropism, routes of virus dissemination and mechanisms of lung and brain injuries in K18-hACE2 mice following SARS-CoV-2 infection. Recent studies have suggested that humans have a higher chance of developing a brain infection if they are infected intranasally with a high dose of virus [40
Alterations in smell and taste are features of COVID-19 disease in humans [8
]. Pathological analyses of human COVID-19 autopsy tissues detected the presence of SARS-CoV-2 proteins in endothelial cells within the olfactory bulb [41
]. Our data indicate that SARS-CoV-2 can productively infect cells within the nasal turbinate, eye and olfactory bulb in intranasally infected K18-hACE2 mice. Virus infection of cells in these tissues in humans may explain the loss of smell associated with some COVID-19 cases [41
]. The detection of virus replication in these tissues suggests that SARS-CoV-2 can access the brain by first infecting the olfactory bulb and then spreading into the brain by infecting connecting brain neuron axons. This hypothesis is consistent with previously published reports that neurotropic coronaviruses infect olfactory neurons and are transmitted to the brain via axonal transportation [8
]. Many viruses, such as HSV-1, Nipah virus, rabies virus, Hendra virus and influenza A virus, have also been shown to enter the CNS via olfactory sensory neurons [44
]. Another route by which a virus can gain access to the brain is via the disruption of the blood–brain barrier (BBB). However, we could not detect any virus in the serum of the infected mice at any time after infection, suggesting a limited role of BBB disruption in SARS-CoV-2 neuroinvasion. This finding is in agreement with previously published studies that detected little or no virus in the blood of K18-hACE2 mice after infection with SARS-CoV-1 or SARS-CoV-2 [22
In summary, we found that intranasal infection of K18-hACE2 mice by SARS-CoV-2 causes severe neurological disease. Our data demonstrate that the CNS is the major target of SARS-CoV-2 infection in K18-hACE2 mice under the conditions used, and that brain infection leads to immune cell infiltration, inflammation and cell death.