key: cord-1039330-4jhrfacx
authors: Kumari, Pratima; Rothan, Hussin A.; Natekar, Janhavi P.; Stone, Shannon; Pathak, Heather; Strate, Philip G.; Arora, Komal; Brinton, Margo A.; Kumar, Mukesh
title: Neuroinvasion and encephalitis following intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice
date: 2020-12-14
journal: bioRxiv
DOI: 10.1101/2020.12.14.422714
sha: 93cf59d8a10a876b44a81d2a3226cacb51071405
doc_id: 1039330
cord_uid: 4jhrfacx

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can cause neurological disease in humans, but little is known about the pathogenesis of SARS-CoV-2 infection in the central nervous system. Herein, using K18-hACE2 mice, we demonstrate that SARS-CoV-2 neuroinvasion and encephalitis is associated with mortality in these mice. Intranasal infection of K18-hACE2 mice with 105 plaque-forming units of SARS-CoV-2 resulted in 100% mortality by day 6 after infection. The highest virus titers in the lungs were observed at day 3 and declined at days 5 and 6 after infection. In contrast, very high levels of infectious virus were uniformly detected in the brains of all the animals at days 5 and 6. Onset of severe disease in infected mice correlated with peak viral levels in the brain. SARS-CoV-2-infected mice exhibited encephalitis hallmarks characterized by production of cytokines and chemokines, leukocyte infiltration, hemorrhage and neuronal cell death. SARS-CoV-2 was also found to productively infect cells within the nasal turbinate, eye and olfactory bulb, suggesting SARS-CoV-2 entry into the brain by this route after intranasal infection. Our data indicate that direct infection of CNS cells together with the induced inflammatory response in the brain resulted in the severe disease observed in SARS-CoV-2-infected K18-hACE2 mice.

Since the K18 promoter is known to be active in the epithelium of multiple organs of K-18-hACE2 mice [27, 36] , we also evaluated viral RNA levels in other peripheral organs. Viral RNA was detected in the heart, kidney, spleen, pancreas and liver on days 1 and 3. There was a slight increase in RNA levels on days 3 and 5 in each of these organs, suggesting limited virus replication at these sites ( Figure 2 ). These data agree with previous reports that also showed the presence of SARS-CoV-2 RNA in these organs [22] [23] [24] [25] 27, 36] . turbinates, olfactory bulbs, eye, serum, kidney, spleen, pancreas, heart and liver was determined on days 1, 3, 5 and 6 after infection by qRT-PCR and expressed as genome copies/µg of RNA. Each data point represents an individual mouse. The solid horizontal lines signify the median.

IFN signaling has a pivotal role in developing an innate and adaptive immune response to viral infection [37, 38] . Therefore, we measured the mRNA and protein levels of IFN-α in the lungs and brain. In the lungs, an increase in both IFN-α mRNA and protein levels was detected on day 1, peaked at day 3 and then decreased on day 6 after infection ( Figures 3A and 3B ). In contrast, an IFN response was not detected in the brain on days 1 and 3 after infection. High levels of IFN-α were detected in the brain only by days 5 and 6 after infection ( Figures 3C and 3D) . Overall, the induction of IFN-α correlated with the SARS-CoV-2 replication kinetics in the lungs and brain. It is interesting to note that relative IFN-α levels were comparatively higher in the lungs compared to the brains of the infected animals despite higher virus replication in the brain. of IFN-α were measured in the lungs (A) and brain (C) by qRT-PCR, and the fold change in the infected tissues compared to the corresponding mock-infected controls was calculated after normalizing to the GAPDH gene. The protein levels of IFN-α were measured in the lungs (B) and brain (D) homogenates using ELISA and expressed as pg/g of tissue. Error bars represent SEM (n = 5 mice per group). *p < 0.05; **p < 0.001. We next examined the mRNA levels of proinflammatory cytokines and chemokines in the lungs and brain of infected mice. SARS-CoV-2 infection resulted in a 10-fold increase on day 1 and a 100fold increase on day 3 in the IL-6 mRNA expression in the lungs ( Figure 4A ). The levels of TNF-α mRNA were elevated ~10-fold in the lungs on day 1 and 3 after infection. The level of IFN-γ mRNA was elevated by 15-fold on day 3. However, the levels of these cytokines had decreased by day 5 after infection. The IL-1β mRNA levels showed no significant increase at any time point after infection. There was a 100-fold increase in the expression of CCL2 on day 3 ( Figure 4B ). However, the levels of CCL2 mRNA had decreased by day 5 after infection. CCL3 mRNA levels increased slightly on day 1 and were undetectable at days 5 and 6 after infection.

In the brain, no increase in the mRNAs of the cytokines or chemokines tested was observed on day 1 after infection. Less than a 10-fold increase was observed in the cytokine mRNA levels on day 3 ( Figures 4C) . There was a 500-fold increase in IL-6 mRNA by day 5 after infection. TNF-α and IFNγ mRNA levels increased by ~ 750-fold in the brain by day 5 after infection. Similarly, IL-1β mRNA levels increased by 400-fold by day 5 after infection. Both CCL2 and CCL3 mRNA levels were elevated by almost 1,000-fold on days 5 and 6 after infection and consistent with the high level of virus in the brain ( Figure 4D ). These results indicate that the inflammatory response was more pronounced in the brain than in the lungs at the later stage of infection. 

We next analyzed the brain sections from infected mice for antigen distribution, infiltration of immune cells and cell death. Immunohistochemical staining for the SARS-CoV-2 spike protein detected cell-associated viral antigen throughout the brain at day 6 after infection. Representative data for sections from the cortex, cerebellum and hippocampus regions are shown in figure 5 . We also detected virus antigen in sections of the olfactory bulb of infected animals on day 6. H&E staining of brain sections from the infected mice demonstrated perivascular hemorrhage and neuronal cell death ( Figures 6A and 6B) . The neurons of infected mice demonstrated shrunken neuron body with light pink cytoplasmic staining representing degenerating neurons ( Figure 6B ). Enhanced leukocyte infiltration was detected within blood vessel walls and in the perivascular space ( Figure 6A ).

Evidence of leukocyte infiltration was confirmed by direct immunohistochemical analysis of the CD45 antigen, which revealed many CD45-positive cells in the brain parenchyma near neurons ( Figure 6C ). SARS-CoV-2-induced cell death was evaluated by direct TUNEL staining of brain tissues. On day 6, infected K18-hACE2 mice had elevated numbers of TUNEL-positive cells in the cortex, hippocampus and cerebellum regions, indicating increased cell death ( Figure 6D ). 

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 the neurons and cause lethal encephalitis in mice [11, 39] . SARS-CoV 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 and immune cell infiltration and CNS cell death. Peak virus titers in the brains were approximately 1,000 times higher than the peak titers in the lungs, suggesting a high replicative potential of SARS-CoV-2 in the brain. The relative up-regulation 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 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] [23] [24] [25] . 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] [23] [24] [25] . 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 and a lower viral dose (10 4 PFU). In our study, six-week-old K18-hACE2 mice were infected with 10 5 PFU. It appears that initial virus dose and age of animals are critical determinants of tissue tropism in this model. More 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.

Alterations in smell and taste are features of COVID-19 disease in humans [8, 40] . Pathological analyses of human COVID-19 autopsy tissues detected the presence of SARS-CoV-2 proteins in endothelial cells within the olfactory bulb [40, 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 [40] . 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, 26, 27, 42] . 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 [43] [44] [45] [46] . 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 tested, 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] [23] [24] [25] 27, 36] .

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. 

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We thank members of the GSU High Containment Core and the Department for Animal Research for assistance with the experiments.

The authors declare no conflict of interest.