Severe Acute Respiratory Syndrome Coronavirus Infection Causes Neuronal Death in the Absence of Encephalitis in Mice Transgenic for Human ACE2^▿

Mice.

Mice Tg for expression of hACE2 (K18-hACE2) were generated as previously described (32). Pathogen-free C57BL/6 mice were purchased the National Cancer Institute. All animal studies were approved by the University of Iowa Animal Care and Use Committee.

Mouse infections.

The Urbani strain of SARS-CoV was obtained from W. Bellini and T. Ksiazek at the Centers for Disease Control, Atlanta, GA. Virus was propagated, and the titer was determined on Vero E6 cells. The titer of virus used for all studies, as determined by plaque assay, was 7.6 × 10⁶ PFU/ml. Mice were lightly anesthetized with isoflurane and infected intranasally with 2.4 × 10⁴ PFU of SARS-CoV in 30 μl of Dulbecco's modified Eagle's medium or intracranially with the indicated doses (see figure legends) of SARS-CoV in 40 μl of Dulbecco's modified Eagle's medium. All work with SARS-CoV was conducted in the University of Iowa BSL3 (biosafety level 3) Laboratory Core Facility. In some experiments, mice were infected intranasally with 5 × 10⁴ to 8 × 10⁴ PFU of the murine CoV, mouse hepatitis virus (MHV) strain JHM (JHMV).

Histology and immunohistochemistry.

Animals were anesthetized and transcardially perfused with phosphate-buffered saline followed by zinc formalin. Organs were removed, fixed in zinc formalin, and paraffin embedded. For routine histology, sagittal or coronal sections were stained with hematoxylin and eosin. SARS-CoV viral antigen was detected using a biotin-conjugated monoclonal antibody (MAb) to SARS-CoV N protein (generously provided by John Nicholls, University of Hong Kong), followed by streptavidin-horseradish peroxidase conjugate (Jackson Immunoresearch, West Grove, PA) and diaminobenzidine (Sigma-Aldrich, St. Louis, MO). JHMV viral antigen was detected using a mouse MAb to the N protein (provided by Michael Buchmeier, University of California at Irvine, Irvine, CA) followed by biotin conjugated goat anti-mouse immunoglobulin G (IgG) (Zymed Laboratories, San Francisco, CA), streptavidin-horseradish peroxidase conjugate, and diaminobenzidine.

Scoring of lung pathology.

Hematoxylin- and eosin-stained lung sections were assessed using the scoring system described in the figure legends. Three animals for each time point were analyzed.

Neuronal counting.

Eight-micrometer brain sections were stained with cresyl violet. Neurons were quantified by manually counting cells with visible nucleoli in a blinded fashion, as previously described (12). For each region of the brain, three adjacent fields were counted at ×40 magnification and averaged. A total of four infected and four uninfected age-matched mice were analyzed.

TUNEL assay.

Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was performed using an In Situ Cell Death Detection Kit (Roche, Indianapolis, IN) according to the manufacturer's instructions.

Confocal microscopy.

Immunofluorescence staining was performed as previously described (23). Briefly, fixed, paraffin-embedded sections were deparaffinized and hydrated. Sections were permeabilized with 0.1% Triton X-100, blocked with 10% horse serum, and incubated sequentially with primary antibody overnight at 4°C and secondary antibody for 1 h at room temperature. The following primary antibodies were used: rabbit anti-mouse glial fibrillary acidic protein (GFAP) (DAKO, Carpinteria, CA), rabbit anti-mouse Iba1 (ionized calcium-binding adaptor molecule 1) (Wako Chemicals USA, Richmond, VA), goat anti-mouse interleukin-6 (IL-6) (R&D Systems, Minneapolis, MN), mouse anti-MHV N and fluorescein isothiocyanate (FITC)-conjugated anti-SARS-CoV N. Secondary antibodies utilized were Cy3-conjugated donkey anti-goat IgG, FITC-conjugated donkey anti-rabbit IgG, Cy3-conjugated donkey anti-mouse IgG, or FITC-conjugated donkey anti-mouse IgG (all purchased from Jackson Immunoresearch). Microscopy was conducted on a Zeiss LSM 510 Confocal Microscope (Carl Zeiss MicroImaging, Thornwood, NY).

Statistical analysis.

A Student's t test was used to analyze differences in mean values between groups. All results are expressed as means ± standard errors of the means. P values of <0.05 were considered statistically significant.

SARS-CoV infects airways and alveoli of K18-hACE2 mice but causes limited pathology.

Although our prior study suggested that infection of the brain was the major contributory factor in a fatal outcome, we also detected changes in the lungs consistent with aspiration, present only in K18-hACE2 mice at day 4 p.i., that hampered efforts to determine the importance of pulmonary infection in disease. Consequently, we initiated an additional set of studies examining a more complete time course of infection in both Tg and non-Tg animals. Our initial studies used hACE2 Tg mice that were generated on a (C57BL/6 × SJL/J)F₂ background and were crossed two to three times to C57BL/6 mice (32). Since then, we have backcrossed the mice an additional five to six times to B6 mice, without observing any changes in susceptibility to SARS-CoV infection. For the studies described in this report, we used only animals from a single line (line 3), the line that exhibited the most prolonged disease time course, with mice surviving until days 5 to 7 p.i. We sacrificed intranasally inoculated Tg mice on a daily basis at days 1 to 6 p.i. Mice began to die by day 5 p.i., and about 30% of mice survived to day 6 p.i. Using a biotin-conjugated anti-SARS-CoV nucleocapsid protein MAb, we confirmed the presence of viral antigen in the conducting airway epithelia of both K18-hACE2 mice and non-Tg controls at days 1 and 2 p.i. (Fig. 1A to D and G), with antigen clearance from these sites in both groups of mice by day 3 (Fig. 1G). In our initial study, lungs past day 2 p.i. were not examined for virus antigen. Therefore, using these newly generated samples, we showed that viral antigen was detected in the alveoli of K18-hACE2 but not non-Tg mice, with maximal levels observed at day 4 p.i. (Fig. 1E, F, and H) before declining at later times (Fig. 1H).

Histological examination of lungs showed mild pathological changes at early times p.i. At day 1 p.i., there was scant evidence of inflammation but a subtle increase in cell bulging/sloughing into the airways in both K18-hACE2 and non-Tg mice (Fig. 2A and I). Starting at day 2, we detected similar amounts of peribronchiolar inflammation and proliferation in both types of mice (Fig. 2B, C, J, and K). In contrast, interstitial disease, characterized by perivascular and septal cellular infiltration, edema, and congestion, was detected in all K18-hACE2 but not non-Tg mice (Fig. 2D). We also detected intense neutrophilic infiltrates in the airways of all K18-hACE2 mice sacrificed later than day 4 p.i. (Fig. 2E), in agreement with our previous results (32). Some affected airways contained foreign debris (Fig. 2F), supporting the conclusion that these infiltrates resulted from aspiration. While some of the interstitial disease observed in K18-hACE2 mice may result from aspiration, some areas of interstitial disease nearly completely overlapped with sites of virus replication (Fig. 2G and H), suggesting that lung disease resulted at least partly from direct virus infection.

Neuronal infection is the major cause of death in K18-hACE2 mice.

To more definitively distinguish between the contributions of lung versus brain infection to a lethal outcome in K18-hACE2 mice, we inoculated mice intracranially with high (3.2 × 10⁴ PFU) and low (3.2 or 320 PFU) doses of SARS-CoV. Mice infected intracranially at all doses became moribund by day 4 p.i. Infection with 3.2 × 10⁴ PFU resulted in a pattern of widespread neuronal infection similar to that observed following intranasal inoculation (Fig. 3A; see also Fig. 4B). Histological examination of the lungs revealed intense neutrophilic infiltrates (Fig. 3B and C) in six of seven cases, with foreign debris/bacteria present in five cases, suggestive of aspiration pneumonia. Low titers of infectious virus were present (Fig. 3G), but we were not able to detect viral antigen in the lungs (Fig. 3C). By contrast, after inoculation with 3.2 or 320 PFU of SARS-CoV, we detected viral antigen throughout the brain (Fig. 3D), although the extent of infection was much less than observed after intranasal or high-dose intracranial inoculation (Fig. 3A; see also Fig. 4B). Unlike mice inoculated intracranially with 3.2 × 10⁴ PFU of SARS-CoV, the lungs appeared normal in all but one of four mice examined (Fig. 3E and F). In this single animal, we observed evidence of mild aspiration (data not shown). As in mice inoculated intracranially with 3.2 × 10⁴ PFU, we detected no viral antigen in the lungs but did detect virus by plaque assay (Fig. 3G). Collectively, these results suggest that SARS-CoV-infected K18-hACE2 mice die primarily as a direct result of CNS, not pulmonary, infection. They also suggest that the airway infiltrates detected in moribund mice are a complication of the brain infection, occur only after extensive neuronal infection, and are not required for a lethal outcome.

As the low-dose intracranially inoculated animals exhibited limited viral antigen distribution but still rapidly succumbed to infection, we reasoned that neurons in a vital region of the brain would be infected. Examination of brain sections from mice inoculated with 3.2 or 320 PFU of SARS-CoV revealed that the dorsal vagal complex (nucleus tractus solitarii, area postrema, and dorsal motor nucleus of the vagus), critical for cardiorespiratory function, was infected in all four samples examined (Fig. 3H). We also examined brains from mice inoculated intracranially (Fig. 3I) or intranasally (Fig. 3J) with high doses of virus and observed that the dorsal vagal complex was infected in all instances. Thus, the fulminant disease occurring in SARS-CoV-infected K18-hACE2 mice may result from infection and functional impairment of this critical brain region.

SARS-CoV spreads in the brain of K18-hACE2 mice transneuronally primarily from the olfactory bulb and induces neuronal loss.

The widespread distribution of viral antigen in the brains of intranasally infected K18-hACE2 mice and the temporal pattern of disease led us to postulate previously that infection occurred via hematogenous spread following lung infection (32). However, from these initial experiments, we could not distinguish transneuronal spread from one or more sites of entry into the CNS from blood-borne spread. To evaluate the kinetics and spread of the neuronal infection, as well as determine the site of entry, we harvested brains from infected animals at days 1 to 6 p.i. Immunohistochemical staining for the SARS-CoV N protein revealed no detectable viral antigen at day 1 or 2 p.i. Viral antigen was first detectable and surprisingly widespread at day 3 p.i. (Fig. 4B). By day 4, we detected virus throughout the brain (Fig. 4C). In mice surviving beyond day 4, viral antigen remained widespread, but small areas in which virus appeared to be cleared became evident (Fig. 4D). These regions of virus clearance were the first ones to be infected, and, as shown below, clearance was accompanied by neuronal loss.

Virus spread rapidly within the brain between days 2 and 3 following intranasal inoculation, making it difficult to determine the initial sites of infection. To determine sites of virus entry, we stained sagittal and coronal sections for viral antigen, using brains harvested at 6-h intervals from 48 to 72 and at 96 h p.i. Virus antigen was first detected at 60 to 66 h (Fig. 4A) p.i. and was most abundant in the olfactory bulb. Regions of the cortex (piriform and infralimbic cortices), basal ganglia (ventral pallidum and lateral preoptic regions), and midbrain (dorsal raphe) were also heavily infected; these regions all have first- or second-order connections with the olfactory bulb. Other areas, such as the parataenial nucleus of the thalamus, paraventricular nucleus of the hypothalamus, and the medial and basolateral amygdala, were also virus antigen positive but less consistently (Table 1). This pattern of antigen distribution is strongly suggestive of entry via the olfactory nerve with subsequent transneuronal spread. Examination of brains harvested from mice at day 3 p.i. reinforced this notion. In these samples, antigen was much more widespread but was largely restricted to first- and second-order connections of the olfactory bulb. By day 4 p.i., nearly all regions of the brain stained positive for SARS-CoV antigen with the exception of the ventral anterior olfactory nucleus, the dentate gyrus, and the cerebellum.

While a number of the areas infected by day 4 p.i. were consistent with spread along olfactory connections, many infected sites were not connected directly with the olfactory bulb. Some of these regions, such as the dorsal vagal complex, nucleus ambiguus, and hypoglossal nucleus, suggested infection via oral uptake, whereas infection of other sites, such as the cochlear nuclei, substantia nigra, and temporal and occipital cortices could not be explained by either olfactory or oral entry/spread. To further assess the role of oral uptake in infection of the CNS, we assayed the small intestine and colon for virus antigen in enterocytes and associated nerve plexuses at day 4 p.i. However, we could not detect viral antigen anywhere in the gut of two infected mice (data not shown), raising the possibility that once virus is established in the brain, it spreads along specific neurotransmitter pathways (such as the serotoninergic dorsal raphe system) that innervate large portions of the brain, or the virus spreads via nonneuronal routes, such as the blood or the Virchow-Robin spaces (13, 52).

Rapid spread throughout the brain appeared to be accompanied by virus clearance at sites of initial infection and concomitant neuronal loss. To assess neuronal loss more precisely, we quantified the number of neurons in several regions of the brain at day 4 p.i. Areas that were infected at earlier times p.i., such as the cingulate and infralimbic cortices and anterior olfactory nucleus exhibited significantly fewer neurons per square millimeter (P values of 0.005, 0.00002, and 0.00006, respectively) than were found in uninfected animals (Fig. 5A). As controls, the numbers of neurons in the dentate gyrus of the hippocampus and deep cerebellar nuclei were quantified, as these structures represent brain regions that were not heavily infected until relatively late times p.i. or that remained largely uninfected. In these regions, neuronal counts were nearly identical in infected and uninfected brains, suggesting that SARS-CoV directly induced neuronal death.

Minimal signs of inflammation are detected in infected K18-hACE2-infected brains.

No evidence of inflammation was detected in the brains of any infected K18-hACE2 mice even when infection was widespread (Fig. 4G and H), and these brains were virtually indistinguishable from uninfected brains (Fig. 4E and F). For comparison, we also examined brains harvested from mice infected with another group 2 CoV, JHMV, which is highly neurotropic and induces extensive cellular infiltration. As expected, JHMV-infected brains displayed perivascular inflammation (Fig. 4I and J, closed arrows) and meningitis (open arrow) when examined at day 6 p.i. The lack of inflammation in SARS-CoV-infected brains raised the possibility that neurons died by apoptosis, a form of cell death associated with minimal cellular infiltration. Therefore, we assayed cells for apoptosis by performing TUNEL staining at days 1 to 6 p.i. TUNEL-positive cells were not detected in any of the SARS-CoV-infected brains (Fig. 5D and E) but were readily detected in DNase 1-treated uninfected brain sections (Fig. 5B and C). Thus, apoptotic death of neurons does not explain the lack of inflammation that we observed.

Another consequence of inflammation in the CNS is activation of astrocytes and microglia and migration to areas of infection, where they exert numerous effector functions involved in pathogen clearance, modulation of the immune response, and tissue repair (reviewed in references 3 and 15). To examine changes in the activation and location of microglia and astrocytes, we costained tissue sections from infected brains with anti-SARS-CoV N antibody and antibodies directed against GFAP or Iba1, which stain astrocytes and microglia/macrophages, respectively. GFAP expression by astrocytes was detected in limited areas in SARS-CoV-infected animals with a distribution and intensity of staining similar to that observed in naïve animals (Fig. 6A). In contrast, JHMV-infected brains displayed upregulation of GFAP, and the GFAP-positive cells were found throughout the brain. These findings suggest that astrocytes in SARS-CoV-infected mice were not activated, despite extensive neuronal infection. However, we did observe similar numbers of Iba1-positive cells in the brains of SARS-CoV and JHMV-infected animals at day 6 p.i. while very few were detected in naïve controls (Fig. 6B).

While the cellular response to SARS-CoV in the brains of K18-hACE2 mice was minimal, proinflammatory cytokines and chemokines are upregulated in the brains of infected mice at day 4 p.i. (32, 49). In JHMV-infected mice and in other CNS infections, cytokines are generally expressed by glia and infiltrating cells, especially astrocytes and macrophages (3, 42, 47). In general, these cytokine-producing cells are uninfected, and cytokines are expressed as part of the proinflammatory response. To determine the cellular source for these cytokines, we examined the localization of a representative cytokine, IL-6, which is highly upregulated in SARS-CoV-infected brains and has both protective and immunopathological roles in the inflamed CNS (9, 34). Abundant amounts of IL-6 were detected in infected brains (Fig. 7A). Astrocytes are known to be a major site of IL-6 synthesis under conditions of inflammation (17), and consistent with this, some of the IL-6-expressing cells were uninfected astrocytes. In contrast, however, the majority of cells that produced IL-6 were SARS-CoV infected and were morphologically neurons. As a control, we showed that although occasional neurons stained positive for IL-6, astrocytes were the major source of IL-6 in the brains of JHMV-infected mice (Fig. 7B).