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Multisystem screening reveals SARS‐CoV‐2 in neurons of the myenteric plexus and in megakaryocytes

Abstract SARS‐CoV‐2, the causative agent of COVID‐19, typically manifests as a respiratory illness, although extrapulmonary involvement, such as in the gastrointestinal tract and nervous system, as well as frequent thrombotic events, are increasingly recognised. How this maps onto SARS‐CoV‐2 organ tropism at the histological level, however, remains unclear. Here, we perform a comprehensive validation of a monoclonal antibody against the SARS‐CoV‐2 nucleocapsid protein (NP) followed by systematic multisystem organ immunohistochemistry analysis of the viral cellular tropism in tissue from 36 patients, 16 postmortem cases and 16 biopsies with polymerase chain reaction (PCR)‐confirmed SARS‐CoV‐2 status from the peaks of the pandemic in 2020 and four pre‐COVID postmortem controls. SARS‐CoV‐2 anti‐NP staining in the postmortem cases revealed broad multiorgan involvement of the respiratory, digestive, haematopoietic, genitourinary and nervous systems, with a typical pattern of staining characterised by punctate paranuclear and apical cytoplasmic labelling. The average time from symptom onset to time of death was shorter in positively versus negatively stained postmortem cases (mean = 10.3 days versus mean = 20.3 days, p = 0.0416, with no cases showing definitive staining if the interval exceeded 15 days). One striking finding was the widespread presence of SARS‐CoV‐2 NP in neurons of the myenteric plexus, a site of high ACE2 expression, the entry receptor for SARS‐CoV‐2, and one of the earliest affected cells in Parkinson's disease. In the bone marrow, we observed viral SARS‐CoV‐2 NP within megakaryocytes, key cells in platelet production and thrombus formation. In 15 tracheal biopsies performed in patients requiring ventilation, there was a near complete concordance between immunohistochemistry and PCR swab results. Going forward, our findings have relevance to correlating clinical symptoms with the organ tropism of SARS‐CoV‐2 in contemporary cases as well as providing insights into potential long‐term complications of COVID‐19. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Introduction SARS-CoV-2 is an enveloped RNA virus, containing an outer lipid membrane derived from the host cell.
such as TMPRSS2, leading to fusion of viral and cellular membranes [1]. TMPRSS2 is expressed in a range of tissues, although TMPRSS2 and ACE2 are coexpressed only in a subset of cells; for example, respiratory epithelial cells, enterocytes, and neurons of the myenteric plexus [1,2]. Additional structural viral proteins include the envelope (E), membrane (M), and nucleocapsid (N) proteins, the latter of which encapsulates the viral RNA. Molecular detection of such proteins and/or viral RNA, via immunohistochemistry and in situ hybridisation, respectively, is emerging as a method whereby the tissue tropism of SARS-COV-2 can be elucidated [3].
Coronavirus disease 2019 (COVID- 19), the clinical disease caused by SARS-CoV-2, primarily affects the respiratory tract, although there is increasing evidence suggesting involvement of multiple organ systems. Clinically, a subset of patients present with alternative symptoms including gastrointestinal; for example, diarrhoea and nausea [4][5][6]. Reported neurological complications following SARS-CoV-2 infection include anosmia, ageusia, headache, impaired consciousness, and cognitive deficits [7]. Moreover, it appears that respiratory failure is not the sole cause of death in COVID-19; thrombosis is emerging as an important cause of morbidity and mortality, particularly in patients with underlying cardiovascular conditions [6]. This is thought to relate to endothelial involvement, given the expression of ACE2 receptor on these cells [4][5][6].
Several histopathological studies have been performed on postmortem tissue from COVID-19 patients, including autopsies of patients who died of severe SARS-CoV-2 infection. Common histological alterations in these patients include diffuse alveolar damage (DAD), thrombotic events, and immune cell depletion [8]. The clinicopathological correlation for several organs potentially affected in COVID-19, such as the kidney, pancreas, and liver are, however, not fully understood. Special attention has been paid to the nervous system in the search for pathological alterations responsible for the neurological symptoms of COVID-19 given the neuroinvasive potential of SARS-CoV-2 [9]. Potential long-term neuropathological sequelae of COVID-19 are also of interest, as there is accumulating evidence that environmental agents including viruses may play a role in the initiation of the most common neurodegenerative diseases [10][11][12][13]. Histopathological investigations of fatal COVID-19 patients with neurological syndromes have so far not shown definitive evidence of viral protein or RNA in the brain, despite some studies reporting neuropathological abnormalities such as microglial activation or focal lymphocytic infiltrates [14][15][16][17]. This suggests that neuroinvasion might not be responsible for neurological symptoms, which could instead be due to inflammatory responses or critical care treatment. Outside of the brain, the enteric nervous system (ENS) has been proposed as an entry point for SARS-CoV-2 into the central nervous system (CNS) via the vagus nerve reaching the medulla, given the expression of the key viral entry receptors (ACE2 and TMPRSS2) in neurons of the myenteric plexus [2].
In this study, we characterise the systemic tropism of SARS-CoV-2 in a cohort of fatal COVID-19 cases as well as in antemortem brain and tracheal biopsies using validated immunohistochemistry against SARS-CoV-2 nucleocapsid protein (NP), a structural protein critical for packaging of the viral genome [18]. We find that overall, the presence of SARS-CoV-2 NP matches the cell types with known high ACE2 receptor expression, and we describe novel extrapulmonary findings that implicate peripheral nervous system involvement (more specifically, the ENS) as well as digestive, genitourinary, and haematopoietic systems.

Cohort selection
We obained tissue from 36 patients to study the cellular tropism of SARS-CoV-2 by immunohistochemistry. The samples comprised tissue from 16 postmortem examinations and 16 antemortem biopsy specimens from the peaks of the pandemic in 2020 and four pre-COVID postmortem controls (supplementary material, Table S1). Of the 16 postmortem cases from 2020 (PM1-PM16, Table 1), 10 had multisystem organ sampling available (nine via full-body autopsies and one via percutaneous biopsy sampling), whilst only brain tissue was available for six. All 16 postmortem cases had SARS-CoV-2 infection confirmed by PCR testing for SARS-CoV-2 RNA in nasopharyngeal swabs antemortem, and clinical courses consistent with COVID-19. Details of patient selection for 14 of the cases have been previously described (PM 1-10 [8], , ).
Of the antemortem biopsy specimens, one was a brain biopsy (BB1, Table 1) and 15 were tracheal biopsies from patients admitted to intensive care during 2020 (TB1-TB15, Table 4). The patient corresponding to sample BB1 had a clinical presentation that was strongly consistent with COVID-19 and has been described in detail elsewhere [20]. For the tracheal biopsies, tissue samples were obtained from tracheal windows excised at surgical tracheostomy, performed in critically ill mechanically ventilated patients as part of their routine intensive care unit (ICU) care. SARS-CoV-2 infection status was determined by reverse transcription polymerase chain reaction (RT-PCR) or multiplex tandem PCR (MT-PCR) of intravitam nasopharyngeal swab samples. Samples were obtained from the Imperial College Healthcare Tissue Bank (ICHTB). The ICHTB is approved by Wales REC3 to release human material for research (17/WA/0161), and the samples for this project (with approvals R20012 and R20027) were issued from subcollection reference number MED_MO_20_011. Additional tissue was obtained from the University College London Hospitals NHS Trust (ref 06-202021-SE) and Brain UK (ref 20-007). For PM1-16, the next of kin gave their consent for tissues taken at coronial autopsy to be used for education, research, and audit. Adapted from (PM1-10) [8], (PM11-12) [14], (PM15-16) [19], and (BB1) [20]. Tissue for PM1-16 was obtained postmortem and BB1 is an antemortem brain biopsy. Detailed case vignettes of PM1-10 can be found within the supplementary material of Hanley et al [8].

Western blotting
Cell lysates derived from SARS-CoV-2-infected Vero E6 cultured cells containing NuPAGE LDS Sample Buffer (Invitrogen, Thermo Fisher Scientific) and 2-mercaptoethanol were boiled for 10 min and then loaded into a NuPAGE 4-12% Bis-Tris gels (Invitrogen) and run with 1X NuPAGE MES Running Buffer at 120 V. Proteins were then transferred onto methanol-activated PVDF membranes (400 mA, 1 h). Blocking nonspecific binding was accomplished using 5% semi-skimmed milk in Trisbuffered saline (TBS) with 0.2% Tween 20 (TBS-T) for 1 h. After washing with TBS-T three times, membranes were incubated with mouse anti-SARS-CoV-2 Nucleoprotein monoclonal antibody (1:1000; Creative Diagnostics, clone 4B21) diluted in 1% bovine serum albumin (BSA) and 0.01% sodium azide in TBS-T overnight at 4 C. The membrane was subsequently washed again three times with TBS-T and then incubated with HRPconjugated secondary mouse antibody (1:3000, Sigma-Aldrich, AP308P) in 5% milk. After 1 h and three TBS-T washes, HRP detection with Pierce ECL western blotting substrate (Thermo Fisher Scientific) allowed for visualisation with the GeneGnome XRQ chemiluminescent imager. The membrane was stripped (ReBlot Plus Strong Antibody Stripping Solution [10Â], SigmaMillipore), blocked again with 5% semi-skimmed milk in TBS-T, and then incubated with anti-beta actin primary antibody (1:25000; Abcam, ab8227) as a loading control. Fixed cells were washed twice in PBS, centrifuged, and the pellet was processed using the Thermo Scientific Shandon Cytoblock system according to the manufacturer's instructions. For protein lysate preparation, infections were performed as above but at an MOI of 0.1. Cells were collected by scraping 48 h postinfection, pelleted, and lysed in 500 μl ice-cold RIPA buffer on ice for 30 min. Lysates were clarified by centrifugation at 4 C for 30 min, then heated at 90 C for 10 min in Laemmli buffer (BioRad, Hercules, CA, USA) with 10% β-mercaptoethanol (Sigma-Aldrich).

Biosafety statement
All work performed at Imperial College London was approved by the local genetic manipulation safety committee of Imperial College London, St. Mary's Campus (centre number GM77), and the Health and Safety Executive of the United Kingdom, under reference CBA1.77.20.1.

Animal models
Tissues from rhesus macaques and Syrian golden hamsters were obtained from experiments carried out at UKHSA Porton Down (Wiltshire, UK). Nonhuman SARS-CoV-2 in myenteric plexus and megakaryocytes 203 primates (NHPs), specifically rhesus macaques (Indian origin) were inoculated via intranasal and intratracheal route with 5 Â 10 6 PFUs of SARS-CoV-2 (hCoV-19/Australia/VIC01/2020) and culled at 4 or 7 days postchallenge [21]. Syrian golden hamsters were inoculated by the intranasal route with 5 Â 10 4 PFUs of the same SARS-CoV-2 isolate. Hamsters were culled at 2 days postchallenge. The infected status of macaques and hamsters was confirmed by RNAScope in situ hybridisation for S-gene [21]. Samples from uninfected animals were obtained from the UKHSA Porton Down Pathology archive. All experimental work performed at UKHSA Porton Down was conducted under the authority of a UK Home Office approved project license (PDC57C033) that had been subject to local ethical review at UKHSA Porton Down by the Animal Welfare and Ethical Review Body (AWERB) and approved as required by the Home Office Animals (Scientific Procedures) Act 1986.

Statistical analysis
All statistical analyses were performed using GraphPad Prism 8 (Graphpad Software, San Diego, CA, USA). Significance of disease duration between negative and positive postmortem cases was determined using Mann-Whitney U tests.

Validation of anti-NP monoclonal antibody
Specificity of the anti-NP monoclonal antibody was evaluated using western blotting and IHC. A single band of 49 kDa, the predicted molecular mass of the NP, was detected in lysates from SARS-CoV-2-infected Vero E6 cells ( Figure 1A, lane 1), whilst no signal was observed in lysates from mock-transfected cells ( Figure 1A, lane 2). By IHC, strong cytoplasmic immunoreactivity was present in SARS-CoV-2-infected cells ( Figure 1B), whilst no staining was observed in mock-infected cells ( Figure 1C). The specificity of the anti-NP antibody for IHC was further validated in animal models of SARS-CoV-2. In the rhesus macaque model of SARS-CoV-2, there was strong positive cytoplasmic immunostaining within the lung parenchyma ( Figure 1D) in contrast to noninfected rhesus macaques, where staining was not apparent ( Figure 1E). In Syrian golden hamsters infected with SARS-CoV-2, similar strong immunoreactivity was observed in the respiratory tract and lung parenchyma ( Figure 1F), but not in technical negative controls ( Figure 1G).
Postmortem cases: clinical characteristics and relationship of tissue SARS-CoV-2 NP presence to disease duration We examined tissue from 16 postmortem cases from the peaks of the COVID-19 pandemic in 2020 (see Table 1 for detailed clinical characteristics In all 16 cases the underlying cause of death (part I of the medical certificate cause of death, MCCD) was attributed to SARS-CoV-2 infection and/or its sequelae (see Table 1 for detailed clinical characteristics).
Of the 10 cases that had tissue available for multisystem analysis ( Table 1, PM1-PM10), four showed SARS-CoV-2 NP by IHC ( Table 2) and in all four, the viral presence was multisystemic. The average time from symptom onset to time of death was significantly shorter in the positive versus the negatively stained cases (mean-= 10.3 days, SD = 3.3 days versus mean = 20.3 days, SD = 6.3 days, Mann-Whitney: U = 2, p = 0.0416), with all positive cases dying 15 days or earlier from symptom onset, whilst no positive staining was seen in patients with a longer disease courses.

Respiratory system
Tissue SARS-CoV-2 was visualised by IHC in the respiratory system of four out of ten cases with multisystem postmortem tissue available. Within the nasal cavities, scattered staining was apparent in stratified squamous epithelium of the vestibule (Figure 2A), as well as in serous and mucinous glands of the lamina propria and the surface nasal epithelium ( Figure 2B). Seromucinous gland staining was commonly patchy, with strongly positive elements near negative ones. Within the larynx, pseudostratified columnar epithelial cells displayed strong staining, as did submucosal seromucous glands ( Figure 2C). A similar pattern of seromucinous gland, epithelial, and endothelial cell staining was observed in the positive tracheal biopsies ( Figure 2D) and postmortem tracheae ( Figure 2E), with appropriate lack of staining shown in a PCR negative biopsy ( Figure 2F). Staining was also apparent in pulmonary parabronchial seromucinous glands ( Figure 2G). At the subcellular level, the staining pattern was predominantly an apical cytoplasmic or perinuclear punctuate pattern, a pattern particularly evident in surface epithelial cells ( Figure 2H,K). Widespread positivity in alveolar pneumocytes was also observed ( Figure 2I). As expected, there was no apparent anti-NP positivity in SARS-CoV-2-negative pre-COVID control lungs ( Figure 2J). Throughout the respiratory tract, hyaline cartilage was consistently negative for anti-NP staining.

Digestive system and myenteric plexus
Moderate intensity staining was observed within the minor salivary glands and tongue epithelium as well as in endothelial cells ( Figure 3A). Definite taste buds were not identified. Strong positive staining was apparent in the pharyngeal and oesophageal epithelium ( Figure 3B,

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S Gray-Rodriguez et al SARS-CoV-2 in myenteric plexus and megakaryocytes 205 C), with scattered staining of underlying endothelial cells ( Figure 3B, arrow) and submucosal glands ( Figure 3D). Weaker scattered positive staining was apparent in parietal cells of the gastric fundus ( Figure 3E). In the small intestine, mucosal staining was preferentially located to the epithelial cells within glandular crypts ( Figure 3F,G), whilst in the colon focal scattered positive staining was noted in colonic glands ( Figure 3H). SARS-CoV-2 NP was present in cholangiocytes of the hepatic bile ducts ( Figure 4A), correlating with the localisation of anti-ACE2 immunostaining ( Figure 4B), with ACE2 and SARS-CoV-2 situated predominantly on the apical end of the cells. Strong diffuse positive staining was seen throughout the hepatobiliary system in the portal tracts ( Figure 4B,D), with focal staining in adjacent hepatocytes in areas with steatosis ( Figure 4C). Endothelial viral presence was also observed in the proximity of positive hepatocytes ( Figure 4C, arrow) and bile ducts ( Figure 4D, arrows). Strong diffuse positive staining was also apparent in columnar epithelial cells of the gallbladder ( Figure 4E) and scattered weak staining in the pancreatic acini ( Figure 4F). Strong ACE2 staining was observed in epithelial cells from bile ducts, gall bladder, and pancreatic ducts in noninfected nonhuman primate tissues (data not shown).
Conspicuous strong staining was identified within neurons of the myenteric plexus, typically located between the smooth muscle layers in the colon ( Figure 5A-C). Paranuclear, vesicle-like punctate staining in the soma of some, but not all, enteric ganglion cells ( Figure 5B, -arrows). Oesophageal myenteric plexus also showed viral positivity within ganglia ( Figure 5D). Supportive glial cells intercalated between the neurons in the myenteric ganglia were unstained ( Figure 5B). The submucosal plexus was not sufficiently represented in the samples available for analysis.
Alpha-synuclein aggregation in Parkinson's disease (PD) is thought to begin in the neurons of the enteric nervous system [22], where cholinergic neurons are particularly susceptible to alpha-synuclein pathology [23,24]. Consequently, we used immunostaining for ChAT (for cholinergic neurons) and nNOS (for nitrergic neurons) to assess any preference of SARS-CoV-2 for a specific neuronal subtype. NP was visualised within both cholinergic and nitrergic neurons (supplementary material, Figure S1). No SARS-CoV-2 staining was seen in the digestive system, including the myenteric plexus, in pre-COVID postmortem controls (supplementary material, Figure S2).

Haematopoietic system and megakaryocytes
Within the bone marrow, there was SARS-CoV-2 NP present within megakaryocytes ( Figure 5E), confirmed by colocalisation with CD61 ( Figure 5F). Similar to other cells, viral protein showed a punctate pattern concentrated in the paranuclear cytoplasmic region. Scattered staining in endothelial cells was seen in spleen and lymph node (supplementary material, Figure S3A, B). No SARS-CoV-2 staining was seen in the Table 2. Summary of SARS-CoV-2 anti-NP immunohistochemical organ localisation in postmortem COVID-19 cases.  SARS-CoV-2 in myenteric plexus and megakaryocytes 207

Genitourinary system
Within the renal cortex, strong positive staining for SARS-CoV-2 NP localised to the epithelium of convoluted tubules ( Figure 6A), but glomeruli were largely negative except for occasional endothelial glomerular labelling. In the renal medulla, a striking viral presence was identified in collecting tubules ( Figure 6B). Scattered positive staining was noted in prostatic acinar cells in urothelial cells in the bladder ( Figure 6C,D).  Figure S4). When present, endothelial staining was widespread in all brain regions sampled in postmortem cases, which included frontal cortex (supplementary material, Figure S4A), medulla, cerebellum (supplementary material, Figure S4B), pons, and olfactory bulb (Table 3). Furthermore, in the full-body postmortem cases, positivity in brain endothelia was restricted to cases exhibiting multiorgan tissue positivity (PM1, 3 and 6). Notably, in the olfactory bulb, a postulated entry site for the virus into the brain, SARS-CoV-2 was restricted to endothelial cells and neurons were negative (supplementary material, Figure S4C). A proportion of cases in our extended brain cohort (PM11-16, BB1), which also included brain-only postmortems and a brain biopsy, displayed widespread endothelial staining. However, there was no apparent relationship between the endothelial labelling and the presence of neurological symptoms, perivascular or parenchymal inflammation, or brain infarction. In the positively stained case showing fatal brainstem encephalitis (PM12), staining was seen both in vessels with perivascular lymphocytic infiltrates (supplementary material, Figure S4G) and in uninflamed vessels (supplementary material, Figure S4D). In contrast, the other case that displayed brainstem encephalitis (PM4) [15] and the case displaying acute disseminated encephalomyelitis (ADEM) in the context of COVID-19 (BB1), displayed negative immunostaining for SARS-CoV-2 NP (supplementary material, Figure S4F). In cases with brain infarction, we found brain endothelial labelling in one case, including in vessels with perivascular lymphocytic infiltrates near the infarction (supplementary material, Figure S4E; PM15) and in uninflamed vessels distant from the infarction, but not in other cases (PM16, PM5). Staining was not apparent within ependymal cells (supplementary material, Figure S4H). No SARS-CoV-2 staining was seen in the brain in pre-COVID postmortem controls (supplementary material, Figure S2).

Endothelium
Endothelial staining, when present, was not only restricted to the brain regions studied, but was multisystemic across several organs (supplementary material, Figure S3). Nevertheless, the distribution of viral staining was variable, with some organs showing broad staining across all vessels (e.g. brain) and others showing focal staining only. In the heart, positivity was observed within endothelial cells but not within any other cell SARS-CoV-2 in myenteric plexus and megakaryocytes 211 type, including myocytes (supplementary material, Figure S3F). No SARS-CoV-2 staining was seen in the endothelia in pre-COVID postmortem controls (supplementary material, Figure S2).

Tracheal biopsies
We also examined tracheal biopsies of 15 individuals who had been admitted to intensive care and required tracheostomy (see Table 4 for detailed clinical Table 3. Summary of SARS-CoV-2 NP localisation in the brain of COVID-19 cases.

Brain regions Cases
Only endothelial cells were observed to show positivity in the brain, but when present, it appeared throughout the brain. As such, E represents positive staining in endothelial cells and EÀ/+ indicates weak positivity in endothelial cells. No definite parenchymal SARS-CoV-2 was seen in any case.  (Table 4, TB1-TB7 and Figure 2D). In five of these (TB1-TB4,  TB7), SARS-CoV-2 was confirmed by RT-PCR nasopharyngeal swab prior to tracheal biopsy. The two remaining cases had persistently negative RT-PCR SARS-CoV-2 nasopharyngeal swabs prior to biopsy (Table 4, TB5 and TB6). Nonetheless, participant TB5 had a respiratory picture which was entirely in keeping with COVID-19, both clinically and radiologically. Participant TB6 did not have clinical features of COVID-19, but required a prolonged admission to intensive care due to unrelated medical issues and may have been infected during this period (the admission was in Winter 2020; a period with high infection rates in the UK).
No definite SARS-CoV-2 NP was detected in the remaining eight tracheal biopsies by IHC ( Figure 2F), and the agreement between negative IHC and nasopharyngeal RT-PCR results was complete (Table 4, participants TB8-TB15). Two of these cases (TB8 and TB9) had a clinical course compatible with COVID-19, but they both had inconsistent SARS-CoV-2 nasopharyngeal swab results, with a documented negative swab result 18 days after and 12 days before, respectively, undergoing tracheal biopsy. Four cases (TB12-TB15) initially presented with positive swab results but, by the time of tracheostomy, their swab results were negative. The remaining two cases (participants TB10 and TB11) were admitted to intensive care for reasons unrelated to COVID-19 and had documented swabnegativity repeatedly before and after tracheostomy, and hence were included as negative controls.

Discussion
Predominantly a respiratory disease, it has been logical for COVID-19 histopathological studies to focus on the respiratory system. Despite this, there is value in examining the histopathological correlates of SARS-COV-2 infection in extrapulmonary organs and tissues, given the increasing spectrum of extrapulmonary symptoms reported. Here, we performed a comprehensive multisystem organ analysis of SARS-CoV-2 cellular tropism in tissues from 36 patients with PCR-confirmed SARS-CoV-2 status using anti-NP IHC, yielding two key findings. First, we report the novel finding of SARS-CoV-2 NP within neurons of the myenteric plexus but not the brain. Our results provide evidence for peripheral neuroinvasion in the digestive tract, but critically, not in the CNS. Furthermore, we have demonstrated viral NP within platelet-producing megakaryocytes, a finding that provides insight into the well-characterised thrombotic sequelae of COVID- 19. It had been previously speculated that, due to the expression of ACE2 in neurons of the myenteric plexus, these cells are possible targets of SARS-CoV-2 infection [2], a hypothesis that we corroborate here for the first time. The long-term consequences of our findings in the myenteric plexus are yet to be established. PD is a neurodegenerative disorder thought to result from the prion-like spread of amyloidogenic alpha-synuclein causing catastrophic motor impairment and other symptoms, such as dementia, as the spread of misfolded protein progresses throughout the brain [25]. However, it is not known if the initial seeding of the misfolded protein conformation is purely a stochastic event or ignited by an internal or external factor, with the exception of 5-10% of cases in which genetic mutations have been shown to impact alpha-synuclein biology [26]. The brain pathology of PD initiates in the olfactory bulb and medulla from where it propagates in a stereotypical pattern [11], and this is anticipated by accumulation of misfolded alpha-synuclein in the ENS. This pattern of pathology progression explains anosmia and gastrointestinal (GI) symptoms such as constipation and nausea [27], appearing early in PD. It has been proposed that an environmental agent reaches the brain from the nose through the olfactory nerves or from the gut through the ENS via retrograde axonal transport along the vagus nerve reaching the medulla [12,28].
Viral infection of myenteric neurons, as implied by our study, could in theory be one such environmental factor. Historically, links between viral infection and Parkinsonism were seen following the 1918 influenzaencephalitis lethargica pandemic [29]. During the COVID-19 pandemic, there have been reports of concurrent neurological complications and encephalitis [20], however, there is currently only one report of postviral Parkinsonism development [30]. Diversity of genetic background, comorbidities, and previous infections all impact susceptibility and likely may only have neurodegenerative ramifications in a subset of people. As we begin to recover from the pandemic, we should nevertheless still be conscious of COVID-19 when assessing future neurological cases.
On the other hand, the extent of SARS-CoV-2's direct invasion of the CNS is uncertain. Throughout the pandemic, neurological symptoms such as headache, ageusia, and anosmia were commonly reported, and the involvement of both the central and peripheral nervous system was hypothesised [31]. Our brain cohort of SARS-CoV-2-infected patients includes patients with clinical and pathological neurological disease, including brainstem encephalitis and cases with no neurological involvement. However, positivity within the brain appeared to be limited to endothelial cells. Crucially, despite observing viral NP in the neurons of the myenteric plexus, we found the origin of the vagus nerve in the medulla oblongata to be devoid of neuronal SARS-CoV-2 NP (supplementary material, Figure S5). This suggests that, in the event of any neurodegenerative disease developing after SARS-CoV-2 infection, consideration should be given to the spread of misfolded protein or other pathophysiological alterations triggered by the virus in ENS neurons, rather than the direct entry of the virus into CNS cells. Previous reports SARS-CoV-2 in myenteric plexus and megakaryocytes 213 about the possibility of viral protein or RNA in the CNS are conflicting [8,14,16,32,33]. One hypothesis postulates that systemic vascular changes are likely behind reported neurological symptoms, as an indirect consequence of lung-derived hypoxia rather than direct viral damage. Additionally, endothelial cell targeting and injury are also considered to be key elements in COVID-19 pathophysiology that have been frequently alluded to [34]. COVID-19 has been established as a prothrombotic disease characterised by coagulopathy, thrombosis, and platelet activation [35,36]. Our results parallel this with findings of platelet-producing megakaryocytes colocalising with the viral NP within the bone marrow. Several publications have also reported the presence of circulating megakaryocytes in multiple organs, including lung, heart, kidney, liver, and brain [14,[37][38][39]. One study demonstrated that SARS-CoV-2 infection can promote procoagulant changes in platelet gene expression [40]. Paradoxically, despite confirming viral presence, the authors also ascertained that megakaryocytes and platelets lack ACE2, by which they suggested an independent route of infection. A recent preprint report that infected calprotectin-expressing megakaryocytes additionally express the ACE2 receptor and TMPRSS2 [41]. Even more intriguing, that study also provided evidence of megakaryocytes harbouring infected pro-platelets. Together with our own observations, it is probable that entry occurs by several mechanisms. The principal molecular route of SARS-CoV-2 entry into megakaryocytes and platelets is therefore still unresolved.
Our results imply appreciable involvement of the GI tract and related digestive organs in COVID-19, correlating with the high prevalence of symptoms such as diarrhoea, abdominal pain, anorexia, and vomiting in COVID-19 symptomatology [42]. Of note, the viral cell tropism in the digestive tract overall mirrors that of the expression profile of ACE2 in cell types such as enterocytes [43]. Preexisting chronic liver diseases, such as steatohepatitis and cirrhosis, have been commonly associated with severe cases of COVID-19, with 2-11% of patients having liver comorbidities [44,45]. With four of ten patients displaying liver disease, this is also a common finding within our own full-body postmortem cohort. In addition to endothelial cells, three cases (PM1, PM6, and PM10) exhibited a striking viral NP presence within parenchymal tissues, including bile duct cholangiocytes, as well as hepatocytes. Studies of the ACE2 receptor in normal liver asserts low expression in hepatocytes; however, mouse models and human samples suggest chronic liver injury may contribute to ACE2 receptor upregulation [43,46]. Cholangiocytes, on the other hand, purportedly have high levels of ACE2, which may explain the strong viral protein detection seen in this study.
We also observed, predictably, a diffuse viral presence throughout the respiratory system. Consistent with other reports, detection of viral NP in the lungs appears to be limited to seromucinous glands, alveolar pneumocytes, and respiratory epithelium [47]. As previously described, all the lungs in the PM1-10 case series present with DAD, a histopathological aberration commonly affiliated with acute respiratory distress syndrome (ARDS) [8]. However, our immunohistochemical analysis revealed parenchymal positivity in only a subset of patients (four of ten). As NP has been canonised as a proxy for replicating SARS-CoV-2, this suggests that only a few patients in our cohort died with active pulmonary infection [48]. Considering the disease duration of these cases, it is likely that our findings capture the early phase of ARDS in which viral activity persists [49][50][51]. Similar observations have been made in other SARS-CoV-2 in situ studies, as well as in SARS-CoV, where viral replication in the lungs is constrained to the early postinfective period ($2 weeks) [47,52,53].
Interestingly, we found that parenchymal SARS-CoV-2 presence in the lungs was associated in all cases with an extrapulmonary viral presence, and that this was also constrained to this $2-week period. Our data, with a universal endothelial presence in these cases, suggests that extrapulmonary dissemination could occur via haematogenous spread. This is supported by increased SARS-CoV-2 RNA load in the blood (RNAemia) to be associated with disease severity in COVID-19 patients [54,55] and by multiorgan damage to be related to high RNAemia [56], although other studies disagree [57]. In the case of GI viral presence, a faecal-oral route of transmission is possible [58], supported by detection of viral RNA in the faeces of COVID-19 patients, even after testing negative in respiratory tissues in a subset of patients [59]. Our data also suggest that beyond this $2-week period, COVID-19 multiorgan complications may be the result of persistence of viral tissue damage and of pathophysiological cascades ignited by the viral infection, including inflammatory activation, rather than continued viral infection. Given that our postmortem samples are from patients who died during the initial first COVID-19 wave in the UK, our study cannot comment on any vaccine-related inflammatory processes.
A strength of this study is the strict validation performed on our immunostaining protocol. Consistently throughout our study we identified a characteristic immunostaining pattern to be indicative of SARS-CoV-2. After gaining entry into host cells, viral RNA and replicative protein components form a reticulovesicular network by reorganising the host endoplasmic reticulum [60]. We have identified that, in most cell types, genuine SARS-CoV-2 positive stain displays a cytoplasmic apical or paranuclear punctate pattern reflecting the viral cycle. In addition, the clinical relevance of this antibody is best illustrated by our tracheal biopsy series in which we used our protocol to show clinicopathological correlations. Out of 15 biopsies, the vast majority of SARS-CoV-2 IHC results were consistent with the timeline of nasopharyngeal swab results, as well as overall symptom presentation. The outliers included one patient who persistently had negative swabs yet showed very strong IHC positivity. As the tracheostomy occurred a considerable time after the last swab, it is likely this patient had been infected during

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S Gray-Rodriguez et al their prolonged admission to the ICU, subsequent to tracheostomy and tracheal biopsy. Our study has limitations. Despite comprehensive sampling, our full-body cohort is of limited size, with some tissues being subjected to autolysis as well as sampling bias. This makes it difficult to speculate on any enteric neuronal loss or cytopathic damage seen in patients who died later in their disease course, although we did find evidence for at least a proportion of the neurons in the myenteric plexus surviving into the postreplicative phase (supplementary material, Figure S6). Second, we cannot completely exclude the possibility of cross-reaction with SARS-CoV-1, although, to our knowledge, this virus is not currently in circulation in our community. Contextualising the patients analysed in this study (e.g. date and location of death), it is highly unlikely that we have misidentified another betacoronavirus, particularly as the cases had confirmed SARS-CoV-2 PCR positivity. Additionally, when considering nucleoprotein homology, the closest sequence (bat coronavirus nucleoprotein) only reveals 75% alignment (supplementary material, Figure S7).
Overall, our investigation has demonstrated the multisystemic tropism of SARS-CoV-2. Concordant with other histopathological publications, we have shown extrapulmonary involvement, with an emphasis on the digestive system, in severe cases. Most significantly, we provide evidence of neuroinvasiveness in the ENS but not the CNS. We have also identified megakaryocytes as potential targets of SARS-CoV-2. Further investigations in gene expression profiling related to these findings will help determine any long-term implications. Figure S1. Presence of SARS-CoV-2 in cholinergic and nitrergic neurons of the myenteric plexus Figure S2. Absence of anti-SARS-CoV-2 immunostaining in pre-COVID controls Figure S3. SARS-CoV-2 tropism to the endothelium is multisystemic Figure S4. Cellular tropism of SARS-CoV-2 in the brain Figure S5. Absence of anti-SARS-CoV-2 immunostaining in dorsal vagal nucleus Figure S6. Immunohistochemistry for anti-SARS-CoV-2 in the myenteric plexus of PM4 Figure S7. Sequence homology between anti-SARS-CoV-2 antibody target peptides Table S1. Clinical characteristics of pre-COVID control cases SARS-CoV-2 in myenteric plexus and megakaryocytes 217