Microgliosis, astrogliosis and loss of aquaporin-4 polarity in frontal cortex of COVID-19 patients (preprint)

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), causing human coronavirus disease 2019 (COVID-19), not only affects the respiratory tract, but also impacts other organs including the brain. A considerable number of COVID-19 patients develop neuropsychiatric symptoms that may linger for weeks and months and contribute to \"long-COVID\". While the neurological symptoms of COVID-19 are well described, the cellular mechanisms of neurologic disorders attributed to the infection are still enigmatic. Here, we studied the effect of an infection with SARS-CoV-2 on the structure and expression of marker proteins of astrocytes and microglial cells in the frontal cortex of patients who died from COVID-19 in comparison to non-COVID-19 controls. Most of COVID-19 patients had microglial cells with retracted processes and rounded and enlarged cell bodies in both gray and white matter, as visualized by anti-Iba1 staining and confocal fluorescence microscopy. In addition, gray matter astrocytes in COVID-19 patients were frequently labeled by intense anti-GFAP staining, whereas in non-COVID-19 controls, most gray matter astrocytes expressed little GFAP. The most striking difference between astrocytes in COVID-19 patients and controls was found by anti-aquaporin-4 (AQP4) staining. In COVID-19 patients, a large number of gray matter astrocytes showed an increase in AQP4. In addition, AQP4 polarity was lost and AQP4 covered the entire cell, including the cell body and all cell processes, while in controls, AQP4 immunostaining was mainly detected in endfeet around blood vessels and did not visualize the cell body. In summary, our data suggest neuroinflammation upon SARS-CoV-2 infection including microgliosis and astrogliosis, including loss of AQP4 polarity.

Vagus nerve inflammation contributes to dysautonomia in COVID-19 (preprint)

Dysautonomia has substantially impacted acute COVID-19 severity as well as symptom burden after recovery from COVID-19 (long COVID), yet the underlying causes remain unknown. Here, we show that SARS-CoV-2 is detectable in postmortem vagus nerve specimen together with inflammatory cell infiltration derived primarily from monocytes. This is associated with a decreased respiratory rate in non-survivors of critical COVID-19. Our data suggest that SARS-CoV-2 induces vagus nerve inflammation followed by autonomic dysfunction.

SARS-CoV-2 Spike Protein Accumulation in the Skull-Meninges-Brain Axis: Potential Implications for Long-Term Neurological Complications in post-COVID-19 (preprint)

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has been associated mainly with a range of neurological symptoms, including brain fog and brain tissue loss, raising concerns about the virus's acute and potential chronic impact on the central nervous system. In this study, we utilized mouse models and human post-mortem tissues to investigate the presence and distribution of the SARS-CoV-2 spike protein in the skull-meninges-brain axis. Our results revealed the accumulation of the spike protein in the skull marrow, brain meninges, and brain parenchyma. The injection of the spike protein alone caused cell death in the brain, highlighting a direct effect on brain tissue. Furthermore, we observed the presence of spike protein in the skull of deceased long after their COVID-19 infection, suggesting that the spike's persistence may contribute to long-term neurological symptoms. The spike protein was associated with neutrophil-related pathways and dysregulation of the proteins involved in the PI3K-AKT as well as complement and coagulation pathway. Overall, our findings suggest that SARS-CoV-2 spike protein trafficking from CNS borders into the brain parenchyma and identified differentially regulated pathways may present insights into mechanisms underlying immediate and long-term consequences of SARS-CoV-2 and present diagnostic and therapeutic opportunities.

Robotic Tissue Sampling for Safe Post-mortem Biopsy in Infectious Corpses (preprint)

In pathology and legal medicine, the histopathological and microbiological analysis of tissue samples from infected deceased is a valuable information for developing treatment strategies during a pandemic such as COVID-19. However, a conventional autopsy carries the risk of disease transmission and may be rejected by relatives. We propose minimally invasive biopsy with robot assistance under CT guidance to minimize the risk of disease transmission during tissue sampling and to improve accuracy. A flexible robotic system for biopsy sampling is presented, which is applied to human corpses placed inside protective body bags. An automatic planning and decision system estimates optimal insertion point. Heat maps projected onto the segmented skin visualize the distance and angle of insertions and estimate the minimum cost of a puncture while avoiding bone collisions. Further, we test multiple insertion paths concerning feasibility and collisions. A custom end effector is designed for inserting needles and extracting tissue samples under robotic guidance. Our robotic post-mortem biopsy (RPMB) system is evaluated in a study during the COVID-19 pandemic on 20 corpses and 10 tissue targets, 5 of them being infected with SARS-CoV-2. The mean planning time including robot path planning is (5.72+-1.67) s. Mean needle placement accuracy is (7.19+-4.22) mm.

Assessing and improving the validity of COVID-19 autopsy studies - a multi center approach to establish essential standards for immunohistochemical and ultrastructural analyses (preprint)

Background Autopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, e.g. by an overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. Methods We assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 spike protein and nucleocapsid, dsRNA, and non-structural protein Nsp3 in cultured cell lines and COVID-19 autopsy tissues. In a multicenter study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 nucleocapsid staining. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. Findings Publications show high variability in the detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. In our study, we show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and also provide recommendations for optimized sampling and analysis. 116 of 122 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM and IHC sections as a reference and for training purposes. Interpretation Since detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2.

Replication of SARS-CoV-2 in Adipose Tissue Determines Organ and Systemic Lipid Metabolism in Hamsters and Humans (preprint)

Obesity increases the risk for poor outcome in patients with coronavirus disease-19 (COVID-19). However, the role of adipose tissue for viral propagation and potential metabolic implications are not understood. We detected SARS-CoV-2 in adipose tissue of overweight but not lean male COVID-19 patients. SARS-CoV-2 replicates to high titres in cultured mature adipocytes, a process depending on lipid accumulation and mobilization. After intranasal inoculation, we observed high viral replication in fat depots of Golden Syrian hamsters, demonstrating dissemination from the respiratory tract and subsequent propagation in adipose tissue. Following induction of pro-inflammatory responses, expression of de novo lipogenesis enzymes was suppressed in adipose tissue. This specific down-regulation was reflected by lipidomic alterations in plasma of SARS-CoV-2 infected hamsters as well as in hospitalized COVID-19 patients. Overall, our study highlights that adipose tissue is an important site of SARS-CoV-2 replication, contributing to dysregulation of systemic lipid metabolism.Funding: This study was supported by a rapid response grant from the Federal Ministry of Health (BMG; ZMV I 1-2520COR501 to GG), by DFG grants SCHE522/4-1 (LS) and SFB1328, project- ID:335447727 (JH). As part of the National Network University Medicine (NUM) funded by the Federal Ministry of Education and Research (BMBF, Germany), this work was funded within the research consortium DEFEAT PANDEMIcs, grant number 01KX2021 (FH, PL, KP, BO).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The Ethics Committee of the Hamburg Chamber of Physicians reviewed and approved the studies (PV7311, 2020-10353-BO-ff, WF-051/20, WF-053/20). For the preparation of primary human white adipocytes, biopsies of subcutaneous and visceral adipose tissues were taken during bariatric surgery at the Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf. All participants signed an informed consent and the study was approved by the Ethics Committee of the Hamburg Chamber of Physicians (PV4889).

Dying From Or With SARS-CoV-2 – Prospective Postmortem Evaluation of 735 Consecutive Death Cases – (preprint)

BackgroundCoronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic with significant mortality. Accurate information on the specific circumstances of death and whether patients died from or with SARS-CoV-2 is scarce.MethodsTo distinguish COVID-19 from non-COVID-19 deaths, we performed a systematic review of 735 SARS-CoV-2-associated deaths in Hamburg, Germany, from March to December 2020, using conventional autopsy, ultrasound-guided minimally invasive autopsy, postmortem computed tomography and medical records. Statistical analyses including multiple logistic regression were used to compare both cohorts.Findings84.1% (n=618) were classified as COVID-19 deaths, 6.4% (n=47) as non-COVID-19 deaths, 9.5% (n=70) remained unclear. Median age of COVID-19 deaths was 83.0 years, 54.4% were male. In the autopsy group (n=283), the majority died of pneumonia and/or diffuse alveolar damage (73.6%; n=187). Thromboses were found in 39.2% (n=62/158 cases), pulmonary embolism in 22.1% (n=56/253 cases). In 2020, annual mortality in Hamburg was about 5.5% higher than in the previous 20 years, of which 3.4% (n=618) represented COVID-19 deaths.InterpretationOur study highlights the need for mortality surveillance and postmortem examinations. The vast majority of individuals who died directly from SARS-CoV-2 infection were of advanced age and had multiple comorbidities.

The Blood-Brain Barrier is Dysregulated in COVID-19 and Serves as a CNS Entry Route for SARS-CoV-2 (preprint)

Neurological complications are common in COVID-19 patients. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in patients’ brain tissues, its entry routes and resulting consequences are not well understood. Here, we report that the blood-brain barrier (BBB) and its microenvironment show pronounced upregulation of interferon signaling pathways in fatal COVID-19. Moreover, human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) were susceptible to SARS-CoV-2 infection and recapitulated the transcriptional changes detected in vivo . While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active transcytosis of the virus across the BBB in vitro . SARS-CoV-2 entry into BCECs could be reduced by anti-spike-, anti-ACE2- and anti-NRP1-specific antibodies or the TMPRSS2 inhibitor nafamostat. Together, our data provide direct evidence for SARS-CoV-2 brain entry across the BBB resulting in an increase in interferon signaling.

Cardiac SARS-CoV-2 infection is associated with distinct transcriptomic changes within the heart (preprint)

1BackgroundAnalyses in hospitalized patients and small autopsy series suggest that severe SARS-CoV-2 infection may affect the heart. We investigated heart tissue by in situ hybridization, immunohistochemistry and RNA sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19. MethodsLeft ventricular tissue from 95 deceased with diagnosed SARS-CoV-2 infection undergoing autopsy was analyzed and clinical data were collected. RNA was isolated to examine virus load of SARS- CoV-2 and its replication in the heart. A virus load >1000 copies per {micro}g RNA was defined as relevant. Viral RNA and inflammatory cells were assessed using histology. RNA sequencing and gene ontology (GO) enrichment were performed in 10 cases with high cardiac virus load and 10 age-matched cases without cardiac infection. ResultsA relevant SARS-CoV-2 virus load was detected in 41 out of 95 deceased (43%). The median cardiac virus load was 7952 copies per {micro}g RNA (IQR 2507, 32 005). In situ hybridization revealed SARS- CoV-2 RNA primarily in the interstitium or interstitial cells. Virus detection was not associated with increased inflammatory cells. Relevant cardiac infection was associated with increased expression of the entry factor TMPRSS2. Cardiac virus replication was found in 14/95 hearts (15%). Remarkably, cardiac virus replication was associated with shorter time between diagnosis and death. RNA sequencing revealed clear activation of immune response pathways to virus infection and destruction of cardiomyocytes. Hearts with high virus load showed activation of the GO term "extracellular exosomes". ConclusionSARS-CoV-2 infection including virus replication and distinct transcriptomic alterations without signs of myocarditis demonstrate a cardiac involvement. In this autopsy series, cardiac replication of SARS-CoV-2 was associated with early death.