2 Abstract:
Stroke and central nervous system dysfunction are cardinal symptoms in critically ill corona virus disease 19 (COVID-19) patients. In an autopsy series of 32 COVID-19 patients, we investigated whether carotid arteries were infected with SARS-CoV-2 by employing genomic, virologic, histochemical and transcriptomic analyses. We show that SARS-CoV-2 productively infects and modulates vascular responses in carotid arteries. This finding has far reaching implications for the understanding and clinical treatment of COVID-19.
In December 2019, a cluster of pneumonia caused by a novel coronavirus (SARS-CoV-2) was reported in Wuhan, China. Corona virus disease 19 (COVID-19) has since spread worldwide affecting more than 7,750,000 people and causing more than 430,000 deaths by June 14, 2020. COVID-19 is characterized by respiratory symptoms with a significant number of patients developing acute respiratory distress syndrome (ARDS) and multi-organ failure, eventually leading to death, especially in older patients with multiple co-morbidities 1-4 . In critically ill patients, who often present with viremia, the virus has been detected in multiple organs 2 . A large autopsy series revealed that pulmonary artery embolism and/or deep vein thrombosis were present in about 40% of cases, indicating that COVID-19 is strongly associated with coagulopathy 3 . Notably, large vessel stroke including one proven case of carotid artery thrombosis was reported in 5 younger COVID-19 patients 5 . In 58 COVID-19 patients with ARDS, neurologic symptoms associated with brain perfusion abnormalities and/or stroke were highly prevalent 6, 7 . Endotheliitis as the potential cause for the COVID-19 induced vascular dysfunction and coagulopathy was inferred by histopathology analysis of various postmortem tissues from a limited number of patients 8,9 . Using real time PCR, we quantified SARS-CoV-2 RNA in Arteria carotis communis (A. carotis, carotid artery) samples and matching lung-and throat samples from 32 COVID-19 patients (basic clinical data see online methods). All patients showed high viral loads in the lung, consistent with viral pneumonia 3 . In 26 (81.25%) of the A. carotis samples viral RNA was detected, with mean viral loads that were comparable to those observed in throat samples ( Fig. 1a) . Histopathologic findings in the carotid arteries were age-appropriate with a moderate degree of inflammation and no obvious signs of vasculitis (supplemental Table S1 ).
On the basis of virus positivity in A. carotis and the availability of additional tissues and organs, we attempted live/infectious virus isolation from A. carotis and 10 matching organ samples (Fig, 1b) . We included 7 patients in this analysis (Fig. 1a , red circles), out of whom 5 had ante-mortem viremia, one had no viremia and one had an unknown viremia status. The clinical histories of the patients are summarized in supplementary Table S2 . Virus isolation most frequently (up to 75%) succeeded in A. carotis and Vena saphena as well as in lung, throat and jejunum, with the latter three tissues being known sites of virus replication 10, 11 . Viral genome analysis revealed identical viral sequences in A. carotis and lung samples from the same patients, whereas a number of single nucleotide variations differentiated sequences from different patients. All SARS-CoV-2 sequences belong to the most prevalent genotype in Europe (supplemental Fig S1) . Isolation of infectious virus from organs required ante-mortem viremia and isolation rates correlated well with viral loads in the respective organs (Fig. 1b) .
Virus isolation from liver, myocardium, spleen and frontal cortex of the brain was unsuccessful. Relative abundance of subgenomic (sg) SARS-CoV-2 RNAs (which are generated during virus replication) were determined for 5 of the 7 carotid arteries (arteries #6 and #7 could not be sequenced due to low RNA quality) and were found to be, on average, comparable or even higher than those observed in lung samples (Fig. 1c, supplementary Table   S3 ). Together these data indicate that SARS-CoV-2 infects and replicates in carotid arteries.
To investigate which cells in the carotid artery wall were infected with the virus, we performed SARS-CoV-2 plus-strand RNA in situ hybridization as well as anti-spike immunofluorescence and -immunohistochemistry in tissue sections. These staining techniques conclusively detected viral RNA and protein in the endothelial layer of the artery wall ( Fig.   2a-d, supplemental Fig. S2 ).
We performed transcriptome analysis in A. carotis tissues from patients 4 and 5 (selected on basis of RNA quality) and two non-COVID-19 control patients in order to characterize the transcriptional response of SARS-CoV-2 infected arteries. Interestingly, type I and type II interferon pathways were down regulated in the SARS-CoV-2 infected arteries, which is in line with recent work demonstrating lowered interferon-related pathways and innate antiviral responses in SARS-CoV-2 infected cell lines and animal models 12 