key: cord-0705803-7w320p9v
authors: Bhatnagar, Julu; Gary, Joy; Reagan-Steiner, Sarah; Estetter, Lindsey B; Tong, Suxiang; Tao, Ying; Denison, Amy M; Lee, Elizabeth; DeLeon-Carnes, Marlene; Li, Yan; Uehara, Anna; Paden, Clinton R; Leitgeb, Brooke; Uyeki, Timothy M; Martines, Roosecelis B; Ritter, Jana M; Paddock, Christopher D; Shieh, Wun-Ju; Zaki, Sherif R
title: Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Replication and Tropism in the Lungs, Airways, and Vascular Endothelium of Patients With Fatal Coronavirus Disease 2019: An Autopsy Case Series
date: 2021-01-27
journal: J Infect Dis
DOI: 10.1093/infdis/jiab039
sha: 5471827ce38551897e1a992943f8d38a1000f6bd
doc_id: 705803
cord_uid: 7w320p9v

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic continues to produce substantial morbidity and mortality. To understand the reasons for the wide-spectrum complications and severe outcomes of COVID-19, we aimed to identify cellular targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and replication in various tissues. METHODS: We evaluated RNA extracted from formalin-fixed, paraffin-embedded autopsy tissues from 64 case patients (age range, 1 month to 84 years; 21 COVID-19 confirmed, 43 suspected COVID-19) by SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR). For cellular localization of SARS-CoV-2 RNA and viral characterization, we performed in situ hybridization (ISH), subgenomic RNA RT-PCR, and whole-genome sequencing. RESULTS: SARS-CoV-2 was identified by RT-PCR in 32 case patients (21 COVID-19 confirmed, 11 suspected). ISH was positive in 20 and subgenomic RNA RT-PCR was positive in 17 of 32 RT-PCR–positive case patients. SARS-CoV-2 RNA was localized by ISH in hyaline membranes, pneumocytes, and macrophages of lungs; epithelial cells of airways; and endothelial cells and vessel walls of brain stem, leptomeninges, lung, heart, liver, kidney, and pancreas. The D614G variant was detected in 9 RT-PCR–positive case patients. CONCLUSIONS: We identified cellular targets of SARS-CoV-2 tropism and replication in the lungs and airways and demonstrated its direct infection in vascular endothelium. This work provides important insights into COVID-19 pathogenesis and mechanisms of severe outcomes.

to severe and fatal outcomes, including acute respiratory distress syndrome and multiorgan failure [4] . Evidence of additional COVID-19-associated complications, including frequent thromboembolic, cardiovascular, and neurological complications, is also emerging [4] [5] [6] [7] [8] [9] .

To understand the reasons for the wide-spectrum clinical manifestations, complications, and severe disease outcomes of COVID-19, it is critical to investigate cellular targets of SARS-CoV-2 tropism, replication, and mechanism of viral dissemination. Autopsy studies can be particularly valuable for answering these questions and providing deeper insight into the underlying pathophysiology of COVID-19. Previous autopsy studies have generally been limited to histopathological analysis and, if SARS-CoV-2-specific tissue-based assays were used, either they were restricted to specific organs or applied to specimens from a small case series of a specific geographic location [6, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . Although a few prior autopsy studies using immunohistochemistry are illustrative of distribution of SARS-CoV-2 antigens in tissues, the presence of antigens does not necessarily indicate virus replication [18, 19] .

The purpose of this work was to identify cellular targets of SARS-CoV-2 replication and tropism by directly localizing viral RNA in tissues from fatal COVID-19 patients, along with indicators of active viral replication, to better understand the severe disease outcomes, wide-spectrum complications, and viral pathogenesis. In addition, to identify the role of coinfections in severe outcomes and to detect alternate etiological pathogens, other respiratory pathogens were also evaluated. Here, we describe a comprehensive analysis of respiratory and nonrespiratory autopsy tissues from case patients with confirmed or suspected COVID-19, comprising both adult and pediatric patients who died after hospitalization or in community settings prior to any testing for SARS-CoV-2.

Formalin-fixed, paraffin-embedded (FFPE) autopsy tissues from 64 case patients, including 21 with confirmed COVID-19 and 43 with suspected COVID-19, from 23 US states were evaluated. These specimens were submitted to the Infectious Diseases Pathology Branch, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, from local and state health departments, medical examiners, and pathologists between 23 January and 4 August 2020 for diagnostic consultation. This activity was reviewed by the CDC and was conducted consistent with applicable federal law and CDC policy (see 45 Code of Federal Regulations [CFR] part 46; 21 CFR part 56; 42 US Code [USC] §241(d); 5 USC §552a; 44 USC §3501 et seq.). All samples and associated medical and autopsy records were provided in the context of diagnostic consultation, a routine public health service provided by the CDC. As such, institutional review was not required for the testing described in this article. In this series, confirmed cases are defined as those with prior laboratory evidence of SARS-CoV-2 by respiratory swab RT-PCR; suspected cases had clinical or epidemiologic suspicion of COVID-19, but respiratory swab testing for SARS-CoV-2 was negative or not performed. FFPE respiratory tissues (lung, trachea, or bronchi) from all case patients and additional FFPE nonrespiratory tissues, including heart, brain, kidney, bladder, adrenal, thyroid, lymph nodes, liver, spleen, pancreas, gastrointestinal (GI), and urogenital tissues, as available, were evaluated by various assays. All tissues were analyzed by routine hematoxylin and eosin (H&E) staining to identify pathological changes. Clinical and histopathological findings from 9 case patients have been previously described [12, 19] .

As a part of the ongoing COVID-19 response effort, we developed 2 conventional RT-PCR (cRT-PCR) assays for detection of SARS-CoV-2 in FFPE tissues, using the newly designed primers targeting the spike gene (S) and nucleocapsid gene (N), as described below: S-gene forward primer 5′-CTT CCC TCA GTC AGC ACC TC-3′; S-gene reverse primer 5′-GTT ACA AAC CAG TGT GTG CCA-3′; N-gene forward primer 5′-ACA TTG GCA CCC GCA ATC-3′; N-gene reverse primer  5′-TGA ACT GTT GCG ACT ACG TGA-3′. For validation,  positive controls consisted of FFPE SARS-CoV-2 cultured  cells while negative controls comprised of FFPE cell culture  controls for RNA was extracted from FFPE tissues from all 64 case patients using RNA extraction protocol as previously described. [20] and evaluated by N-gene and S-gene cRT-PCR assays. The assays were performed using the OneStep RT-PCR Kit (Qiagen, Valencia, California) and 5 µL of RNA template, using the manufacturer's instructions. The thermocycling conditions for N-gene and S-gene RT-PCR assays were as follows: 1 cycle at 50°C for 30 minutes, 1 cycle at 95°C for 15 minutes, then 40 cycles of incubation at 94°C, 56°C, and 72°C for 1 minute each, followed by 1 cycle of final extension at 72°C for 10 minutes. The N-gene (150 bp) and S-gene (162 bp) PCR-positive amplicons were directly sequenced by Sanger sequencing on a GenomeLab GeXP sequencer (AB SCIEX, Redwood City, California). The search for homologies to known sequences was performed by using the BLAST nucleotide database (http://blast.ncbi.nlm. nih.gov/Blast.cgi). Validation results showed that the S-gene cRT-PCR was specific for SARS-CoV-2 while N-gene cRT-PCR amplified both SARS-CoV-2 and SARS-CoV controls. However, sequencing of amplicons clearly differentiated SARS-CoV-2 from SARS-CoV. A SARS-CoV-2-positive result was inferred only when an amplicon sequence showed ≥99% nucleotide identity with SARS-CoV-2.

To demonstrate evidence of active viral replication, an additional subgenomic RNA RT-PCR was performed on cRT-PCR-positive case patients, as described previously [21] . Additionally, SARS-CoV-2 real-time RT-PCR (rRT-PCR) assay was performed on tissues from cRT-PCR-positive case patients to evaluate cycle threshold (Ct) values for each tissue type for correlative assessment of viral load [22] (Supplementary Data).

RNA extracts from FFPE respiratory tissues of 64 case patients were tested by rRT-PCR for influenza A and B viruses, respiratory syncytial virus, and human parainfluenza virus (HPIV) types 1-4, as previously described [23, 24] . DNA was extracted from FFPE respiratory tissues of case patients with clinical history and/or histopathology consistent with a possible respiratory bacterial infection and tested by PCR assays for S. pneumoniae, S. pyogenes, Streptococcus species, and Staphylococcus aureus, and by wide-range eubacterial 16S rRNA gene PCR [25, 26] .

Two ISH assays, using RNAscope probes (Advanced Cell Diagnostics, Newark, California) targeting the N-and S-genes of SARS-CoV-2, were developed and validated. For validation, positive controls consisted of FFPE SARS-CoV-2 cultured cells while negative controls comprised of FFPE cell culture controls for alphacoronavirus (HCoV-NL63), betacoronaviruses (HCoV-HKU1, MERS-CoV, SARS-CoV), and FFPE tissues from RT-PCR-confirmed cases of other viral infections, including influenza viruses A and B, HPIV-3, enterovirus, and Zika virus. To directly localize SARS-CoV-2 RNA in tissues, newly developed ISH assays were performed on respiratory and nonrespiratory tissues from 32 SARS-CoV-2 cRT-PCR-positive case patients, using the manufacturer's instructions (Advanced Cell Diagnostics). To further characterize specific cell types, ISH using RNAscope probes, including the SFTPB probe for type II pneumocytes, CD68 for macrophages, and MUC5B and MUC5AC probes for goblet cells, were performed on serial sections from SARS-CoV-2 ISH-positive tissue blocks. ISH RNAscope probes targeting MERS-CoV and Zika virus were used as negative control probes on FFPE tissues from confirmed SARS-CoV-2 cases and cell cultures. The quality of RNA in tissues was assessed by using RNAscope human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probes.

Whole-genome sequencing (WGS) was performed on available RNA extracts from FFPE tissues of SARS-CoV-2 cRT-PCRpositive case patients at the Respiratory Viruses Branch, CDC. The primary method utilized multiplex RT-PCR with a panel of overlapping sets of SARS-CoV-2 primers, employing either the Oxford Nanopore MinION or Illumina MiSeq sequencers [27] . Consensus sequences were generated with Minimap 2.17 and Samtools 1.9. Representative full-genome sequences with collection dates up until September 2020 were downloaded from GISAID for sequence analysis.

Of all 64 case patients evaluated, the median age was 50 years (range, 1 month-84 years) and 54 (84%) were adults (≥18 years of age). Forty-seven (73%) had 1 or more underlying medical conditions. For those with known illness onset date available (n = 58), the median duration of illness (DOI) was 6 days (range, 0-40 days), and 34 (53%) died at home or in the emergency department. Demographic and clinical features of case patients are summarized in Table 1 .

SARS-CoV-2 RNA was detected in respiratory tissues of 32 of 64 case patients by N-gene and S-gene cRT-PCR assays and sequencing of amplicons, showing 99%-100% nucleotide identity with SARS-CoV-2. SARS-CoV-2 cRT-PCR assays were positive in all 21 previously confirmed COVID-19 case patients and provided the first evidence of SARS-CoV-2 in 11 of 43 (26%) suspected COVID-19 case patients (Table 2) , including the first 2 US COVID-19 deaths. Subgenomic RNA transcripts, indicating active SARS-CoV-2 replication, were detected by subgenomic RNA RT-PCR in lung or tracheal tissues of 17 of 32 (53%) cRT-PCR-positive case patients. In these 17 case patients, the DOI was 2-18 days. Additionally, our findings suggested a relationship between location of viral replication in respiratory tract tissues and DOI (Table 3 ). For instance, in a case patient (CP 12) with a 2-day DOI, replicative viral RNA was detected only in trachea, while in 4 case patients with DOI 7-18 days, replicative RNA was detected only in lung tissues. In addition, replicative viral RNA was detected in lung or trachea of 10 of 11 cRT-PCR-positive long-term care facility residents (DOI 2-18 days; median age, 74 years [range, 54-91 years]), indicating more extensive infection in this high-risk group of older patients with underlying conditions. Case-by-case results of all assays performed on cRT-PCR-positive case patients are summarized in Table 3 .

SARS-CoV-2 tissue rRT-PCR was also positive for 30 of 32 (94%) cRT-PCR-positive case patients ( , indicating higher viral load in lung tissues. Although both cRT-PCR and rRT-PCR were positive for heart, GI, kidney, brain, liver/spleen, or pancreas tissues for 14 (44%) case patients, SARS-CoV-2 rRT-PCR Ct values were generally higher for these, in comparison to respiratory tissues, suggesting lower viral load in nonrespiratory tissues (Supplementary Figure 3) .

Coinfection of SARS-CoV-2 with other viral or bacterial pathogens, including influenza B, HPIV-3, S. aureus, and S. pneumoniae, were identified in respiratory tissues of 10 of 32 (31%) SARS-CoV-2 cRT-PCR-positive case patients (Tables 3  and 4 ). Other non-SARS-CoV-2 respiratory pathogens were also identified in respiratory tissues from 14 of 32 (44%) SARS-CoV-2 cRT-PCR-negative case patients (Table 4 ).

SARS-CoV-2 ISH assays using N-gene and/or S-gene probes were performed on multiple FFPE tissues blocks from 32 cRT-PCR-positive case patients; 20 (63%) had at least 1 block of lung or trachea positive by ISH (Table  2 ). In the lungs, ISH staining demonstrating SARS-CoV-2 RNA was observed in hyaline membranes ( Figure 1A and 1B), as well as within intra-alveolar cells, including alveolar macrophages and sloughed pneumocytes ( Figure 1A2 , 1 A4, and 1B2). Staining was also seen in pneumocytes lining alveoli ( Figure 1A3 and 1A4) and in bronchiolar epithelium. Staining in pneumocytes and macrophages was determined by cell morphology, as well as by ISH staining for cell markers SFTPB and CD68 in the serial sections of SARS-CoV-2-positive lung blocks (Supplementary Figure  2A1 and 2A2).

SARS-CoV-2 RNA was also observed in trachea/mainstem bronchi from 9 cRT-PCR-positive case patients, showing multifocal ISH staining in tracheal epithelium in both ciliated epithelial cells and goblet cells ( Figure 2A2 ). Goblet cell staining was determined by cell morphology, as well as by cell marker for MUC5B and MUC5AC in serial sections of trachea (Supplementary Figure 2A3 and 2A4). SARS-CoV-2 RNA was noted in the tracheal submucosal glands of 3 case patients ( Figure 2A4 ). Multifocal ISH signals were also seen within the medullary sinuses of a hilar lymph node from an immunosuppressed individual, likely in circulating macrophages (CP 17, Figure 2B1 and 2B2). In 1 case (CP 30), ISH staining demonstrating SARS-CoV-2 RNA was seen within the endothelium, tunica media of vessels, and within vessel lumens in multiple tissues, including in the leptomeninges of the cerebellum, brain stem parenchyma, lung, heart, liver, kidney, and pancreas ( Figure 3A , 3B1, and 3B2). SARS-CoV-2 RNA was observed in endothelial cells in the pulmonary vessels, adjacent to a thrombus, from another case patient (CP 22, Figure  3B3 and 3B4). ISH images for positive and negative controls (Supplementary Figure 1) and GAPDH RNA quality control (Supplementary Figure 2B1 and 2B2) are described in the Supplementary Data. The S-gene ISH assay was specific for SARS-CoV-2 while N-gene ISH assay cross-reacted with both SARS-CoV and SARS-CoV-2 cell culture controls. However, the staining by N-gene probe ISH was more abundant compared with the S-gene probe ISH. Histologic findings for 32 SARS-CoV-2 cRT-PCR-positive case patients ( Figure 2A1 and 2A3) . The most predominant histological feature identified in the lungs of 21 of these 32 (66%) case patients was diffuse alveolar damage (DAD) with various levels of progression and severity, including both exudative and proliferative stages. Interestingly, none of the 4 pediatric case patients had DAD (DOI ≤3 days). Of 32 SARS-CoV-2 cRT-PCR-positive case patients, pulmonary thrombi or emboli were detected in the lungs of 8 (25%), 6 (19%) had mild interstitial pneumonitis, and 10 (31%) had bronchopneumonia. Moderate lymphoplasmacytic myocarditis was seen in 1 case (CP 5) and multifocal lymphohistiocytic and eosinophilic myocarditis was seen in the second (CP 9). However, SARS-CoV-2 RT-PCR and ISH were negative in heart tissues from these patients.

WGS was performed on respiratory tissues from 27 case patients; we obtained SARS-CoV-2 full and nearly full genome sequences from 19 case patients and partial genome sequences from 7 case patients. WGS did not work for 1 patient. Phylogenetic analysis showed sequences representing all 3 groups (groups A-C) and fall into clades 19A, 19B, 20A, and 20C. Spike amino acid variant D614G was identified in 9 of 26 (35%) case patients; all 9 of them died between 19 March and 1 July 2020. In contrast, all 15 case patients without the D614G variant (with full genome sequences obtained) died between 24 February and 22 March 2020. A summary of common amino acid mutations, along with their respective clades, is described in Supplementary Table 1 . No significant amino acid mutation was identified that was consistent within these 19 full genomes. Identical sequences were obtained from the 7 case patients (CP 13-18 and 20) who were part of a long-term care facility outbreak [19] .

This work provides direct evidence of SARS-CoV-2 replication in lungs and trachea of COVID-19 case patients. SARS-CoV-2 was identified by cRT-PCR in 32 case patients (21 confirmed and 11 suspected) and subgenomic RNA transcripts, demonstrating active viral replication, were detected in lungs or trachea from 17 of 32 RT-PCR-positive case patients. Furthermore, we localized SARS-CoV-2 RNA by ISH in hyaline membranes, pneumocytes, and macrophages of lungs and epithelial cells of airways, and identified cellular targets of SARS-CoV-2 tropism and replication. Replicative viral RNA was detected in lungs Wide-range and specific real-time/conventional PCR assays for bacteria were performed only on the cases that had clinical and/or histopathological suspicion of these infections or if they were positive by immunohistochemical assay.

Mixed infections of other respiratory pathogens were identified in 7 SARS-CoV-2-negative case patients.

and trachea within the areas of histopathological changes, suggesting direct virus-induced injury and inflammation. Importantly, we also demonstrated direct infection of SARS-CoV-2 in vascular endothelium by localizing viral RNA in endothelial cells and tunica media of the vessels in multiple tissues, including lungs, brain stem, cerebellar leptomeninges, heart, liver, kidney, and pancreas. Both case patients with endothelial infection had comorbidities (cardiovascular disease and diabetes) that are characterized by preexisting vascular dysfunction with altered endothelial cell metabolism [28] . Additionally, in 1 of these case patients, SARS-CoV-2 RNA was observed in endothelial cells of the pulmonary vessels, adjacent to a thrombus. In both case patients, relatively lower SARS-CoV-2 rRT-PCR Ct values, consistent with high viral load, was also noted in the lungs. A few recent studies have also reported SARS-CoV-2 infection of endothelial cells using electron microscopy [6, 9] ; however, interpretation of electron microscopy is complex [29] . Cellular localization of SARS-CoV-2 RNA in endothelial cells by ISH provides strong evidence of endothelial infection and supports the hypothesis that direct infection of SARS-CoV-2 in endothelial cells may cause vascular dysfunction that leads to hypercoagulation and thrombosis, possibly triggering subsequent immune response and severe disease outcomes. Further investigation into the pathophysiology of virusinduced injury, thrombosis, and associated host inflammatory response is warranted. Our findings also indicate early tropism and replication of SARS-CoV-2 in the upper respiratory tract. Notably, in SARS-CoV-2 cRT-PCR-positive case patients who had short DOI (1-5 days), active viral replication was detected in trachea of 33% case patients and in lungs of only 17% case patients (Table 3) . Conversely, in cRT-PCR-positive case patients with longer DOI (≥10 days), active viral replication was detected in lungs of 69% case patients and in trachea of only 45% case patients (Table 3 ). In these case patients with longer DOI, lung specimens showed relatively lower SARS-CoV-2 rRT-PCR Ct values in comparison to trachea, suggesting higher viral load in lungs. This may indicate progression of infection from airway to lungs over time. Additionally, we noted prolonged detection (up to 26 days) and replication (up to 18 days) of SARS-CoV-2 RNA in the lungs. Similar findings were reported for SARS-CoV in which viral replication was commonly seen in lung tissues up to 20 days after symptom onset [30] . Our histopathological findings show tracheobronchitis in 86% of case patients with trachea available. We also detected viral replication in tracheal submucosal glands of 3 case patients. These findings, along with early tropism and replication in trachea, may help to possibly explaining more efficient transmission of SARS-CoV-2. Our findings in lung tissues from adult case patients support previous autopsy reports, showing DAD as the most prominent finding [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . However, in all 4 pediatric case patients, including 3 infants, a marked absence of DAD was observed in the lungs; all 4 patients had short DOI (≤3 days) and were positive only by RT-PCR. More studies are needed to elucidate the pathogenesis of COVID-19 in children.

We also detected SARS-CoV-2 RNA by cRT-PCR and rRT-PCR in heart, kidney, brain, small intestine, colon, liver, spleen, and pancreas in 14 of 32 respiratory tissue RT-PCR-positive case patients; however, SARS-CoV-2 rRT-PCR Ct values were higher in nonrespiratory tissues than in respiratory tissues (Supplementary Figure 3) . Moreover, we did not detect evidence of active viral replication or ISH positivity in nonrespiratory tissues. Thus, SARS-CoV-2 RT-PCR-positive results in nonrespiratory tissues may represent residual genomic viral RNA in blood that was detected by RT-PCR. Higher SARS-CoV-2 RNA levels in blood have been reported in critically ill patients [31] . Another possible explanation of our RT-PCR results could be due to introduction of SARS-CoV-2 RNA from respiratory tissues to nonrespiratory tissues during procurement of postmortem tissues. Some recent studies have identified SARS-CoV-2 RNA by RT-PCR in kidney, colon, spleen, skin, heart, and brain [10, 13, 14] . Nevertheless, extrapulmonary dissemination of SARS-CoV-2 is not well understood.

In addition, we detected coinfection of SARS-CoV-2 with other bacterial or viral pathogens in respiratory tissues of 10 of 32 (31%) cRT-PCR-positive case patients. Streptococcus spp, including S. pneumoniae, was identified in the lungs of 50% of these 10 case patients; all had histologic evidence of bronchopneumonia. Bacterial coinfections in viral pneumonia are generally associated with increased mortality [32] . Although data are limited, SARS-CoV-2 and community-acquired invasive bacterial coinfection appears to be infrequent [33, 34] . Interestingly, we also detected other pathogens in respiratory tissues from 14 of 32 (44%) SARS-CoV-2 cRT-PCR-negative case patients. Thus, this work highlights the importance of fixed tissue analysis for retrospective diagnosis of infection by SARS-CoV-2 and other respiratory pathogens, particularly for fatal cases in which no previous testing for SARS-CoV-2 was performed. By tissue analysis, we provided the first evidence of SARS-CoV-2 in 11 of 43 (26%) case patients with suspected COVID-19 and identified the first 2 US COVID-19 deaths retrospectively [35] . Furthermore, we performed genetic characterization of SARS-CoV-2 from fixed tissues of fatal cases and identified D614G spike variant in 9 of 26 (35%) RT-PCR-positive case patients. These 9 case patients had a lower median SARS-CoV-2 rRT-PCR Ct value, indicating higher viral load, in lung tissues, in comparison to the 17 case patients without this variant. However, other factors, such as DOI and underlying medical conditions, could also contribute to relatively higher viral load in these case patients. Nonetheless, a recent study showed a correlation between D614G and higher viral load [36] .

In summary, this work provides new insights into cellular targets of SARS-CoV-2 tropism and replication, and enhances the understanding of viral pathogenesis, transmissibility, and the mechanisms of severe outcomes of COVID-19, which may have important implications for patient management. SARS-CoV-2 replication in the upper airways during the early disease course, followed by active replication in the lungs for up to 2 weeks, suggests that there may be a wider window of opportunity for interventional antiviral therapy. Furthermore, our work suggests that direct infection of SARS-CoV-2 in vascular endothelial cells may play a crucial role in modulating endothelial dysfunction and thrombosis, triggering subsequent host immune response, and causing vascular complications and severe disease outcomes. Taken together, COVID-19 has a complex pathogenesis and much remains to be explored.

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

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We sincerely thank the staff of the Centers for Disease Control and Prevention's (CDC) Infectious Diseases Pathology Branch teams, including Epidemiology and Operations, Histology, Immunohistochemistry, Molecular Pathology, and Microbiology teams for accessioning the cases, processing and sectioning the tissues, and preparing the control blocks. We also thank CDC's Natalie Thornburg, Jennifer L. Harcourt, and Azaibi Tamin for providing SARS-CoV-2 culture controls, and Triona Henderson-Samuel for facilitating specimen submission and data collection for the cases. We also gratefully acknowledge the tireless work and support of the CDC COVID-19 Response Teams and thank all state and local health departments, clinicians, pathologists, medical examiners, and coroners who submitted specimens to the CDC.Disclaimer. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC.Financial support. This work was supported by the Centers for Disease Control and Prevention, Atlanta, Georgia. 12