key: cord-0691695-6vmo93nh
authors: El Jamal, Siraj M.; Pujadas, Elisabet; Ramos, Irene; Bryce, Clare; Grimes, Zachary M.; Amanat, Fatima; Tsankova, Nadejda M.; Mussa, Zarmeen; Olson, Sara; Salem, Fadi; Miorin, Lisa; Aydillo, Teresa; Schotsaert, Michael; Albrecht, Randy A.; Liu, Wen-Chun; Marjanovic, Nada; Francoeur, Nancy; Sebra, Robert; Sealfon, Stuart C.; García-Sastre, Adolfo; Fowkes, Mary; Cordon-Cardo, Carlos; Westra, William H.
title: Tissue-Based SARS-Cov-2 Detection in Fatal COVID-19 Infections: Sustained Direct Viral-Induced Damage is Not Necessary to Drive Disease Progression
date: 2021-05-04
journal: Hum Pathol
DOI: 10.1016/j.humpath.2021.04.012
sha: 2ae8bdb32755db46a67ffbc1d7ac8dfaeeeeb798
doc_id: 691695
cord_uid: 6vmo93nh

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although viral infection is known to trigger inflammatory processes contributing to tissue injury and organ failure, it is unclear whether direct viral damage is needed to sustain cellular injury. An understanding of pathogenic mechanisms has been handicapped by the absence of optimized methods to visualize the presence and distribution of SARS-CoV-2 in damaged tissues. METHODS: We first developed a positive control cell line (Vero E6) to validate SARS-CoV-2 detection assays. We then evaluated multiple organs (lungs, kidneys, heart, liver, brain, intestines, lymph nodes and spleen) from fourteen COVID-19 autopsy cases using immunohistochemistry (IHC) for the spike and the nucleoprotein proteins, and RNA in-situ hybridization (RNA ISH) for the spike protein mRNA. Tissue detection assays were compared with quantitative PCR (qPCR)-based detection. RESULTS: SARS-CoV-2 was histologically detected in the Vero E6 positive cell line control, 1 of 14 (7%) lungs, and none (0%) of the other 59 organs. There was perfect concordance between the IHC and RNA ISH results. qPCR confirmed high viral load in the SARS-CoV-2 ISH-positive lung tissue, and absent or low viral load in all ISH-negative tissues. CONCLUSIONS: In patients who die of COVID-19-related organ failure, SARS-CoV-2 is largely not detectable using tissue-based assays. Even in lungs showing widespread injury, SARS-CoV-2 viral RNA or proteins were detected in only a small minority of cases. This observation supports the concept that viral infection is primarily a trigger for multiple organ pathogenic pro-inflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.

COVID-19 is an ongoing pandemic caused by the SARS-CoV-2 virus. To date, almost 73 million cases have been reported worldwide resulting in more than 1.62 million deaths (coronavirus.jhu.edu; checked in December 2020). Clearly, there is an urgent need to better understand the pathogenesis of the disease in a way that will inform preventive and therapeutic strategies. Progress could be accelerated by detection assays that define the tissue distribution of the virus in infected patients. Instead, the capriciousness of tissue-based detection assays has thwarted insight into the role and timing of SARS-CoV-2 in disease progression. While some studies have consistently visualized the virus in organs such as lungs, brain, heart, and gastrointestinal tract of infected patients (1-11), and have used these findings as direct evidence of tissue specific tropism and viral-mediated tissue injury, others have been unable to detect virus widely in these same tissues, suggesting alternative mechanisms of organ damage that do not require persistent active viral infection (12) (13) (14) (15) .

Access to tissues for investigative purposes has largely come through the post-mortem examinations of fatal cases. The value of these samples has been restricted by two factors that have mired tissue-based research. First, post-mortem samples are highly susceptible to tissue degradation, limiting the effectiveness of detection assays that rely on the integrity of viral protein, DNA and RNA. Second, there has been a conspicuous absence of a positive control to guide interpretation of detection assays rendering detection assays vulnerable to bias and erroneous interpretation. In the absence of positive controls to establish standards of interpretation, even electron microcopy -the historic benchmark for confirming cellular viral infections -has led to erroneous affirmation of SARS-CoV-2 due to misinterpretation of non-J o u r n a l P r e -p r o o f specific ultrastructural findings (16) (17) (18) (19) . For tissue based platforms, discordant detection rates across studies may underscores the absence of well-defined and universally applied standards for probe validity and test interpretation (10) . These two factors, taken together, have obscured the ability to understand fundamental aspects of cell injury and organ dysfunction such as the distribution of SARS-CoV-2 in human tissues. It remains unclear, for example, whether the consistent inability to detect virus using conventional immunohistochemical and in-situ hybridization assays in damaged tissues reflects post-mortem viral degradation, ineffectiveness of detection assays, absence of tropism towards a particular tissue, low viral load or effective viral clearance. Conversely, it is not always clear whether the ready identification of SARS-CoV-2 in various tissue reflects the true presence of virus, or the misinterpretation of nonspecific changes. Resolution of key mechanisms of viral pathogenesis and, more specifically, the relative importance of direct viral injury versus subsequent inflammatory damage, cannot move forward until a positive control is established to guide the interpretation of detection assays. To more definitively define the tissue distribution of SARS-CoV-2 in fatal cases of COVID-19, we 1) developed a positive cell line control to guide interpretation of various detection assays for detection of viral protein and RNA, 2) used these detection assays to look for SARS-CoV-2 RNA or proteins in tissues from multiple organs, and then 3) compared histologic detection with a highly sensitive PCR-based detection assay.

Generation of SARS-CoV-2 infected Vero E6 cells A SARS-CoV-2 infected cell line was generated as previously described in detail (20) .

Briefly, 1,000,000 Vero E6 cells (ATCC CRL-1586) per well were seeded in a 6-well cell culture plate and maintained in Dulbecco's modified Eagle medium (DMEM; Life Technologies) J o u r n a l P r e -p r o o f supplemented with 10% fetal bovine serum (FBS), 10 ml of Pen-Strep (Gibco; catalog #15140122) and 10 ml of HEPES buffer (Gibco, catalog #15630080). After a day, the medium was removed, and SARS-CoV-2 isolate USA-WA1/2020 (obtained from BEI Resources NR-52281) was added to the cells at a multiplicity of infection of 0.01. The SARS-CoV-2 stock was diluted and added to the cells in 1X MEM that was supplemented with 2% FBS. The viruscontaining cell media was left on the cells and cells were scraped off and collected in PBS at three intervals: 24 hours, 48 hours, and 72 hours. Cells were fixed with 10% formaldehyde for 24 hours before use.

Ten ml of fixed Vero cell culture fluid suspension was centrifuged at 1600 rpm for 10 minutes resulting in a well-formed pellet. The tube was decanted and 5 drops of Histogel was added to the pellet. This mixture was vortexed for 3 seconds and refrigerated for 5 minutes to harden the pellet. The hardened pellet was then wrapped in biopsy tissue paper, placed in a tissue cassette, and fixed in formalin. Transmission electron microscopy was used to confirm the presence of SARS-CoV-2 viral particles within the infected cell line.

Uninfected Vero E6 cells were used as a control to compare Day 3 morphology for the presence of cytopathic effect and as a negative control for the IHC, ISH, and IF.

Fourteen COVID-19 autopsy cases were included: nine full autopsies and five additional lungonly cases. All patients had been admitted to the Mount Sinai for progressing COVID-related signs and symptoms, and a positive nasopharyngeal swap PCR SARS-CoV2 test. Autopsies were preferentially selected on the basis of short intervals from time of death to autopsy and/or short time intervals from onset of symptoms to death. To assess tissue-specific tropism, J o u r n a l P r e -p r o o f formalin-fixed and paraffin-embedded tissue blocks were selected from multiple organs including the lung (multiple samples from each lung), heart, liver, kidney, small intestine, brain (frontal lobe), spleen and lymph nodes. Paired fresh frozen specimens were collected at the time of autopsy from seven of the cases for qPCR analysis. Archived autopsy blocks from 2019 (i.e.

pre-COVID cases) were used as negative tissue controls Immunohistochemistry Four µm-thick sections were cut from the FFPE autopsy blocks and the SARS-CoV-2 infected cell pellets. Immunostaining was performed using antibodies against the SARS-CoV To minimize the problem of non-specific amplification, two independent primers (i.e. E gene and IP2) were used with positive detection requiring the amplification of both primers within 40 amplification cycles. The limit of detection of this assay for both primers was determined to be 1-5 RNA copies/reaction. Virus RNA was considered "detected" in a specific tissue when RNA amplification was found with both E and IP2 primer sets at a cycle threshold

[Ct] value lower than 40 amplification cycles. Cases where viral RNA was detected by only one of the primers sets were considered inconclusive. Figure 1 . Viral immunostaining of the nucleoprotein ( Figure 1B ) and spike protein ( Figure 1C ) was equally intense at 24, 48, and 72 hour preparations. In these suspensions of cultured cells, the intense staining together with cytoplasmic overlay of the nucleus made it difficult to confirm cytoplasmic restriction of the signals. RNA ISH with the sense strand RNA probe (S) showed abundant dot-like hybridization signals in the cytoplasm that coalesced into brown globules that effaced cellular and nuclear details ( Figure 1D ). The antisense RNA ISH probe (SS), specific for the SARS-CoV-2 spike protein negative RNA strand, showed dot-like hybridization signals confined to the cytoplasm, a pattern indicative of active viral replication within the infected cells ( Figure 1E ).

Immunofluorescence using the identical SARS-CoV-2 nucleoprotein primary antibody used for immunohistochemistry but at 1:500 concentration showed strong cytoplasmic staining within the majority of SARS-CoV-2 infected Vero cells at 24 and 48hrs of infection ( Figure 1F ).

Transmission electron microscopy confirmed the presence of densely packed viral particles within the infected cells ( Figure 1F, insert) . The presence of dot-like signals in infected cells is consistent with the formation of replication-transcription complexes during infection by coronaviruses and other positive sense RNA viruses (24) (25) (26) .

Uninfected Vero cells showed no recognizable nucleoprotein immunoreactivity and minimal to no background staining was detected using secondary only and non-specific monoclonal IgG isotype controls. All of the detection assays were negative in the uninfected Vero E6 cells used as a negative control. The general RNA ISH probe confirmed the presence of intact RNA in the Vero cell lines.

The autopsy cases were from eight (57%) females and six (43%) males (Table 1) . Their ages ranged from 32 to 94 years (median, 66.5 years; mean, 67 years). Eleven (79%) of the autopsies were performed within 24 hours of death (median post-mortem interval, 7.5 hours). The duration of disease from the onset symptoms to death ranged from 1 to 34 days (median, 7.5 days; mean, 10 days). All patients had chronic medical conditions known to increase the risk of severe COVID-19 illness including chronic hypertension (n=10), chronic kidney disease (n= 4), asthma (n=4), diabetes (n=4), cirrhosis (n=2), dementia (n=2) and/or severe obesity.

A summary of the major pathological findings at the time of autopsy are tabulated in Table   2 . The histopathologic findings have been previously reported as part of a larger Mount Sinai COVID-19 autopsy cohort (27) . Most of the injury was in the lungs. Diffuse alveolar damage (DAD) was present in all cases, predominantly in the acute phase, and judged to be the major factor contributing to patient death. All cases showed some degree of hypertensive changes in the heart manifesting as myocyte hypertrophy and interstitial fibrosis. Two cases showed end stage renal disease. Two cases showed liver cirrhosis. Five cases showed thrombi in the brain 

Correlation between qPCR testing and the histologic detection assays is shown in Table 3 .

Using qPCR, virus RNA was detected at a low amplification cycle threshold (Ct 24.07) in the lungs from case 7. In this same patient, virus RNA was also detected in a hilar lymph node, but at higher amplification cycle (34.06). For the other seven cases tested, virus RNA was detected in three lungs (cases 1, 5 and 6), 1 spleen (case 3), and 1 lymph node (case 5) with Ct values ranging from 31.06 to 36.75 (Table 3) . Of note, only the lungs in case 7 showed positivity for SARS-CoV-2 by IHC (for spike and NP proteins) and by ISH for S strand. None of the other tissues in this case or the other cases in which the virus detected by PCR showed positivity by IHC or ISH.

In this study, we have investigated the tissue distribution of SARS-CoV-2 in fatal cases of COVID-19 using multiple detection platforms. Following construction of a SARS-CoV-2 infected Vero cell line to guide interpretation of assay results, we found that tissue damage in the lungs and other sites does not always directly correlate with the histologic presence of virus.

This disconnect between the presence of virus and tissue damage points to other mechanisms of cellular injury not requiring the sustained presence of SARS-Co-V2.

Although direct viral cytotoxic effects may play some role in initiating a chain of events culminating in patient death, our findings suggest that the sustained presence of SARS-CoV-2 is not necessary to drive disease progression. Of the 73 organs we tested from 14 patients who died of COVID-19, we were able to histologically detect the presence of SARS-CoV-2 protein and RNA in the lungs of only a single patient. In this single positive case, the uncoupling of sense and anti-sense spike protein RNA expression suggests low levels of RNA replication at the time J o u r n a l P r e -p r o o f of staining. Admittedly, SARS-CoV-2 was identified by qPCR -albeit at relatively high cycle thresholds, likely indicative of low viral loads -in the lungs, spleen and lymph nodes from four patients where virus products were not detected using tissue-based assays. This uncoupling of qPCR-based and tissue-based detection does not invalidate methods of histologic detection, but only underscores that virus may be present at levels below histologic detection thresholds. Low level viral RNA detection by qPCR align with emerging models of disease progression where SARS-CoV-2 RNA is shed into the blood (15, 28) . In fact, PCR-based detection could simply reflect virus in transit and should not be taken as unequivocal evidence of organ-specific tropism.

The uncoupling of tissue injury SARS-CoV-2 with the ability to consistently visualize the presence of virus using histologic detection assays has been intimated in smaller studies. (14, (29) (30) (31) . This phenomena is not unique to the coronavirus family but has also been reported influenza virus infections (32) (33) (34) .

Even though sustained viral presence may not be required to drive disease progression, tissue infection with cytotoxic injury may be still be required to trigger the cascade of diffuse J o u r n a l P r e -p r o o f endothelial injury, hypercoagulability and a hyper-inflammatory state culminating in respiratory demise, multi-organ failure and death (14, 35, 36) . Histologic detection of SARS-CoV-2 could help define the temporal sequence of these events. In the progression of COVID-19, the temporal role of SARS-CoV-2 as a triggering event in the lung infections appears to be very early and transitory as some have suggested that SARS-CoV-2 tissue infection occurs at an early acute phase of disease progression (1, 37). Some have suggested that SARS-CoV-2 tissue infection occurs at an early acute phase of disease progression (1, 37) . With this in mind, we selectively evaluated post-mortem lungs from five cases with short intervals disease intervals (mean 3 days). SARS-CoV-2 was not detected in any of these cases. In light of these findings, the temporal role of SARS-CoV-2 as a triggering event in the lungs lung infections appears to be very early and transitory.

In summary, what was initially conceptualized as a primarily respiratory viral disease, COVID-19 is now recognized as a heterogeneous illness with a diverse array of symptoms and complications that may not necessarily be linked to direct viral injury. Our findings support the view that this complex process is likely related to ongoing and progressive immune dysregulation rather than persistent viral replication within the lung and other tissues and may advocate to focus on attenuating the pathological host response rather than targeting the actual virus in managing and treating COVID-19 patients. 

In situ detection of SARS-CoV-2 in lungs and airways of patients with COVID-19

Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China

Myocardial localization of coronavirus in COVID-19 cardiogenic shock

Evidence for Gastrointestinal Infection of SARS-CoV-2

Multiorgan and Renal Tropism of SARS-CoV-2

SARS-CoV-2 renal tropism associates with acute kidney injury

Persistence of viral RNA, pneumocyte syncytia and thrombosis are hallmarks of advanced COVID-19 pathology

Pathology and Pathogenesis of SARS-CoV-2 Associated with Fatal Coronavirus Disease, United States

COVID-19 pulmonary pathology: a multi-institutional autopsy cohort from Italy and New York City

Identification of Immunohistochemical Reagents for In Situ Protein Expression Analysis of Coronavirus-associated Changes in Human Tissues

Insights into pathogenesis of fatal COVID-19 pneumonia from histopathology with immunohistochemical and viral RNA studies

Postmortem Kidney Pathology Findings in Patients with COVID-19

Comparison of RNA In Situ Hybridization and Immunohistochemistry Techniques for the Detection and Localization of SARS-CoV-2 in Human Tissues

Tissue-Specific Immunopathology in Fatal COVID-19

Evidence of SARS-CoV-2 Replication and Tropism in the Lungs, Airways and Vascular Endothelium of Patients with Fatal COVID-19: An Autopsy Case-Series

Electron microscopy of SARS-CoV-2: a challenging task

Visualization of putative coronavirus in kidney

Difficulties in Differentiating Coronaviruses from Subcellular Structures in Human Tissues by Electron Microscopy

Why misinterpretation of electron micrographs in SARS-CoV-2-infected tissue goes viral

A serological assay to detect SARS-CoV-2 seroconversion in humans

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Performance Assessment of SARS-CoV-2 PCR Assays Developed by WHO Referral Laboratories

Protocol: Real-Time RT-PCR Assays for the Detection of SARS-CoV-2

SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro

Krijnse-Locker, Modification of intracellular membrane structures for virus replication

Pathophysiology of SARS-CoV-2: the Mount Sinai COVID-19 autopsy experience

Neuropathological Features of Covid-19

Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases

Immunohistochemical study of severe acute respiratory syndrome-associated coronavirus in tissue sections of patients

Time course and cellular localization of SARS-CoV nucleoprotein and RNA in lungs from fatal cases of SARS

Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs

Histopathologic and immunohistochemical features of fatal influenza virus infection in children during the 2003-2004 season

pandemic influenza A (H1N1): pathology and pathogenesis of 100 fatal cases in the United States

COVID-19: Staging of a New Disease

Extrapulmonary manifestations of COVID-19

Viral presence and immunopathology in patients with lethal COVID-19: a prospective autopsy cohort study

J o u r n a l P r e -p r o o f