Characterization of SARS-CoV-2 and host entry factors distribution in a COVID-19 autopsy series.

Background: SARS-CoV-2 is a highly contagious virus that causes the disease COVID-19. We have recently reported that androgens regulate the expression of SARS-CoV-2 host entry factors ACE2 and TMPRSS2, and androgen receptor (AR) in lung epithelial cells. We also demonstrated that the transcriptional repression of the AR enhanceosome inhibited SARS-CoV-2 infection in vitro.
Methods: To better understand the various sites of SARS-CoV-2 infection, and presence of host entry factors, we extensively characterized the tissue distribution and localization of SARS-CoV-2 virus, viral replication, and host entry factors in various anatomical sites sampled via autopsy. We applied RNA in-situ-hybridization (RNA-ISH), immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) approaches. We also assessed histopathological changes in SARS-CoV-2 infected tissues.
Results: We detect SARS-CoV-2 virus and viral replication in pulmonary tissues by RNA-ISH and IHC and a variety of non-pulmonary tissues including kidney, heart, liver, spleen, thyroid, lymph node, prostate, uterus, and colon by qRT-PCR. We observe heterogeneity in viral load and viral cytopathic effects among various organ systems, between individuals and within the same patient. In a patient with a history of kidney transplant and under immunosuppressant therapy, we observe an unusually high viral load in lung tissue by RNA-ISH, IHC and qRT-PCR. SARS-CoV-2 virus is also detected in this patent's kidney, liver and uterus. We find ACE2, TMPRSS2 and AR expression to overlap with the infection sites. Conclusions: This study portrays the impact of dispersed SARS-CoV-2 infection in diverse organ systems, thereby facilitating avenues for systematic therapeutic approaches.

Introduction

Coronavirus disease-19 (COVID-19), an infectious disease caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic by the World Health Organization on March 11, 2020. As of May 21st, 2021, 165,874,001 diagnosed cases and 3,438,383 deaths have been reported worldwide (https://coronavirus.jhu.edu/), with the United States of America bearing the highest disease impact in terms of morbidity and mortality (33,084,872 cases and 589,222 deaths) followed by India where is currently undergoing a massive surge of cases. COVID-19 is an established multiorgan disease in humans with the greatest involvement and derangement involving the respiratory, cardiovascular, renal, and immune systems[1].

Coronaviruses are a group of enveloped viruses with a single-stranded RNA genome. In rare instances, animal coronaviruses such as severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) in 2002, Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in 2012, and currently SARS-CoV-2, can infect humans with variable clinical complications and impact. All three of these coronaviruses are transmitted zoonotically and spread among humans through close contact[2]. SARS-CoV-2 is most commonly transmitted from person to person via respiratory droplets. Most patients experience mild symptoms. However, a significant subset of patients may experience severe disease outcomes including long term health sequelae and death[3]. The SARS-CoV-2 genome shares ~80% sequence identity with SARS-CoV and MERS-CoV, although the sequence similarity varies among genes encoding structural proteins and essential enzymes. SARS-CoV-2 is considered to be more pathogenic than the MERS-CoV and SARS-CoV[4,5]. Structurally, SARS-CoV-2 is comprised of four structural proteins: spike (S), envelope (E), membrane glycoprotein (M), and nucleocapsid phosphoprotein (N) proteins[5]. SARS-CoV-2 entry into host cells depends on binding of viral spike proteins to the host receptor protein angiotensin-converting enzyme 2 (ACE2) and priming by the host serine protease, the cell surface transmembrane protease serine 2 (TMPRSS2)[6,7]. In our recent study detailing transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2, we described co-expression of the androgen receptor (AR), TMPRSS2, and ACE2 in bronchial and alveolar cells in human and murine lung tissues; demonstrated that androgens regulate the expression of ACE2, TMPRSS2, and AR in subpopulations of lung epithelial cells; and reported that transcriptional repression of the AR enhanceosome by AR antagonists inhibited SARS-CoV2 infection in vitro[8]. To further investigate transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19, we set out to characterize the distribution and localization of SARS-CoV-2 viral particles, viral replication, and the host entry factor receptor ACE2, priming protease TMPRSS2 and transcriptional regulator AR by RNA in situ hybridization (RNA-ISH), immunohistochemistry (IHC) and qRT-PCR in various pulmonary and nonpulmonary tissues obtained from a series of six clinical autopsies performed on patients who succumbed to COVID-19 disease. We also correlated the presence of SARS-CoV-2 viruses and host entry machinery with clinicopathologic findings in the affected organ systems. We detect SARS-CoV-2 viral RNA and viral replication events in both pulmonary tissues and nonpulmonary tissues. We observe heterogeneity in viral load and viral cytopathic effects among various organ systems, between individuals and within the same patient. We also find the presence of host factors, ACE2, TMPRSS2, and AR, to overlap with the infection sites.

Methods

Patient selection

This study was performed under ethical approval protocols of the Institutional Review Boards of the University of Michigan Medical School (IRBMED). Six patients with SARS-CoV-2 infection who died from COVID-19 disease with clinical autopsies performed at Michigan Medicine were included in the study. The legal next of kin of patients provided the informed consent prior to autopsy. All the patients tested positive for SARS-CoV-2 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) performed in a clinical laboratory. This study was conducted prior to the FDA approval of emergency use of COVID-19 vaccines and none of the patients in this study received COVID-19 vaccination. During autopsy, multiple tissue samples were harvested from various pulmonary and nonpulmonary sites for clinicopathologic characterization. For the purposes of this study, representative FFPE tissue blocks (n = 74) were selected from 16 different anatomical sites by two pathologists (R. Mannan and R. Mehra) based on the evaluation of hematoxylin and eosin (H&E) stained FFPE slides with brightfield microscopy.

Histopathological evaluation

The pathological appraisal of tissue samples was performed for pulmonary and nonpulmonary organs. The histopathological assessment for the respiratory region was carried out on the trachea, bronchus, and pulmonary parenchymal tissues. For nonrespiratory sites, we evaluated myocardial tissues, hematological tissues (lymph node and spleen), liver, and tissues from the gastrointestinal (esophagus, stomach, small intestine, and colon) tract, endocrine (thyroid, pancreas, and adrenal) glands, and genitourinary (kidney, prostate, and uterus) tract. A systematic evaluation for histo-morphological and pathological changes were noted and recorded for each sample.

RNA in situ hybridization (RNA-ISH)

RNA-ISH was performed on 4 µm FFPE tissue sections using RNAscope 2.5 HD Brown kit, duplex kit and target probes against SARS-CoV-2 spike (S) gene (SARS-CoV-2-S probe), minus strand of SARS-CoV-2-S gene (SARS-CoV-2-S-sense), SARS-CoV-2 nucleocapsid (N) gene (SARS-CoV-2-N-O1-C2), human ACE2, TMPRSS2, and AR genes (Supplementary Table 1) (Advanced Cell Diagnostics, Newark, CA). The SARS-CoV-2-S-sense probe detects the minus strand viral RNA, which is present during active viral replication. RNA quality was assessed using Hs-PPIB probe as positive control and assay background was monitored using DapB probe as negative control. In addition, tissue sections from one normal lung and one H1N1 influenza patient lung were used as negative control samples for SARS-CoV-2 probes. RNA-ISH assay was performed as previously described[9-11]. After deparaffinization, FFPE sections were pretreated with hydrogen peroxide followed by heat-induced target retrieval and protease, and subsequently hybridized with target probe followed by a series of signal replications. Finally, chromogenic detection was performed using DAB and counterstained with 50% Gill’s hematoxylin I (Fisher Scientific, Rochester, NY). To confirm that the RNA-ISH signals were amplified from viral RNA, tissue sections were treated with 5 mg/ml RNase A for 30 min at 40 °C prior to target probe hybridization.

Immunohistochemistry (IHC)

IHC was performed on 4 µm FFPE tissue sections on the Ventana Discovery XT automated slide staining system (Roche-Ventana, AZ) using the ChromoMap diaminobenzidine (DAB) detection kit (760-500, Roche-Ventana, AZ). SARS-CoV-2 nucleocapsid IHC was performed using mouse monoclonal antibody (R&D system MAB10474) at 1:100 dilutions with heat-induced epitope retrieval with TRIS antigen retrieval buffer.

RNA extraction and quantitative RT-PCR (qRT-PCR)

Total RNA was extracted from two 5 µm FFPE tissue sections using the miRNeasy FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and eluted into 30 µl H2O.

For SARS-CoV-2 RNA detection, 8 µl RNA template was used per qRT-PCR reaction using the GenePath CoViDx One kit (Maharashtra, India), which contains premixed RT-PCR primer sets for SARS-CoV-2 Envelop (E) gene, Nucleocapsid (N) gene, RNA-dependent RNA polymerase (RdRP) gene and the human RNaseP, an internal extraction and amplification control. Samples were classified as SARS-CoV-2 positive when at least one of the three SARS-CoV-2 genes were detected with a Ct value < 40. Each reaction was performed in replicates. Tissues from one normal lung, one H1N1 influenza patient lung and one normal prostate were selected as negative control. For undetermined data points, Ct value was set to 40 to calculate the ∆Ct.

For viral replication detection, we performed two-step strand-specific RT-PCR as previously described[12]. Frist, strand-specific primers were used to reverse transcribe SARS-CoV-2 viral RNA to cDNA in two separate reactions: a forward E gene primer to generate minus strand cDNA representing viral replication and a reverse E gene primer to generate plus strand cDNA representing the single-strand virus. In addition, a separate set of human RNaseP primers were added to each set of reactions to serve as internal amplification control. Second, each cDNA sample was amplified by real-time PCR to detect either SARS-CoV-2 E gene or human RNaseP gene by specific primer sets using SYBR Green (Bio-Rad, Hercules, CA).

The RT-PCR data are available in the supplementary information files. All other data that support the findings of this study are available from the corresponding author upon reasonable request.