Bat pluripotent stem cells reveal unusual entanglement between host and viruses.

Bats are distinctive among mammals due to their ability to fly, use laryngeal echolocation, and tolerate viruses. However, there are currently no reliable cellular models for studying bat biology or their response to viral infections. Here, we created induced pluripotent stem cells (iPSCs) from two species of bats: the wild greater horseshoe bat (Rhinolophus ferrumequinum) and the greater mouse-eared bat (Myotis myotis). The iPSCs from both bat species showed similar characteristics and had a gene expression profile resembling that of cells attacked by viruses. They also had a high number of endogenous viral sequences, particularly retroviruses. These results suggest that bats have evolved mechanisms to tolerate a large load of viral sequences and may have a more intertwined relationship with viruses than previously thought. Further study of bat iPSCs and their differentiated progeny will provide insights into bat biology, virus host relationships, and the molecular basis of bats' special traits.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

Dietary fructose improves intestinal cell survival and nutrient absorption.

Fructose consumption is linked to the rising incidence of obesity and cancer, which are two of the leading causes of morbidity and mortality globally1,2. Dietary fructose metabolism begins at the epithelium of the small intestine, where fructose is transported by glucose transporter type 5 (GLUT5; encoded by SLC2A5) and phosphorylated by ketohexokinase to form fructose 1-phosphate, which accumulates to high levels in the cell3,4. Although this pathway has been implicated in obesity and tumour promotion, the exact mechanism that drives these pathologies in the intestine remains unclear. Here we show that dietary fructose improves the survival of intestinal cells and increases intestinal villus length in several mouse models. The increase in villus length expands the surface area of the gut and increases nutrient absorption and adiposity in mice that are fed a high-fat diet. In hypoxic intestinal cells, fructose 1-phosphate inhibits the M2 isoform of pyruvate kinase to promote cell survival5-7. Genetic ablation of ketohexokinase or stimulation of pyruvate kinase prevents villus elongation and abolishes the nutrient absorption and tumour growth that are induced by feeding mice with high-fructose corn syrup. The ability of fructose to promote cell survival through an allosteric metabolite thus provides additional insights into the excess adiposity generated by a Western diet, and a compelling explanation for the promotion of tumour growth by high-fructose corn syrup.

FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires.

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.

LINE-1 promotes tumorigenicity and exacerbates tumor progression via stimulating metabolism reprogramming in non-small cell lung cancer.

BACKGROUND: Long Interspersed Nuclear Element-1 (LINE-1, L1) is increasingly regarded as a genetic risk for lung cancer. Transcriptionally active LINE-1 forms a L1-gene chimeric transcript (LCTs), through somatic L1 retrotransposition (LRT) or L1 antisense promoter (L1-ASP) activation, to play an oncogenic role in cancer progression. METHODS: Here, we developed Retrotransposon-gene fusion estimation program (ReFuse), to identify and quantify LCTs in RNA sequencing data from TCGA lung cancer cohort (n = 1146) and a single cell RNA sequencing dataset then further validated those LCTs in an independent cohort (n = 134). We next examined the functional roles of a cancer specific LCT (L1-FGGY) in cell proliferation and tumor progression in LUSC cell lines and mice. RESULTS: The LCT events correspond with specific metabolic processes and mitochondrial functions and was associated with genomic instability, hypomethylation, tumor stage and tumor immune microenvironment (TIME). Functional analysis of a tumor specific and frequent LCT involving FGGY (L1-FGGY) reveal that the arachidonic acid (AA) metabolic pathway was activated by the loss of FGGY through the L1-FGGY chimeric transcript to promote tumor growth, which was effectively targeted by a combined use of an anti-HIV drug (NVR) and a metabolic inhibitor (ML355). Lastly, we identified a set of transcriptomic signatures to stratify the LUSC patients with a higher risk for poor outcomes who may benefit from treatments using NVR alone or combined with an anti-metabolism drug. CONCLUSIONS: This study is the first to characterize the role of L1 in metabolic reprogramming of lung cancer and provide rationale for L1-specifc prognosis and potential for a therapeutic strategy for treating lung cancer. TRIAL REGISTRATION: Study on the mechanisms of the mobile element L1-FGGY promoting the proliferation, invasion and immune escape of lung squamous cell carcinoma through the 12-LOX/Wnt pathway, Ek2020111. Registered 27 March 2020 - Retrospectively registered.

Molecular Profiling of Coronavirus Disease 2019 (COVID-19) Autopsies Uncovers Novel Disease Mechanisms.

Current understanding of coronavirus disease 2019 (COVID-19) pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multiscale, next-generation RNA-sequencing methods (bulk, single-nuclei, and spatial transcriptomics) to provide unprecedented molecular resolution of COVID-19-induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin receptor-like receptor, or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin-converting enzyme 2 was rarely expressed, whereas basigin showed diffuse expression, and alanyl aminopeptidase, membrane, was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptoms (one had died after a month-long hospitalization with multiorgan involvement, and the other had died after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger data sets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis.

The dynamic nature of gene regulatory networks allows cells to rapidly respond to environmental change. However, the underlying temporal connections are missed, even in kinetic studies, as transcription factor (TF) binding within at least one time point is required to identify primary targets. The TF-regulated but unbound genes are dismissed as secondary targets. Instead, we report that these genes comprise transient TF-target interactions most relevant to rapid signal transduction. We temporally perturbed a master TF (Basic Leucine Zipper 1, bZIP1) and the nitrogen (N) signal it transduces and integrated TF regulation and binding data from the same cell samples. Our enabling approach could identify primary TF targets based solely on gene regulation, in the absence of TF binding. We uncovered three classes of primary TF targets: (i) poised (TF-bound but not TF-regulated), (ii) stable (TF-bound and TF-regulated), and (iii) transient (TF-regulated but not TF-bound), the largest class. Unexpectedly, the transient bZIP1 targets are uniquely relevant to rapid N signaling in planta, enriched in dynamic N-responsive genes, and regulated by TF and N signal interactions. These transient targets include early N responders nitrate transporter 2.1 and NIN-like protein 3, bound by bZIP1 at 1-5 min, but not at later time points following TF perturbation. Moreover, promoters of these transient targets are uniquely enriched with cis-regulatory motifs coinherited with bZIP1 binding sites, suggesting a recruitment role for bZIP1. This transient mode of TF action supports a classic, but forgotten, "hit-and-run" transcription model, which enables a "catalyst TF" to activate a large set of targets within minutes of signal perturbation.

STIM1 participates in the contractile rhythmicity of HL-1 cells by moderating T-type Ca(2+) channel activity.

STIM1 plays a crucial role in Ca(2+) homeostasis, particularly in replenishing the intracellular Ca(2+) store following its depletion. In cardiomyocytes, the Ca(2+) content of the sarcoplasmic reticulum must be tightly controlled to sustain contractile activity. The presence of STIM1 in cardiomyocytes suggests that it may play a role in regulating the contraction of cardiomyocytes. The aim of the present study was to determine how STIM1 participates in the regulation of cardiac contractility. Atomic force microscopy revealed that knocking down STIM1 disrupts the contractility of cardiomyocyte-derived HL-1 cells. Ca(2+) imaging also revealed that knocking down STIM1 causes irregular spontaneous Ca(2+) oscillations in HL-1 cells. Action potential recordings further showed that knocking down STIM1 induces early and delayed afterdepolarizations. Knocking down STIM1 increased the peak amplitude and current density of T-type voltage-dependent Ca(2+) channels (T-VDCC) and shifted the activation curve toward more negative membrane potentials in HL-1 cells. Biotinylation assays revealed that knocking down STIM1 increased T-VDCC surface expression and co-immunoprecipitation assays suggested that STIM1 directly regulates T-VDCC activity. Thus, STIM1 is a negative regulator of T-VDCC activity and maintains a constant cardiac rhythm by preventing a Ca(2+) overload that elicits arrhythmogenic events.

Involvement of phosphoinositide 3-kinase and PTEN protein in mechanism of activation of TRPC6 protein in vascular smooth muscle cells.

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry after the stimulation of a G(q)-protein-coupled or tyrosine-kinase receptor. TRPC6 translocates to the plasma membrane upon stimulation and remains there as long as the stimulus is present. However, the mechanism that regulates the trafficking and activation of TRPC6 are unclear. In this study we showed phosphoinositide 3-kinase and its antagonistic phosphatase, PTEN, are involved in the activation of TRPC6. The inhibition of PI3K by PIK-93, LY294002, or wortmannin decreased carbachol-induced translocation of TRPC6 to the plasma membrane and carbachol-induced net Ca(2+) entry into T6.11 cells. Conversely, a reduction of PTEN expression did not affect carbachol-induced externalization of TRPC6 but increased Ca(2+) entry through TRPC6 in T6.11 cells. We also showed that the PI3K/PTEN pathway regulates vasopressin-induced translocation of TRPC6 to the plasma membrane and vasopressin-induced Ca(2+) entry into A7r5 cells, which endogenously express TRPC6. In summary, we provided evidence that the PI3K/PTEN pathway plays an important role in the translocation of TRPC6 to the plasma membrane and may thus have a significant impact on Ca(2+) signaling in cells that endogenously express TRPC6.

The Breast Cancer Single-Cell Atlas: Defining cellular heterogeneity within model cell lines and primary tumors to inform disease subtype, stemness, and treatment options.

PURPOSE: Breast Cancer (BC) is the most diagnosed cancer in women; however, through significant research, relative survival rates have significantly improved. Despite progress, there remains a gap in our understanding of BC subtypes and personalized treatments. This manuscript characterized cellular heterogeneity in BC cell lines through scRNAseq to resolve variability in subtyping, disease modeling potential, and therapeutic targeting predictions. METHODS: We generated a Breast Cancer Single-Cell Cell Line Atlas (BSCLA) to help inform future BC research. We sequenced over 36,195 cells composed of 13 cell lines spanning the spectrum of clinical BC subtypes and leveraged publicly available data comprising 39,214 cells from 26 primary tumors. RESULTS: Unsupervised clustering identified 49 subpopulations within the cell line dataset. We resolve ambiguity in subtype annotation comparing expression of Estrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor 2 genes. Gene correlations with disease subtype highlighted S100A7 and MUCL1 overexpression in HER2 + cells as possible cell motility and localization drivers. We also present genes driving populational drifts to generate novel gene vectors characterizing each subpopulation. A global Cancer Stem Cell (CSC) scoring vector was used to identify stemness potential for subpopulations and model multi-potency. Finally, we overlay the BSCLA dataset with FDA-approved targets to identify to predict the efficacy of subpopulation-specific therapies. CONCLUSION: The BSCLA defines the heterogeneity within BC cell lines, enhancing our overall understanding of BC cellular diversity to guide future BC research, including model cell line selection, unintended sample source effects, stemness factors between cell lines, and cell type-specific treatment response.

Isoform-resolved transcriptome of the human preimplantation embryo.

Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.

Long-read RNA-seq atlas of novel microglia isoforms elucidates disease-associated genetic regulation of splicing.

Microglia, the innate immune cells of the central nervous system, have been genetically implicated in multiple neurodegenerative diseases. We previously mapped the genetic regulation of gene expression and mRNA splicing in human microglia, identifying several loci where common genetic variants in microglia-specific regulatory elements explain disease risk loci identified by GWAS. However, identifying genetic effects on splicing has been challenging due to the use of short sequencing reads to identify causal isoforms. Here we present the isoform-centric microglia genomic atlas (isoMiGA) which leverages the power of long-read RNA-seq to identify 35,879 novel microglia isoforms. We show that the novel microglia isoforms are involved in stimulation response and brain region specificity. We then quantified the expression of both known and novel isoforms in a multi-ethnic meta-analysis of 555 human microglia short-read RNA-seq samples from 391 donors, the largest to date, and found associations with genetic risk loci in Alzheimer's disease and Parkinson's disease. We nominate several loci that may act through complex changes in isoform and splice site usage.

Molecular states during acute COVID-19 reveal distinct etiologies of long-term sequelae.

Post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are debilitating, clinically heterogeneous and of unknown molecular etiology. A transcriptome-wide investigation was performed in 165 acutely infected hospitalized individuals who were followed clinically into the post-acute period. Distinct gene expression signatures of post-acute sequelae were already present in whole blood during acute infection, with innate and adaptive immune cells implicated in different symptoms. Two clusters of sequelae exhibited divergent plasma-cell-associated gene expression patterns. In one cluster, sequelae associated with higher expression of immunoglobulin-related genes in an anti-spike antibody titer-dependent manner. In the other, sequelae associated independently of these titers with lower expression of immunoglobulin-related genes, indicating lower non-specific antibody production in individuals with these sequelae. This relationship between lower total immunoglobulins and sequelae was validated in an external cohort. Altogether, multiple etiologies of post-acute sequelae were already detectable during SARS-CoV-2 infection, directly linking these sequelae with the acute host response to the virus and providing early insights into their development.

A microRNA activity map of human mesenchymal tumors: connections to oncogenic pathways; an integrative transcriptomic study.

BACKGROUND: MicroRNAs (miRNAs) are nucleic acid regulators of many human mRNAs, and are associated with many tumorigenic processes. miRNA expression levels have been used in profiling studies, but some evidence suggests that expression levels do not fully capture miRNA regulatory activity. In this study we integrate multiple gene expression datasets to determine miRNA activity patterns associated with cancer phenotypes and oncogenic pathways in mesenchymal tumors - a very heterogeneous class of malignancies. RESULTS: Using a computational method, we identified differentially activated miRNAs between 77 normal tissue specimens and 135 sarcomas and we validated many of these findings with microarray interrogation of an independent, paraffin-based cohort of 18 tumors. We also showed that miRNA activity is imperfectly correlated with miRNA expression levels. Using next-generation miRNA sequencing we identified potential base sequence alterations which may explain differential activity. We then analyzed miRNA activity changes related to the RAS-pathway and found 21 miRNAs that switch from silenced to activated status in parallel with RAS activation. Importantly, nearly half of these 21 miRNAs were predicted to regulate integral parts of the miRNA processing machinery, and our gene expression analysis revealed significant reductions of these transcripts in RAS-active tumors. These results suggest an association between RAS signaling and miRNA processing in which miRNAs may attenuate their own biogenesis. CONCLUSIONS: Our study represents the first gene expression-based investigation of miRNA regulatory activity in human sarcomas, and our findings indicate that miRNA activity patterns derived from integrated transcriptomic data are reproducible and biologically informative in cancer. We identified an association between RAS signaling and miRNA processing, and demonstrated sequence alterations as plausible causes for differential miRNA activity. Finally, our study highlights the value of systems level integrative miRNA/mRNA assessment with high-throughput genomic data, and the applicability of paraffin-tissue-derived RNA for validation of novel findings.

Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection.

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance.

Involvement of Rab9 and Rab11 in the intracellular trafficking of TRPC6.

TRPC proteins become involved in Ca2+ entry following the activation of Gq-protein coupled receptors. TRPC6 is inserted into the plasma membrane upon stimulation and remains in the plasma membrane as long as the stimulus is present. However, the mechanism that regulates the trafficking of TRPC6 is unclear. In the present study, we highlighted the involvement of two Rab GTPases in the trafficking of TRPC6. Rab9 co-localized in vesicular structures with TRPC6 in HeLa cells and co-immunoprecipitated with TRPC6. When co-expressed with TRPC6, Rab9(S21N), a dominant negative mutant, caused an increase in the level of TRPC6 at the plasma membrane and in TRPC6-mediated Ca2+ entry upon activation by a muscarinic receptor agonist. Similarly, the expression of Rab11 also caused an increase in TRPC6 expression at the cell surface and an increase in TRPC6-mediated Ca2+ entry. The co-expression of TRPC6 with the dominant negative mutant Rab11(S25N) abolished CCh-induced TRPC6 activation and reduced the level of TRPC6 at the plasma membrane. This study demonstrates that the trans-Golgi network and recycling endosomes are involved in the intracellular trafficking of TRPC6 by regulating channel density at the cell surface.

Long read, isoform aware sequencing of mouse nucleus accumbens after chronic cocaine treatment.

To better understand the full-length transcriptome of the nucleus accumbens (NAc)-a key brain reward region-in chronic cocaine treatment, we perform the first single molecule, long-read sequencing analysis using the Iso-seq method to detect 42,114 unique transcripts from mouse NAc polyadenylated RNA. Using GENCODE annotation as a reference, we find that over half of the Iso-seq derived transcripts are annotated, while 46% of them harbor novel splicing events in known genes; around 1% of them correspond to other types of novel transcripts, such as fusion, antisense and intergenic. Approximately 34% of the novel transcripts are matched with a compiled transcriptome assembled from published short-read data from various tissues, with the remaining 69% being unique to NAc. These data provide a more complete picture of the NAc transcriptome than existing annotations and can serve as a comprehensive reference for future transcriptomic analyses of this important brain reward region.

Identification of Alternative Polyadenylation in Cyanidioschyzon merolae Through Long-Read Sequencing of mRNA.

Alternative polyadenylation (APA) is widespread among metazoans and has been shown to have important impacts on mRNA stability and protein expression. Beyond a handful of well-studied organisms, however, its existence and consequences have not been well investigated. We therefore turned to the deep-branching red alga, Cyanidioschyzon merolae, to study the biology of polyadenylation in an organism highly diverged from humans and yeast. C. merolae is an acidothermophilic alga that lives in volcanic hot springs. It has a highly reduced genome (16.5 Mbp) and has lost all but 27 of its introns and much of its splicing machinery, suggesting that it has been under substantial pressure to simplify its RNA processing pathways. We used long-read sequencing to assess the key features of C. merolae mRNAs, including splicing status and polyadenylation cleavage site (PAS) usage. Splicing appears to be less efficient in C. merolae compared with yeast, flies, and mammalian cells. A high proportion of transcripts (63%) have at least two distinct PAS's, and 34% appear to utilize three or more sites. The apparent polyadenylation signal UAAA is used in more than 90% of cases, in cells grown in both rich media or limiting nitrogen. Our documentation of APA for the first time in this non-model organism highlights its conservation and likely biological importance of this regulatory step in gene expression.

Staphylococcus aureus induces an itaconate-dominated immunometabolic response that drives biofilm formation.

Staphylococcus aureus is a prominent human pathogen that readily adapts to host immune defenses. Here, we show that, in contrast to Gram-negative pathogens, S. aureus induces a distinct airway immunometabolic response dominated by the release of the electrophilic metabolite, itaconate. The itaconate synthetic enzyme, IRG1, is activated by host mitochondrial stress, which is induced by staphylococcal glycolysis. Itaconate inhibits S. aureus glycolysis and selects for strains that re-direct carbon flux to fuel extracellular polysaccharide (EPS) synthesis and biofilm formation. Itaconate-adapted strains, as illustrated by S. aureus isolates from chronic airway infection, exhibit decreased glycolytic activity, high EPS production, and proficient biofilm formation even before itaconate stimulation. S. aureus thus adapts to the itaconate-dominated immunometabolic response by producing biofilms, which are associated with chronic infection of the human airway.

Tissue-based SARS-CoV-2 detection in fatal COVID-19 infections: Sustained direct viral-induced damage is not necessary to drive disease progression.

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. 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 polymerase chain reaction (qPCR)-based detection. 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. 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 proinflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.