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1.
Kidney360 ; 2(4): 639-652, 2021 04 29.
Article in English | MEDLINE | ID: covidwho-1776889

ABSTRACT

Background: Kidney damage has been reported in patients with COVID-19. Despite numerous reports about COVID-19-associated nephropathy, the factual presence of the SARS-CoV-2 in the renal parenchyma remains controversial. Methods: We consecutively performed 16 immediate (≤3 hours) postmortem renal biopsies in patients diagnosed with COVID-19. Kidney samples from five patients who died from sepsis not related to COVID-19 were used as controls. Samples were methodically evaluated by three pathologists. Virus detection in the renal parenchyma was performed in all samples by bulk RNA RT-PCR (E and N1/N2 genes), immunostaining (2019-nCOV N-Protein), fluorescence in situ hybridization (nCoV2019-S), and electron microscopy. Results: The mean age of our COVID-19 cohort was 68.2±12.8 years, most of whom were male (69%). Proteinuria was observed in 53% of patients, whereas AKI occurred in 60% of patients. Acute tubular necrosis of variable severity was found in all patients, with no tubular or interstitial inflammation. There was no difference in acute tubular necrosis severity between the patients with COVID-19 versus controls. Congestion in glomerular and peritubular capillaries was respectively observed in 56% and 88% of patients with COVID-19, compared with 20% of controls, with no evidence of thrombi. The 2019-nCOV N-Protein was detected in proximal tubules and at the basolateral pole of scattered cells of the distal tubules in nine out of 16 patients. In situ hybridization confirmed these findings in six out of 16 patients. RT-PCR of kidney total RNA detected SARS-CoV-2 E and N1/N2 genes in one patient. Electron microscopy did not show typical viral inclusions. Conclusions: Our immediate postmortem kidney samples from patients with COVID-19 highlight a congestive pattern of AKI, with no significant glomerular or interstitial inflammation. Immunostaining and in situ hybridization suggest SARS-CoV-2 is present in various segments of the nephron.


Subject(s)
Acute Kidney Injury , COVID-19 , Acute Kidney Injury/diagnosis , Aged , Aged, 80 and over , COVID-19/complications , Capillaries/pathology , Humans , In Situ Hybridization, Fluorescence , Kidney Glomerulus/pathology , Male , Middle Aged , Necrosis , SARS-CoV-2
2.
J Med Virol ; 94(7): 3203-3222, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1750409

ABSTRACT

Circular RNAs (circRNAs) are a newly recognized component of the transcriptome with critical roles in autoimmune diseases and viral pathogenesis. To address the importance of circRNA in RNA viral transcriptome, we systematically identified and characterized circRNAs encoded by the RNA genomes of betacoronaviruses using both bioinformatical and experimental approaches. We predicted 351, 224, and 2764 circRNAs derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus, respectively. We experimentally identified 75 potential SARS-CoV-2 circRNAs from RNA samples extracted from SARS-CoV-2-infected Vero E6 cells. A systematic comparison of viral and host circRNA features, including abundance, strand preference, length distribution, circular exon numbers, and breakpoint sequences, demonstrated that coronavirus-derived circRNAs had a spliceosome-independent origin. We further showed that back-splice junctions (BSJs) captured by inverse reverse-transcription polymerase chain reaction have different level of resistance to RNase R. Through northern blotting with a BSJ-spanning probe targeting N gene, we identified three RNase R-resistant bands that represent SARS-CoV-2 circRNAs that are detected cytoplasmic by single-molecule and amplified fluorescence in situ hybridization assays. Lastly, analyses of 169 sequenced BSJs showed that both back-splice and forward-splice junctions were flanked by homologous and reverse complementary sequences, including but not limited to the canonical transcriptional regulatory sequences. Our findings highlight circRNAs as an important component of the coronavirus transcriptome, offer important evaluation of bioinformatic tools in the analysis of circRNAs from an RNA genome, and shed light on the mechanism of discontinuous RNA synthesis.


Subject(s)
COVID-19 , Middle East Respiratory Syndrome Coronavirus , Humans , In Situ Hybridization, Fluorescence , Middle East Respiratory Syndrome Coronavirus/genetics , RNA, Circular/genetics , SARS-CoV-2/genetics , Spliceosomes/genetics
3.
Life Sci Alliance ; 5(4)2022 04.
Article in English | MEDLINE | ID: covidwho-1614505

ABSTRACT

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.


Subject(s)
COVID-19/diagnosis , In Situ Hybridization, Fluorescence/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Caco-2 Cells , Cell Line, Tumor , Chlorocebus aethiops , Humans , In Situ Hybridization/methods , Microscopy, Electron/methods , RNA, Viral/ultrastructure , Reproducibility of Results , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Sensitivity and Specificity , Vero Cells , Virus Release/drug effects , Virus Release/genetics , Virus Release/physiology , Virus Replication/drug effects , Virus Replication/physiology
4.
Am J Obstet Gynecol MFM ; 3(6): 100468, 2021 11.
Article in English | MEDLINE | ID: covidwho-1525659

ABSTRACT

OBJECTIVE: This study aimed to report the spectrum of placental pathology findings in pregnancies complicated by SARS-CoV-2 infection. DATA SOURCES: MEDLINE, Embase, Google Scholar, and the Web of Science databases were searched up to August 11, 2021. STUDY ELIGIBILITY CRITERIA: Histopathologic anomalies included maternal vascular malperfusion, fetal vascular malperfusion, acute inflammatory pathology, chronic inflammatory pathology, increased perivillous fibrin, and intervillous thrombosis. Moreover, subanalyses of symptomatic women only and high-risk pregnancies were performed. METHODS: Histopathologic analysis of the placenta included gross examination, histopathology on hematoxylin and eosin, immunohistochemistry, fluorescence in situ hybridization, quantitative reverse transcription-polymerase chain reaction on placental tissue, and transmission electron microscope. Random-effect meta-analyses were used to analyze the data. RESULTS: A total of 56 studies (1008 pregnancies) were included. Maternal vascular malperfusion was reported in 30.7% of placentas (95% confidence interval, 20.3-42.1), whereas fetal vascular malperfusion was observed in 27.08 % of cases (95% confidence interval, 19.2-35.6). Acute and chronic inflammatory pathologies were reported in 22.68% (95% confidence interval, 16.9-29.0) and 25.65% (95% confidence interval, 18.4-33.6) of cases, respectively. Increased perivillous fibrin was observed in 32.7% (95% confidence interval, 24.1-42.0) of placentas undergoing histopathologic analysis, whereas intervillous thrombosis was observed in 14.6% of cases (95% confidence interval, 9.7-20.2). Other placental findings, including a basal plate with attached myometrial fibers, microscopic accretism, villous edema, increased circulating nucleated red blood cells, or membranes with hemorrhage, were reported in 37.5% of cases (95% confidence interval, 28.0-47.5), whereas only 17.5% of cases (95% confidence interval, 10.9-25.2) did not present any abnormal histologic findings. The subanalyses according to maternal symptoms owing to SARS-CoV-2 infection or the presence of a high-risk pregnancy showed a similar distribution of the different histopathologic anomalies to that reported in the main analysis. Moreover, the risk of placental histopathologic anomalies was higher when considering only case-control studies comparing women with SARS-CoV-2 infection with healthy controls. CONCLUSION: In pregnant women with SARS-CoV-2 infection, a significant proportion of placentas showed histopathologic findings, suggesting placental hypoperfusion and inflammation. Future multicenter prospective blinded studies are needed to correlate these placental lesions with pregnancy outcomes.


Subject(s)
COVID-19 , Pregnancy Complications, Infectious , Female , Humans , In Situ Hybridization, Fluorescence , Multicenter Studies as Topic , Placenta , Pregnancy , Pregnancy Outcome , Prospective Studies , SARS-CoV-2
5.
Sci Rep ; 11(1): 19579, 2021 10 01.
Article in English | MEDLINE | ID: covidwho-1447327

ABSTRACT

The increasing risk from viral outbreaks such as the ongoing COVID-19 pandemic exacerbates the need for rapid, affordable and sensitive methods for virus detection, identification and quantification; however, existing methods for detecting virus particles in biological samples usually depend on multistep protocols that take considerable time to yield a result. Here, we introduce a rapid fluorescence in situ hybridization (FISH) protocol capable of detecting influenza virus, avian infectious bronchitis virus and SARS-CoV-2 specifically and quantitatively in approximately 20 min, in virus cultures, combined nasal and throat swabs with added virus and likely patient samples without previous purification. This fast and facile workflow can be adapted both as a lab technique and a future diagnostic tool in enveloped viruses with an accessible genome.


Subject(s)
In Situ Hybridization, Fluorescence/methods , RNA, Viral/isolation & purification , Viruses/isolation & purification , Viruses/genetics
6.
Am J Clin Pathol ; 157(1): 54-63, 2022 01 06.
Article in English | MEDLINE | ID: covidwho-1379433

ABSTRACT

OBJECTIVES: Respiratory failure is the major cause of death in coronavirus disease 2019 (COVID-19). Autopsy-based reports describe diffuse alveolar damage (DAD), organizing pneumonia, and fibrotic change, but data on early pathologic changes and during progression of the disease are rare. METHODS: We prospectively enrolled three patients with COVID-19 and performed full clinical evaluation, including high-resolution computed tomography. We took transbronchial biopsy (TBB) specimens at different time points and autopsy tissue samples for histopathologic and ultrastructural evaluation after the patients' death. RESULTS: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed by reverse transcription polymerase chain reaction and/or fluorescence in situ hybridization in all TBBs. Lung histology showed reactive pneumocytes and capillary congestion in one patient who died shortly after hospital admission with detectable virus in one of two lung autopsy samples. SARS-CoV-2 was detected in two of two autopsy samples from another patient with a fulminant course and very short latency between biopsy and autopsy, showing widespread organizing DAD. In a third patient with a prolonged course, autopsy samples showed extensive fibrosis without detectable virus. CONCLUSIONS: We report the course of COVID-19 in paired biopsy specimens and autopsies, illustrating vascular, organizing, and fibrotic patterns of COVID-19-induced lung injury. Our results suggest an early spread of SARS-CoV-2 from the upper airways to the lung periphery with diminishing viral load during disease.


Subject(s)
COVID-19 , SARS-CoV-2 , Autopsy , Biopsy , Humans , In Situ Hybridization, Fluorescence , Lung
7.
PLoS One ; 16(8): e0256378, 2021.
Article in English | MEDLINE | ID: covidwho-1360649

ABSTRACT

Saliva is a matrix which may act as a vector for pathogen transmission and may serve as a possible proxy for SARS-CoV-2 contagiousness. Therefore, the possibility of detection of intracellular SARS-CoV-2 in saliva by means of fluorescence in situ hybridization is tested, utilizing probes targeting the antisense or sense genomic RNA of SARS-CoV-2. This method was applied in a pilot study with saliva samples collected from healthy persons and those presenting with mild or moderate COVID-19 symptoms. In all participants, saliva appeared a suitable matrix for the detection of SARS-CoV-2. Among the healthy, mild COVID-19-symptomatic and moderate COVID-19-symptomatic persons, 0%, 90% and 100% tested positive for SARS-CoV-2, respectively. Moreover, the procedure allows for simultaneous measurement of viral load ('presence', sense genomic SARS-CoV-2 RNA) and viral replication ('activity', antisense genomic SARS-CoV-2 RNA) and may yield qualitative results. In addition, the visualization of DNA in the cells in saliva provides an additional cytological context to the validity and interpretability of the test results. The method described in this pilot study may be a valuable diagnostic tool for detection of SARS-CoV-2, distinguishing between 'presence' (viral load) and 'activity' (viral replication) of the virus. Moreover, the method potentially gives more information about possible contagiousness.


Subject(s)
COVID-19/diagnosis , In Situ Hybridization, Fluorescence/methods , RNA, Viral/analysis , SARS-CoV-2/genetics , Saliva/virology , COVID-19/pathology , COVID-19/virology , Case-Control Studies , Genomics , Humans , RNA, Antisense/genetics , RNA, Antisense/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Severity of Illness Index , Viral Load , Virus Replication
8.
RNA ; 27(11): 1318-1329, 2021 11.
Article in English | MEDLINE | ID: covidwho-1329126

ABSTRACT

The transcriptional induction of interferon (IFN) genes is a key feature of the mammalian antiviral response that limits viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Here, we performed single-molecule RNA visualization to examine the expression and localization of host mRNAs during SARS-CoV-2 infection. Our data show that the biogenesis of type I and type III IFN mRNAs is inhibited at multiple steps during SARS-CoV-2 infection. First, translocation of the interferon regulatory factor 3 (IRF3) transcription factor to the nucleus is limited in response to SARS-CoV-2, indicating that SARS-CoV-2 inhibits RLR-MAVS signaling and thus weakens transcriptional induction of IFN genes. Second, we observed that IFN mRNAs primarily localize to the site of transcription in most SARS-CoV-2 infected cells, suggesting that SARS-CoV-2 either inhibits the release of IFN mRNAs from their sites of transcription and/or triggers decay of IFN mRNAs in the nucleus upon exiting the site of transcription. Lastly, nuclear-cytoplasmic transport of IFN mRNAs is inhibited during SARS-CoV-2 infection, which we propose is a consequence of widespread degradation of host cytoplasmic basal mRNAs in the early stages of SARS-CoV-2 replication by the SARS-CoV-2 Nsp1 protein, as well as the host antiviral endoribonuclease, RNase L. Importantly, IFN mRNAs can escape SARS-CoV-2-mediated degradation if they reach the cytoplasm, making rescue of mRNA export a viable means for promoting the immune response to SARS-CoV-2.


Subject(s)
COVID-19/genetics , Host-Pathogen Interactions/genetics , Interferons/genetics , RNA Stability , SARS-CoV-2/pathogenicity , Viral Nonstructural Proteins/genetics , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Cell Line , Endoribonucleases/genetics , Endoribonucleases/metabolism , Humans , In Situ Hybridization, Fluorescence/methods , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferons/metabolism , RNA, Messenger/metabolism , Single Molecule Imaging
9.
Int J Cardiovasc Imaging ; 37(11): 3279-3283, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1279469

ABSTRACT

Frequent clinical presentations have been reported in patients with Coronavirus disease 2019 (COVID-19). It may be associated with multi-organ and cardiovascular involvements such as myocarditis and clot formation. Hypereosinophilic syndrome (HES) is a rare disease diagnosed with idiopathic eosinophilia and organ involvement. Here, we report a patient with COVID-19 who presented with clot formation and myocarditis. One month after discharge, regarding persistent peripheral/bone marrow hypereosinophilia and clot in echocardiography, fluorescent in situ hybridization (FISH) analysis was done that showed FIP1L1-CHIC2 fusion (PDGFRɑ rearrangement) in 18% of scored cells and PDGFRß rearrangement in 12% of scored cells, which confirmed HES diagnosis. Clot formation may be a late manifestation of COVID-19 or myocarditis due to COVID-19, or the first manifestation of HES that COVID-19 might provoke in this rare syndrome.


Subject(s)
COVID-19 , Hypereosinophilic Syndrome , Myocarditis , Humans , Hypereosinophilic Syndrome/complications , Hypereosinophilic Syndrome/diagnostic imaging , Hypereosinophilic Syndrome/drug therapy , In Situ Hybridization, Fluorescence , Myocarditis/diagnostic imaging , Myocarditis/etiology , Oncogene Proteins, Fusion/genetics , Predictive Value of Tests , SARS-CoV-2
12.
Sci Transl Med ; 12(574)2020 12 16.
Article in English | MEDLINE | ID: covidwho-1207479

ABSTRACT

Lung transplantation can potentially be a life-saving treatment for patients with nonresolving COVID-19-associated respiratory failure. Concerns limiting lung transplantation include recurrence of SARS-CoV-2 infection in the allograft, technical challenges imposed by viral-mediated injury to the native lung, and the potential risk for allograft infection by pathogens causing ventilator-associated pneumonia in the native lung. Additionally, the native lung might recover, resulting in long-term outcomes preferable to those of transplant. Here, we report the results of lung transplantation in three patients with nonresolving COVID-19-associated respiratory failure. We performed single-molecule fluorescence in situ hybridization (smFISH) to detect both positive and negative strands of SARS-CoV-2 RNA in explanted lung tissue from the three patients and in additional control lung tissue samples. We conducted extracellular matrix imaging and single-cell RNA sequencing on explanted lung tissue from the three patients who underwent transplantation and on warm postmortem lung biopsies from two patients who had died from COVID-19-associated pneumonia. Lungs from these five patients with prolonged COVID-19 disease were free of SARS-CoV-2 as detected by smFISH, but pathology showed extensive evidence of injury and fibrosis that resembled end-stage pulmonary fibrosis. Using machine learning, we compared single-cell RNA sequencing data from the lungs of patients with late-stage COVID-19 to that from the lungs of patients with pulmonary fibrosis and identified similarities in gene expression across cell lineages. Our findings suggest that some patients with severe COVID-19 develop fibrotic lung disease for which lung transplantation is their only option for survival.


Subject(s)
COVID-19/surgery , Lung Transplantation , Lung/surgery , Pulmonary Fibrosis/surgery , Adult , Aged, 80 and over , COVID-19/diagnosis , COVID-19/physiopathology , COVID-19/virology , COVID-19 Nucleic Acid Testing , Databases, Factual , Disease Progression , Female , Humans , In Situ Hybridization, Fluorescence , Lung/physiopathology , Lung/virology , Male , Middle Aged , Pulmonary Fibrosis/diagnosis , Pulmonary Fibrosis/physiopathology , Pulmonary Fibrosis/virology , RNA-Seq , Recovery of Function , Retrospective Studies , Severity of Illness Index , Single-Cell Analysis , Treatment Outcome
13.
Mol Syst Biol ; 17(4): e10232, 2021 04.
Article in English | MEDLINE | ID: covidwho-1204403

ABSTRACT

Exacerbated pro-inflammatory immune response contributes to COVID-19 pathology. However, despite the mounting evidence about SARS-CoV-2 infecting the human gut, little is known about the antiviral programs triggered in this organ. To address this gap, we performed single-cell transcriptomics of SARS-CoV-2-infected intestinal organoids. We identified a subpopulation of enterocytes as the prime target of SARS-CoV-2 and, interestingly, found the lack of positive correlation between susceptibility to infection and the expression of ACE2. Infected cells activated strong pro-inflammatory programs and produced interferon, while expression of interferon-stimulated genes was limited to bystander cells due to SARS-CoV-2 suppressing the autocrine action of interferon. These findings reveal that SARS-CoV-2 curtails the immune response and highlights the gut as a pro-inflammatory reservoir that should be considered to fully understand SARS-CoV-2 pathogenesis.


Subject(s)
Intestines/immunology , SARS-CoV-2/physiology , Single-Cell Analysis , COVID-19/virology , Gastrointestinal Microbiome , Humans , In Situ Hybridization, Fluorescence , Organoids/metabolism , Sequence Analysis, RNA
14.
J Vis Exp ; (166)2020 12 23.
Article in English | MEDLINE | ID: covidwho-1022040

ABSTRACT

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , COVID-19/virology , RNA, Viral , Respiratory Mucosa/virology , SARS-CoV-2/genetics , Animals , Chlorocebus aethiops , Fluorescence , Humans , In Situ Hybridization, Fluorescence , Respiratory System , Vero Cells
15.
Chembiochem ; 21(15): 2214-2218, 2020 08 03.
Article in English | MEDLINE | ID: covidwho-186217

ABSTRACT

The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid-19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single-fluorophore-containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off-target binding effects that create background noise. Here, we used click chemistry and alkyne-modified DNA oligonucleotides to prepare multiple-fluorophore-containing probes. We found that these multiple-dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5-10) of probe strands. The new method enabled the in situ detection of viral transcripts as early as 4 hours after infection.


Subject(s)
Click Chemistry/methods , Early Diagnosis , In Situ Hybridization, Fluorescence/methods , Oligonucleotide Probes/chemistry , RNA, Messenger/analysis , RNA, Viral/analysis , Alkynes/chemistry , Betacoronavirus/genetics , COVID-19 , Coronavirus Infections/diagnosis , Humans , Oligodeoxyribonucleotides/chemistry , Pandemics , Pneumonia, Viral/diagnosis , SARS-CoV-2
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