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Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals.

ABSTRACT: The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity among samples. Mixed allelic frequencies along the 20kb ORF1ab gene in one sample, suggested the presence of a defective viral RNA species subpopulation maintained in mixture with functional RNA in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

SUBMITTER: Doddapaneni H

PROVIDER: S-EPMC8386831 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals.

Project description:The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

| S-EPMC7402036 | biostudies-literature

Oligonucleotide Capture Sequencing of the SARS-CoV-2 Genome and Subgenomic Fragments from COVID-19 Individuals.

| S-EPMC7743067 | biostudies-literature

SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19

Project description:ACE2 on epithelial cells is the SARS-CoV-2 entry receptor. Single-cell RNA-sequencing data derived from two COVID-19 cohorts revealed that MAP4K3/GLK-positive epithelial cells were increased in patients. SARS-CoV-2-induced GLK overexpression in epithelial cells correlated with COVID-19 severity and vesicle secretion. GLK overexpression induced the epithelial cell-derived exosomes containing ACE2; the GLK-induced exosomes transported ACE2 proteins to recipient cells, facilitating pseudovirus infection. Consistently, ACE2 proteins were increased in the serum exosomes from another COVID-19 cohort. Remarkably, SARS-CoV-2 spike protein stimulated GLK, and GLK stabilized ACE2 in epithelial cells. Mechanistically, GLK phosphorylated ACE2 at two serine residues (Ser776, Ser783), leading to dissociation of ACE2 from its E3 ligase UBR4. Reduction of UBR4-induced Lys48-linked ubiquitination at three lysine residues (Lys26, Lys112, Lys114) of ACE2 prevented its degradation. Furthermore, SARS-CoV-2 pseudovirus or live virus infection in humanized ACE2 mice induced GLK and ACE2 protein levels, as well as ACE2-containing exosomes. Collectively, ACE2 stabilization by SARS-CoV-2-induced MAP4K3/GLK may contribute to the pathogenesis of COVID-19.

| S-SCDT-EMM-2022-15904 | biostudies-other

Characterization of two SARS-CoV-2 subgenomic RNA dynamics in severe COVID-19 patients

Project description:Little is known about Subgenomic RNA (sgRNA) dynamics in patients with Coronavirus diseases 2019 (COVID-19). We collected 147 throat swabs, 74 gut swabs and 46 plasma samples from 117 COVID-19 patients recruited in the LOTUS China trial (ChiCTR2000029308) and compared E and orf7a sgRNA load in patients with different illness duration, outcome, and comorbidities. Both sgRNAs were detected in all the three types of samples, with longest duration of 25, 13, and 17 days for E sgRNA, and 32, 28, and 17 days for orf7a sgRNA in throat, gut, and plasma, respectively. A total of 95% (57/60) of patients had no E sgRNA detected after 10 days post treatment, though 86% of them were still E RNA positive. High correlation on titer was observed between sgRNA encoding E and orf7a gene. sgRNA showed similar variation in the standard care and Lopinavir-Ritonavir group. Patients with diabetes and heart diseases showed higher pharyngeal E sgRNA at the first day (P = 0.016 and 0.013, respectively) but no difference at five days after treatment, compared with patients without such commodities. Patients with hypertension and cerebrovascular diseases showed no difference in the pharyngeal sgRNA levels at both one and five days after treatment, compared with patients without these two commodities. E sgRNA levels in the initial infection showed no correlation with the serum antibody against spike, nucleoprotein, and receptor binding domains at ten days later. sgRNA lasted a long period in COVID-19 patients and might have little effect on humoral response.

| S-EPMC8762994 | biostudies-literature

Subgenomic RNA identification in SARS-CoV-2 genomic sequencing data.

Project description:We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "subgenomic RNAs." sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5' UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5' end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

| S-EPMC8015849 | biostudies-literature

Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients with acute respiratory distress syndrome

Project description: Not available

| S-BSST955 | biostudies-other

Project description:Bat sarbecovirus BANAL-236 is highly related to SARS-CoV-2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. BANAL-236 replicates efficiently and pauci-symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS-CoV-2. BANAL-236 infection leads to protection against superinfection by a virulent strain. We find no evidence of antibodies recognizing bat sarbecoviruses in populations in close contact with bats in which the virus was identified, indicating that such spillover infections, if they occur, are rare. Six passages in humanized mice or in human intestinal cells, mimicking putative early spillover events, select adaptive mutations without appearance of a furin cleavage site and no change in virulence. Therefore, acquisition of a furin site in the spike protein is likely a pre-spillover event that did not occur upon replication of a SARS-CoV-2-like bat virus in humans or other animals. Other hypotheses regarding the origin of the SARS-CoV-2 should therefore be evaluated, including the presence of sarbecoviruses carrying a spike with a furin cleavage site in bats.

| S-SCDT-10_15252-EMBR_202256055 | biostudies-other

Comparative Evaluation of the Thermo Fisher TaqPath COVID-19 Combo Kit with the Cepheid Xpert® Xpress SARS-CoV-2 Assay for Detecting SARS-CoV-2 in Nasopharyngeal Specimens

Project description: Not available

| S-BSST645 | biostudies-other

Low avidity T cell responses to SARS-CoV-2 in unexposed individuals and severe COVID-19

Project description:Pre-existing memory T-cells against SARS-CoV-2 are present in a fraction of unexposed individuals and their induction by common cold corona viruses (CCCoVs) infection is suggested. Here we demonstrate that SARS-CoV-2-reactive T-cells were present in the memory compartment of virtually all unexposed individuals but possessed only low functional avidity. They harbored multiple, highly variable cross-reactivities that were not restricted to CCCoVs. Cross-reactivity to CCCoV was almost absent in COVID-19 patients. This was irrespective of strong T-cell memory against CCCoV in all donors. In severe but not mild COVID-19, SARS-CoV-2-specific T-cells also displayed low functional avidity and reduced clonal expansion, despite strongly increased frequencies. Our data question a major protective role of CCCoV for COVID-19. Instead, we suggest that a low avidity pre-existing T-cell memory may contribute to the excessive but low avidity T-cell responses, which we identified here as a hallmark of severe COVID-19.

2020-12-23 | GSE162086 | GEO

Diagnostic usefulness of subgenomic RNA detection of viable SARS-CoV-2 in patients with COVID-19.

Project description:ObjectivesThe development of a rapid diagnostic test for viable SARS-CoV-2 is important for infection control. Real-time RT-PCR assays detect non-viable virus, and cell culture differentiates viable virus but it takes several weeks and is labour-intensive. Subgenomic RNAs may reflect replication-competent virus. We therefore evaluated the usefulness of subgenomic RNAs for diagnosing viable SARS-CoV-2 in patients with COVID-19.MethodsPatients with various severities of confirmed COVID-19 were enrolled at a tertiary hospital between February and December 2020. RT-PCR assay results for genomic and subgenomic RNA of SARS-CoV-2 from nasopharyngeal swab, sputum and saliva specimens were compared with cell culture results.ResultsA total 189 specimens from 20 COVID-19 patients were tested in genomic and subgenomic PCR assays and cultured on Vero cells. Of these 189 samples, 62 (33%) gave positive culture results, 93 (49%) negative results and the remaining 34 (18%) indeterminate results. Compared with cell culture results, the sensitivities of genomic RNA and subgenomic RNA of the N and S genes were comparable at 100%, but the specificity of subgenomic RNA (N, 65% and S, 68%) was higher than that of genomic RNA (N, 23% and S, 17%, p < 0.001). The mean durations of positive culture and subgenomic RNA were 11.39 ± 10.34 and 13.75 ± 11.22 days after symptom onset (p 0.437), respectively, while that of genomic RNA was 22.85 ± 11.83 days after symptom onset (p < 0.001).DiscussionOur comparison of subgenomic RNA detection with symptom duration and SARS-CoV-2 culture positivity provides a significant advancement on the transmissibility-based approach beyond the detection of SARS-CoV-2 genomic RNA, and warrants further studies on the development of better diagnostic strategy.

| S-EPMC8360988 | biostudies-literature

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