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1.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-320953

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pneumonia in humans. The virus is enzootic in dromedary camels across the Middle East and Africa. It is acquired through animal contact and undergoes limited onward transmission particularly in hospitals. Because of this initial potential for human-to-human transmission, we monitor the virus for phenotypic changes related to its pandemic potential. Potential phenotypic changes have been suspected since the year 2015, when a novel recombinant clade (MERS-CoV lineage 5) caused large nosocomial outbreaks in Saudi Arabia and South Korea that effectively swept other, hitherto co-circulating viral lineages. To this day, lineage 5 remains the only circulating MERS-CoV lineage on the Arabian Peninsula. In spite of available sequence data, no studies of viral phenotype have been carried out to date. Here we performed a comprehensive in-vitro and ex-vivo comparison of live virus isolates taken in Saudi Arabia immediately before and after the shift toward lineage 5. We characterized seven isolates representing the recombination-parental lineage 3, eight isolates representing parental lineage 4, as well as eight isolates representing lineage 5. Replication of lineage 5 viruses is significantly increased over isolates from parental lineages in cell culture and ex-vivo lung models. Transcriptional profiling by real-time RT-PCR shows that several key immune genes (IFNb1, CCL5, IFNL1) are significantly less induced in lung cells infected with lineage 5 MERS-CoV compared to parental strains. In IFN receptor knock out cells, as well as under chemical inhibition of IFN signalling, the differences in replication level between lineage 5 and parental lineages are reduced, suggesting that phenotypic differences may be determined by IFN antagonism. Concordantly, lineage 5 shows increased resilience against interferon (IFN) pre-treatment of Calu-3 cells and maintains a 10-fold higher replication level under low and high concentrations of IFN. Reduced immune activation combined with enhanced virus replication and IFN resilience may explain the dominance of lineage 5 on the Arabian Peninsula. This phenotypic difference is highly relevant with regard to pandemic potential, and has remained undiscovered in spite of viral sequence surveillance.

2.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-327696

ABSTRACT

Cell-intrinsic responses mounted in vivo in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or are, at least partially, resulting from physical interaction with virus particles, remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. Bulk and single cell RNA-sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes, but not pro-inflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG base-line profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, non-productive physical interaction of PBMCs with SARS-CoV-2- but not SARS-CoV particles stimulates JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.

3.
Nat Commun ; 12(1): 3818, 2021 06 21.
Article in English | MEDLINE | ID: covidwho-1279876

ABSTRACT

Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13 µM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.


Subject(s)
COVID-19/metabolism , COVID-19/virology , SARS-CoV-2/metabolism , Animals , Antinematodal Agents/pharmacology , Autophagosomes/metabolism , Autophagy , Autophagy-Related Proteins/metabolism , COVID-19/drug therapy , COVID-19/pathology , Cells, Cultured , Chlorocebus aethiops , Cricetinae , Disease Models, Animal , Humans , Lung/metabolism , Lung/pathology , Lung/virology , Metabolome , Niclosamide/pharmacology , Organoids , SARS-CoV-2/isolation & purification , Spermidine/pharmacology , Spermine/pharmacology
4.
Nat Commun ; 12(1): 2117, 2021 04 09.
Article in English | MEDLINE | ID: covidwho-1174671

ABSTRACT

To estimate the seroprevalence and temporal course of SARS-CoV-2 neutralizing antibodies, we embedded a multi-tiered seroprevalence survey within an ongoing community-based cohort study in Bonn, Germany. We first assessed anti-SARS-CoV-2 immunoglobulin G levels with an immunoassay, followed by confirmatory testing of borderline and positive test results with a recombinant spike-based immunofluorescence assay and a plaque reduction neutralization test (PRNT). Those with a borderline or positive immunoassay result were retested after 4 to 5 months. At baseline, 4771 persons participated (88% response rate). Between April 24th and June 30th, 2020, seroprevalence was 0.97% (95% CI: 0.72-1.30) by immunoassay and 0.36% (95% CI: 0.21-0.61) when considering only those with two additional positive confirmatory tests. Importantly, about 20% of PRNT+ individuals lost their neutralizing antibodies within five months. Here, we show that neutralizing antibodies are detectable in only one third of those with a positive immunoassay result, and wane relatively quickly.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/diagnosis , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , COVID-19/prevention & control , COVID-19/virology , Cohort Studies , Female , Germany , Humans , Immunoassay/methods , Male , Middle Aged , Neutralization Tests/methods , Population Surveillance/methods , SARS-CoV-2/physiology , Seroepidemiologic Studies
5.
Lancet Microbe ; 2(5): e210-e218, 2021 05.
Article in English | MEDLINE | ID: covidwho-1117258

ABSTRACT

BACKGROUND: The COVID-19 agent, SARS-CoV-2, is conspecific with SARS-CoV, the causal agent of the severe acute respiratory syndrome epidemic in 2002-03. Although the viruses share a completely homologous repertoire of proteins and use the same cellular entry receptor, their transmission efficiencies and pathogenetic traits differ. We aimed to compare interferon antagonism by SARS-CoV and SARS-CoV-2. METHODS: For this functional study, we infected Vero E6 and Calu-3 cells with strains of SARS-CoV and SARS-CoV-2. We studied differences in cell line-specific replication (Vero E6 vs Calu-3 cells) and analysed these differences in relation to TMPRSS2-dependent cell entry based on inhibition with the drug camostat mesilate. We evaluated viral sensitivity towards type I interferon treatment and assessed cytokine induction and type I interferon signalling in the host cells by RT-PCR and analysis of transcription factor activation and nuclear translocation. Based on reverse genetic engineering of SARS-CoV, we investigated the contribution of open reading frame 6 (ORF6) to the observed phenotypic differences in interferon signalling, because ORF6 encodes an interferon signalling antagonist. We did a luciferase-based interferon-stimulated response element promotor activation assay to evaluate the antagonistic capacity of SARS-CoV-2 wild-type ORF6 constructs and three mutants (Gln51Glu, Gln56Glu, or both) that represent amino acid substitutions between SARS-CoV and SARS-CoV-2 protein 6 in the carboxy-terminal domain. FINDINGS: Overall, replication was higher for SARS-CoV in Vero E6 cells and for SARS-CoV-2 in Calu-3 cells. SARS-CoV-2 was reliant on TMPRSS2, found only in Calu-3 cells, for more efficient entry. SARS-CoV-2 was more sensitive to interferon treatment, less efficient in suppressing cytokine induction via IRF3 nuclear translocation, and permissive of a higher level of induction of interferon-stimulated genes MX1 and ISG56. SARS-CoV-2 ORF6 expressed in the context of a fully replicating SARS-CoV backbone suppressed MX1 gene induction, but this suppression was less efficient than that by SARS-CoV ORF6. Mutagenesis showed that charged amino acids in residues 51 and 56 shift the phenotype towards more efficient interferon antagonism, as seen in SARS-CoV. INTERPRETATION: SARS-CoV-2 ORF6 interferes less efficiently with human interferon induction and interferon signalling than SARS-CoV ORF6. Because of the homology of the genes, onward selection for fitness could involve functional optimisation of interferon antagonism. Charged amino acids at positions 51 and 56 in ORF6 should be monitored for potential adaptive changes. FUNDING: Bundesministerium für Bildung und Forschung, EU RECOVER project.


Subject(s)
COVID-19 , Interferon Type I , SARS Virus , Amino Acids/genetics , Antiviral Agents/pharmacology , COVID-19/drug therapy , Humans , Interferon Type I/genetics , Reverse Genetics , SARS Virus/genetics , SARS-CoV-2/genetics , Viral Proteins/chemistry
6.
iScience ; 24(3): 102151, 2021 Mar 19.
Article in English | MEDLINE | ID: covidwho-1065235

ABSTRACT

Detailed knowledge of the molecular biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is crucial for understanding of viral replication, host responses, and disease progression. Here, we report gene expression profiles of three SARS-CoV- and SARS-CoV-2-infected human cell lines. SARS-CoV-2 elicited an approximately two-fold higher stimulation of the innate immune response compared to SARS-CoV in the human epithelial cell line Calu-3, including induction of miRNA-155. Single-cell RNA sequencing of infected cells showed that genes induced by virus infections were broadly upregulated, whereas interferon beta/lambda genes, a pro-inflammatory cytokines such as IL-6, were expressed only in small subsets of infected cells. Temporal analysis suggested that transcriptional activities of interferon regulatory factors precede those of nuclear factor κB. Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 activity resulted in a reduction of viral replication and pro-inflammatory cytokine expression in primary human airway epithelial cells.

7.
Cell ; 183(4): 1058-1069.e19, 2020 11 12.
Article in English | MEDLINE | ID: covidwho-785287

ABSTRACT

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from 10 COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb, CV07-209, neutralized authentic SARS-CoV-2 with an IC50 value of 3.1 ng/mL. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2-neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss, and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Betacoronavirus/metabolism , Coronavirus Infections/pathology , Pneumonia, Viral/pathology , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Viral/therapeutic use , Antigen-Antibody Reactions , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , Binding Sites , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Cricetinae , Crystallography, X-Ray , Disease Models, Animal , Humans , Kinetics , Lung/immunology , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Molecular Dynamics Simulation , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
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