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1.
mBio ; 13(2): e0370521, 2022 04 26.
Article in English | MEDLINE | ID: covidwho-1714363

ABSTRACT

Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Two independent cell-to-cell membrane fusion assays showed that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low-micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19. IMPORTANCE The lack of effective antiviral treatments against SARS-CoV-2 is a significant limitation in the fight against the COVID-19 pandemic. Single-drug regimens have so far yielded limited results, indicating that combinations of antivirals might be required, as previously seen for other RNA viruses. Our work introduces the drug booster cobicistat, which is approved by the FDA and typically used to potentiate the effect of anti-HIV protease inhibitors, as a candidate inhibitor of SARS-CoV-2 replication. Beyond its direct activity as an antiviral, we show that cobicistat can enhance the effect of remdesivir, which was one of the first drugs proposed for treatment of SARS-CoV-2. Overall, the dual action of cobicistat as a direct antiviral and a drug booster can provide a new approach to design combination therapies and rescue the activity of compounds that are only partially effective in monotherapy.


Subject(s)
COVID-19 , Hepatitis C, Chronic , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Cobicistat , Cricetinae , Disease Progression , Humans , Mesocricetus , Pandemics , SARS-CoV-2 , Viral Load
2.
Cell Host Microbe ; 28(6): 853-866.e5, 2020 12 09.
Article in English | MEDLINE | ID: covidwho-1385263

ABSTRACT

Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.


Subject(s)
COVID-19/genetics , Endoplasmic Reticulum/ultrastructure , SARS-CoV-2/ultrastructure , Viral Replication Compartments/ultrastructure , COVID-19/diagnostic imaging , COVID-19/pathology , COVID-19/virology , Cell Death/genetics , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/virology , Humans , Microscopy, Electron , Pandemics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Viral Replication Compartments/metabolism , Virus Replication/genetics
3.
Viruses ; 13(5)2021 04 24.
Article in English | MEDLINE | ID: covidwho-1201582

ABSTRACT

The emerging SARS-CoV-2 pandemic entails an urgent need for specific and sensitive high-throughput serological assays to assess SARS-CoV-2 epidemiology. We, therefore, aimed at developing a fluorescent-bead based SARS-CoV-2 multiplex serology assay for detection of antibody responses to the SARS-CoV-2 proteome. Proteins of the SARS-CoV-2 proteome and protein N of SARS-CoV-1 and common cold Coronaviruses (ccCoVs) were recombinantly expressed in E. coli or HEK293 cells. Assay performance was assessed in a COVID-19 case cohort (n = 48 hospitalized patients from Heidelberg) as well as n = 85 age- and sex-matched pre-pandemic controls from the ESTHER study. Assay validation included comparison with home-made immunofluorescence and commercial enzyme-linked immunosorbent (ELISA) assays. A sensitivity of 100% (95% CI: 86-100%) was achieved in COVID-19 patients 14 days post symptom onset with dual sero-positivity to SARS-CoV-2 N and the receptor-binding domain of the spike protein. The specificity obtained with this algorithm was 100% (95% CI: 96-100%). Antibody responses to ccCoVs N were abundantly high and did not correlate with those to SARS-CoV-2 N. Inclusion of additional SARS-CoV-2 proteins as well as separate assessment of immunoglobulin (Ig) classes M, A, and G allowed for explorative analyses regarding disease progression and course of antibody response. This newly developed SARS-CoV-2 multiplex serology assay achieved high sensitivity and specificity to determine SARS-CoV-2 sero-positivity. Its high throughput ability allows epidemiologic SARS-CoV-2 research in large population-based studies. Inclusion of additional pathogens into the panel as well as separate assessment of Ig isotypes will furthermore allow addressing research questions beyond SARS-CoV-2 sero-prevalence.


Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Viral/blood , Antibody Formation , COVID-19/blood , COVID-19/virology , Cohort Studies , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Female , HEK293 Cells , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Middle Aged , Open Reading Frames , Pandemics , Phosphoproteins , Proteome/immunology , SARS-CoV-2/genetics , Young Adult
4.
J Virol ; 95(4)2021 01 28.
Article in English | MEDLINE | ID: covidwho-1117221

ABSTRACT

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.


Subject(s)
COVID-19/virology , Dengue Virus/isolation & purification , Dengue/virology , SARS-CoV-2/isolation & purification , A549 Cells , Animals , COVID-19/diagnosis , COVID-19/metabolism , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Dengue/diagnosis , Dengue/metabolism , Dengue/pathology , Dengue Virus/genetics , Dengue Virus/metabolism , Genes, Reporter , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Nuclear Localization Signals/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Vero Cells , Viral Nonstructural Proteins/metabolism , Virus Replication
5.
Cell Microbiol ; 23(7): e13319, 2021 07.
Article in English | MEDLINE | ID: covidwho-1087999

ABSTRACT

The ongoing SARS-CoV-2 pandemic with over 80 million infections and more than a million deaths worldwide represents the worst global health crisis of the 21th century. Beyond the health crisis, the disruptions caused by the COVID-19 pandemic have serious global socio-economic consequences. It has also placed a significant pressure on the scientific community to understand the virus and its pathophysiology and rapidly provide anti-viral treatments and procedures in order to help the society and stop the virus spread. Here, we outline how advanced microscopy technologies such as high-throughput microscopy and electron microscopy played a major role in rapid response against SARS-CoV-2. General applicability of developed microscopy technologies makes them uniquely positioned to act as the first line of defence against any emerging infection in the future.


Subject(s)
COVID-19 , Microscopy/methods , SARS-CoV-2 , Antibodies, Viral/blood , Antiviral Agents/pharmacology , COVID-19/diagnosis , COVID-19/drug therapy , COVID-19/pathology , COVID-19/virology , COVID-19 Serological Testing , COVID-19 Vaccines , Cryoelectron Microscopy , Drug Development , High-Throughput Screening Assays , Humans , Microscopy, Electron , Pandemics , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/physiology , SARS-CoV-2/ultrastructure , Virus Replication
6.
JAMA Pediatr ; 175(6): 586-593, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1044436

ABSTRACT

Importance: School and daycare closures were enforced as measures to confine the novel coronavirus disease 2019 (COVID-19) pandemic, based on the assumption that young children may play a key role in severe acute respiratory coronavirus 2 (SARS-CoV-2) spread. Given the grave consequences of contact restrictions for children, a better understanding of their contribution to the COVID-19 pandemic is of great importance. Objective: To describe the rate of SARS-CoV-2 infections and the seroprevalence of SARS-CoV-2 antibodies in children aged 1 to 10 years, compared with a corresponding parent of each child, in a population-based sample. Design, Setting, and Participants: This large-scale, multicenter, cross-sectional investigation (the COVID-19 BaWü study) enrolled children aged 1 to 10 years and a corresponding parent between April 22 and May 15, 2020, in southwest Germany. Exposures: Potential exposure to SARS-CoV-2. Main Outcomes and Measures: The main outcomes were infection and seroprevalence of SARS-CoV-2. Participants were tested for SARS-CoV-2 RNA from nasopharyngeal swabs by reverse transcription-polymerase chain reaction and SARS-CoV-2 specific IgG antibodies in serum by enzyme-linked immunosorbent assays and immunofluorescence tests. Discordant results were clarified by electrochemiluminescence immunoassays, a second enzyme-linked immunosorbent assay, or an in-house Luminex-based assay. Results: This study included 4964 participants: 2482 children (median age, 6 [range, 1-10] years; 1265 boys [51.0%]) and 2482 parents (median age, 40 [range, 23-66] years; 615 men [24.8%]). Two participants (0.04%) tested positive for SARS-CoV-2 RNA. The estimated SARS-CoV-2 seroprevalence was low in parents (1.8% [95% CI, 1.2-2.4%]) and 3-fold lower in children (0.6% [95% CI, 0.3-1.0%]). Among 56 families with at least 1 child or parent with seropositivity, the combination of a parent with seropositivity and a corresponding child with seronegativity was 4.3 (95% CI, 1.19-15.52) times higher than the combination of a parent who was seronegative and a corresponding child with seropositivity. We observed virus-neutralizing activity for 66 of 70 IgG-positive serum samples (94.3%). Conclusions and Relevance: In this cross-sectional study, the spread of SARS-CoV-2 infection during a period of lockdown in southwest Germany was particularly low in children aged 1 to 10 years. Accordingly, it is unlikely that children have boosted the pandemic. This SARS-CoV-2 prevalence study, which appears to be the largest focusing on children, is instructive for how ad hoc mass testing provides the basis for rational political decision-making in a pandemic.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , COVID-19/epidemiology , SARS-CoV-2/isolation & purification , Adult , Age Distribution , Age Factors , Aged , COVID-19/blood , COVID-19 Serological Testing , Child , Child, Preschool , Cross-Sectional Studies , Germany/epidemiology , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Middle Aged , Parents , Prevalence , Seroepidemiologic Studies
7.
SSRN; 2020.
Preprint | SSRN | ID: ppcovidwho-5070

ABSTRACT

Background: School and day-care closures were enforced as measures to confine the COVID-19 pandemic based on the assumption that young children may play a key role in SARS-CoV-2 spreading. However, infection prevalence in children under 10 years of age is not very well analysed. Methods: The COVID-19 BaWü study is a large-scale multicentre cross-sectional investigation of children aged 1–10 years and one of their parents, both not diagnosed with COVID-19 before, in southwest Germany. We tested for SARS-CoV-2 RNA from nasopharyngeal swabs by RT-PCR and for SARS-CoV-2 specific IgG antibodies in serum by ELISA and immunofluorescence. Discordant results were clarified by ECLIA, a second ELISA or an in-house Luminex-based assay. We used mixed effects logistic regression to estimate the seroprevalence and to analyse the association between SARS-CoV-2 seropositivity and covariates. Findings: Between April 22nd and May 15th, 2020, we enrolled 4964 subjects, 2482 children and 2482 corresponding parents. 0•04% tested positive for SARS-CoV-2 RNA. The estimated SARS-CoV-2 seroprevalence was low in parents (1•8%;95% CI, 1•2–2•4%) and 3-fold lower in children (0•6%;95% CI, 0•3–1•0%). We observed virus-neutralizing activity for 66 of 70 IgG-positive sera (94•3%). Interpretation: The spread of SARS-CoV-2 infection during a period of lock-down in southwest Germany was particularly low in children aged 1–10 years. Accordingly, it is unlikely that children have boosted the pandemic. This largest reported SARS-CoV-2 prevalence study focussing on children is instructive for how ad hoc mass testing provides the basis for rational political decision making in a pandemic setting. Funding: Grant from the Federal State of Baden-Württemberg, Germany Declaration of Interests: All authors state no conflict of interest. Ethics Approval Statement: The study protocol was approved by the independent Ethics committees of each centre. The study was conducted according to the Declaration of Helsinki. Written informed consent was obtained from all parents/guardians, with assent from children when appropriate for their age.

8.
SSRN; 2020.
Preprint | SSRN | ID: ppcovidwho-3774

ABSTRACT

Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation dominated cytokine response and virus-induced cell perturbation causing cell death. Here we employed an integrative light and electron microscopy based imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2 infected human lung epithelial cells. We report 3D reconstructions of whole-cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodelling of cytoskeleton elements;pharmacological inhibition of their dynamics strongly suppresses SARS-CoV-2 replication. Taken together, we provide critical insights into virus-induced cytopathic effects, while alongside presenting a comprehensive and publicly available repository of 3D data-sets of SARS-CoV-2 infected cells. Funding: This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project number 240245660 – SFB 1129 (TP11, TP13, TP14 and project Z2) to R.B., A.R., S.B. and Y.S. R.B. was supported in addition by the German Center for Infection Research (DZIF), project numbers 8029801806 and 8029705705, and the DFG, project number 272983813 – TRR 179.SB and MS received additional financial support from the DFG, project number 415089553 (Heisenberg) and 272983813 (TRR179) to S.B. and project number 416072091 to MS.

9.
Bioessays ; 43(3): e2000257, 2021 03.
Article in English | MEDLINE | ID: covidwho-995863

ABSTRACT

Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Immunoassay , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Microscopy/methods , SARS-CoV-2/immunology , COVID-19/immunology , COVID-19/virology , COVID-19 Testing/methods , Fluorescent Antibody Technique , High-Throughput Screening Assays , Humans , Image Processing, Computer-Assisted/statistics & numerical data , Immune Sera/chemistry , Machine Learning , Sensitivity and Specificity
10.
J Virol ; 95(4)2021 01 28.
Article in English | MEDLINE | ID: covidwho-952556

ABSTRACT

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.


Subject(s)
COVID-19/virology , Dengue Virus/isolation & purification , Dengue/virology , SARS-CoV-2/isolation & purification , A549 Cells , Animals , COVID-19/diagnosis , COVID-19/metabolism , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Dengue/diagnosis , Dengue/metabolism , Dengue/pathology , Dengue Virus/genetics , Dengue Virus/metabolism , Genes, Reporter , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Nuclear Localization Signals/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Vero Cells , Viral Nonstructural Proteins/metabolism , Virus Replication
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