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1.
J Proteome Res ; 2022 Jan 04.
Article in English | MEDLINE | ID: covidwho-1605127

ABSTRACT

Severe acute respiratory syndrome (SARS)-CoV and SARS-CoV-2 infections are characterized by remarkable differences, including infectivity and case fatality rate. The underlying mechanisms are not well understood, illustrating major knowledge gaps of coronavirus biology. In this study, protein expression of the SARS-CoV- and SARS-CoV-2-infected human lung epithelial cell line Calu-3 was analyzed using data-independent acquisition-mass spectrometry. This resulted in a comprehensive map of infection-related proteome-wide expression changes in human cells covering the quantification of 7478 proteins across four time points. Most notably, the activation of interferon type-I response was observed, which is surprisingly absent in several proteome studies. The data reveal that SARS-CoV-2 triggers interferon-stimulated gene expression much stronger than SARS-CoV, which reflects the already described differences in interferon sensitivity. Potentially, this may be caused by the enhanced abundance of the viral M protein of SARS-CoV in comparison to SARS-CoV-2, which is a known inhibitor of type I interferon expression. This study expands the knowledge on the host response to SARS-CoV-2 infections on a global scale using an infection model, which seems to be well suited to analyze the innate immunity.

2.
Preprint in English | EuropePMC | ID: ppcovidwho-295601

ABSTRACT

The reliable detection of SARS-CoV-2 genomic RNA and infectious virus particles from patient samples requires a good sample quality. This is especially critical when the sample has to be transported to the analysing laboratory which can take several days. To determine optimal transport conditions, we simulated oropharyngeal swab samples using defined virus amounts and stored the samples at 4 °C or at room temperature for up to four days. Moreover, we analysed the influence of dry swabs in comparison to swabs stored in transport medium. Our results show that care should be taken when analysing samples for infectious SARS-CoV-2 particles since infectivity is strongly influenced by sample storage.

3.
Preprint in English | Other preprints | ID: ppcovidwho-294848

ABSTRACT

SARS-CoV and SARS-CoV-2 infections are characterized by remarkable differences, including contagiosity and case fatality rate. The underlying mechanisms are not well understood, illustrating major knowledge gaps of coronavirus biology. In this study, protein expression of SARS-CoV- and SARS-CoV-2-infected human lung epithelial cell line Calu-3 was analysed using data-independent acquisition mass spectrometry (DIA-MS). This resulted in the so far most comprehensive map of infection-related proteome-wide expression changes in human cells covering the quantification of 7478 proteins across 4 time points. Most notably, the activation of interferon type-I response was observed, which surprisingly is absent in other recent proteome studies, but is known to occur in SARS-CoV-2-infected patients. The data reveal that SARS-CoV-2 triggers interferon-stimulated gene (ISG) expression much stronger than SARS-CoV, which reflects the already described differences in interferon sensitivity. Potentially, this may be caused by the enhanced expression of viral M protein of SARS-CoV in comparison to SARS-CoV-2, which is a known inhibitor of type I interferon expression. This study expands the knowledge on the host response to SARS-CoV-2 infections on a global scale using an infection model, which seems to be well suited to analyse innate immunity.

4.
Euro Surveill ; 26(44)2021 11.
Article in English | MEDLINE | ID: covidwho-1504591

ABSTRACT

IntroductionThe detection of SARS-CoV-2 with rapid diagnostic tests (RDT) has become an important tool to identify infected people and break infection chains. These RDT are usually based on antigen detection in a lateral flow approach.AimWe aimed to establish a comprehensive specimen panel for the decentralised technical evaluation of SARS-CoV-2 antigen rapid diagnostic tests.MethodsWhile for PCR diagnostics the validation of a PCR assay is well established, there is no common validation strategy for antigen tests, including RDT. In this proof-of-principle study we present the establishment of a panel of 50 pooled clinical specimens that cover a SARS-CoV-2 concentration range from 1.1 × 109 to 420 genome copies per mL of specimen. The panel was used to evaluate 31 RDT in up to six laboratories.ResultsOur results show that there is considerable variation in the detection limits and the clinical sensitivity of different RDT. We show that the best RDT can be applied to reliably identify infectious individuals who present with SARS-CoV-2 loads down to 106 genome copies per mL of specimen. For the identification of infected individuals with SARS-CoV-2 loads corresponding to less than 106 genome copies per mL, only three RDT showed a clinical sensitivity of more than 60%.ConclusionsSensitive RDT can be applied to identify infectious individuals with high viral loads but not to identify all infected individuals.


Subject(s)
COVID-19 , SARS-CoV-2 , Antigens, Viral , Diagnostic Tests, Routine , Humans , Sensitivity and Specificity , Serologic Tests
5.
Proteomics ; 21(7-8): e2000226, 2021 04.
Article in English | MEDLINE | ID: covidwho-1384280

ABSTRACT

A major part of the analysis of parallel reaction monitoring (PRM) data is the comparison of observed fragment ion intensities to a library spectrum. Classically, these libraries are generated by data-dependent acquisition (DDA). Here, we test Prosit, a published deep neural network algorithm, for its applicability in predicting spectral libraries for PRM. For this purpose, we targeted 1529 precursors derived from synthetic viral peptides and analyzed the data with Prosit and DDA-derived libraries. Viral peptides were chosen as an example, because virology is an area where in silico library generation could significantly improve PRM assay design. With both libraries a total of 1174 precursors were identified. Notably, compared to the DDA-derived library, we could identify 101 more precursors by using the Prosit-derived library. Additionally, we show that Prosit can be applied to predict tandem mass spectra of synthetic viral peptides with different collision energies. Finally, we used a spectral library predicted by Prosit and a DDA library to identify SARS-CoV-2 peptides from a simulated oropharyngeal swab demonstrating that both libraries are suited for peptide identification by PRM. Summarized, Prosit-derived viral spectral libraries predicted in silico can be used for PRM data analysis, making DDA analysis for library generation partially redundant in the future.


Subject(s)
COVID-19/virology , Proteomics/methods , SARS-CoV-2/chemistry , Viral Proteins/analysis , Amino Acid Sequence , Humans , Neural Networks, Computer , Peptide Library , Peptides/analysis , Tandem Mass Spectrometry/methods
6.
J Proteome Res ; 20(9): 4598-4602, 2021 09 03.
Article in English | MEDLINE | ID: covidwho-1371586

ABSTRACT

Mass spectrometry-based proteomics is applied in SARS-CoV-2 research and is, moreover, being discussed as a novel method for SARS-CoV-2 diagnostics. However, the safe inactivation of coronaviruses by proteomics lysis buffers has not been systematically analyzed yet. Hence, for safety reasons a heating step prior to sample preparation is often performed. This step could be omitted once the safe inactivation with the typical buffers is proven. Here we test five different proteomics lysis buffers-4% SDS, 1% SDC, TFA, 6 M GdmCl, and 8 M urea-for their inactivation capacity of coronaviruses. Two representative human coronaviruses, namely HCoV-229E and HCoV-OC43, were used as surrogate for SARS-CoV-2. Lysis was performed at room temperature and at 95 °C for 5 min. Inactivation was confirmed by the absence of a cytopathic effect in MRC-5 cells, and equivocal results were further confirmed by serial passaging and quantitative real-time PCR. While at room temperature SDS, SDC, and TFA inactivated both coronaviruses, and GdmCl and urea resulted in partially incomplete inactivation. This demonstrates that care should be taken when choosing lysis buffers for proteomics analysis of coronaviruses, because some buffers do not ensure inactivation and, hence, biosafety during the further sample preparation.


Subject(s)
COVID-19 , Coronavirus 229E, Human , Coronavirus OC43, Human , Humans , Proteomics , SARS-CoV-2
7.
Virol J ; 18(1): 110, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1255943

ABSTRACT

BACKGROUND: The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. AIM: Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). METHODS: Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. RESULTS: Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. CONCLUSION: The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Coronavirus Envelope Proteins/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Limit of Detection , Polyproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Viral Proteins/genetics
8.
Viruses ; 13(4)2021 03 24.
Article in English | MEDLINE | ID: covidwho-1154529

ABSTRACT

Since the SARS-CoV-2 pandemic started in late 2019, the search for protective vaccines and for drug treatments has become mandatory to fight the global health emergency. Travel restrictions, social distancing, and face masks are suitable counter measures, but may not bring the pandemic under control because people will inadvertently or at a certain degree of restriction severity or duration become incompliant with the regulations. Even if vaccines are approved, the need for antiviral agents against SARS-CoV-2 will persist. However, unequivocal evidence for efficacy against SARS-CoV-2 has not been demonstrated for any of the repurposed antiviral drugs so far. Amantadine was approved as an antiviral drug against influenza A, and antiviral activity against SARS-CoV-2 has been reasoned by analogy but without data. We tested the efficacy of amantadine in vitro in Vero E6 cells infected with SARS-CoV-2. Indeed, amantadine inhibited SARS-CoV-2 replication in two separate experiments with IC50 concentrations between 83 and 119 µM. Although these IC50 concentrations are above therapeutic amantadine levels after systemic administration, topical administration by inhalation or intranasal instillation may result in sufficient amantadine concentration in the airway epithelium without high systemic exposure. However, further studies in other models are needed to prove this hypothesis.


Subject(s)
Amantadine/pharmacology , Antiviral Agents/pharmacology , COVID-19/virology , SARS-CoV-2/drug effects , Animals , COVID-19/drug therapy , Chlorocebus aethiops , Humans , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Vero Cells , Virus Replication/drug effects
9.
J Proteome Res ; 19(11): 4380-4388, 2020 11 06.
Article in English | MEDLINE | ID: covidwho-889125

ABSTRACT

One of the most widely used methods to detect an acute viral infection in clinical specimens is diagnostic real-time polymerase chain reaction. However, because of the COVID-19 pandemic, mass-spectrometry-based proteomics is currently being discussed as a potential diagnostic method for viral infections. Because proteomics is not yet applied in routine virus diagnostics, here we discuss its potential to detect viral infections. Apart from theoretical considerations, the current status and technical limitations are considered. Finally, the challenges that have to be overcome to establish proteomics in routine virus diagnostics are highlighted.


Subject(s)
Coronavirus Infections/diagnosis , Mass Spectrometry/methods , Pneumonia, Viral/diagnosis , Proteomics/methods , Virology/methods , Betacoronavirus/chemistry , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Infections/virology , Humans , Pandemics , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , Virus Diseases/diagnosis , Virus Diseases/virology
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