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1.
J Infect Dis ; 222(4): 551-555, 2020 07 23.
Article in English | MEDLINE | ID: covidwho-704462

ABSTRACT

We simulated 3 transmission modes, including close-contact, respiratory droplets and aerosol routes, in the laboratory. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be highly transmitted among naive human angiotensin-converting enzyme 2 (hACE2) mice via close contact because 7 of 13 naive hACE2 mice were SARS-CoV-2 antibody seropositive 14 days after being introduced into the same cage with 3 infected-hACE2 mice. For respiratory droplets, SARS-CoV-2 antibodies from 3 of 10 naive hACE2 mice showed seropositivity 14 days after introduction into the same cage with 3 infected-hACE2 mice, separated by grids. In addition, hACE2 mice cannot be experimentally infected via aerosol inoculation until continued up to 25 minutes with high viral concentrations.


Subject(s)
Betacoronavirus , Coronavirus Infections/transmission , Pneumonia, Viral/transmission , Aerosols , Anal Canal/virology , Animals , Antibodies, Viral/blood , Betacoronavirus/genetics , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , Chlorocebus aethiops , Female , Humans , Immunoglobulin G/blood , Lung/pathology , Lung/virology , Male , Mice , Mice, Transgenic , Pandemics , Peptidyl-Dipeptidase A/genetics , Pharynx/virology , RNA, Viral/isolation & purification , Respiratory System/virology , Risk , Specific Pathogen-Free Organisms , Time Factors , Vero Cells , Viral Load , Weight Loss
2.
J Exp Med ; 217(11)2020 11 02.
Article in English | MEDLINE | ID: covidwho-697830

ABSTRACT

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.


Subject(s)
Antibodies, Neutralizing/analysis , Antibodies, Viral/analysis , Betacoronavirus/immunology , Coronavirus Infections/immunology , Immunoassay/methods , Pneumonia, Viral/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/genetics , Cell Line , Chimera/genetics , Chimera/immunology , Chlorocebus aethiops , Coronavirus Infections/virology , HEK293 Cells , HIV-1/genetics , HIV-1/immunology , Humans , Neutralization Tests/methods , Pandemics , Pneumonia, Viral/virology , Recombination, Genetic , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/immunology
3.
Int J Mol Sci ; 21(12)2020 Jun 26.
Article in English | MEDLINE | ID: covidwho-692289

ABSTRACT

In the 21st century, three highly pathogenic betacoronaviruses have emerged, with an alarming rate of human morbidity and case fatality. Genomic information has been widely used to understand the pathogenesis, animal origin and mode of transmission of coronaviruses in the aftermath of the 2002-2003 severe acute respiratory syndrome (SARS) and 2012 Middle East respiratory syndrome (MERS) outbreaks. Furthermore, genome sequencing and bioinformatic analysis have had an unprecedented relevance in the battle against the 2019-2020 coronavirus disease 2019 (COVID-19) pandemic, the newest and most devastating outbreak caused by a coronavirus in the history of mankind. Here, we review how genomic information has been used to tackle outbreaks caused by emerging, highly pathogenic, betacoronavirus strains, emphasizing on SARS-CoV, MERS-CoV and SARS-CoV-2. We focus on shared genomic features of the betacoronaviruses and the application of genomic information to phylogenetic analysis, molecular epidemiology and the design of diagnostic systems, potential drugs and vaccine candidates.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Genome, Viral , Pandemics/prevention & control , Pneumonia, Viral/virology , Animals , Betacoronavirus/immunology , Coronavirus Infections/diagnosis , Coronavirus Infections/drug therapy , Drug Design , Genes, Viral , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Molecular Epidemiology , Phylogeny , Pneumonia, Viral/diagnosis , Pneumonia, Viral/drug therapy , SARS Virus/genetics , Severe Acute Respiratory Syndrome/virology , Viral Vaccines/genetics , Viral Vaccines/immunology
4.
Euro Surveill ; 25(30)2020 07.
Article in English | MEDLINE | ID: covidwho-690919

ABSTRACT

We analysed consecutive RT-qPCR results of 537 symptomatic coronavirus disease (COVID-19) patients in home quarantine. Respectively 2, 3, and 4 weeks after symptom onset, 50%, 25% and 10% of patients had detectable RNA from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In patients with mild COVID-19, RNA detection is likely to outlast currently known periods of infectiousness by far and fixed time periods seem more appropriate in determining the length of home isolation than laboratory-based approaches.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus/genetics , Pandemics , Pneumonia, Viral , RNA Replicase/genetics , Real-Time Polymerase Chain Reaction/methods , Viral Nonstructural Proteins/genetics , Adult , Aged , Aged, 80 and over , Betacoronavirus/isolation & purification , Coronavirus/isolation & purification , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Germany/epidemiology , Humans , Middle Aged , Patient Isolation , Pneumonia, Viral/diagnosis , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Quarantine , Survival Analysis , Time Factors
5.
Virulence ; 11(1): 964-967, 2020 12.
Article in English | MEDLINE | ID: covidwho-690823

ABSTRACT

Currently, testing for coronavirus is performed with time and personnel consuming PCR assays. The aim of this study was to evaluate the sensitivity, specificity and capacity of a fully automated, random access high-throughput real-time PCR-based diagnostic platform for the detection of SARS-CoV-2. The NeuMoDx N96 system displayed an equal or better detection rate for SARS-CoV-2 compared with the LightCycler 480II system and showed a specificity of 100%. The median PCR run time for all 28 PCR runs was 91 (IQR 84-97) minutes. The capacity of the NeuMoDx N96 could easily surpass the capacity of most currently used molecular test systems and significantly reduce the turn-around time.


Subject(s)
Betacoronavirus/isolation & purification , High-Throughput Nucleotide Sequencing/methods , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , High-Throughput Nucleotide Sequencing/instrumentation , High-Throughput Nucleotide Sequencing/standards , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/instrumentation , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Sensitivity and Specificity , Time Factors
6.
Front Immunol ; 11: 1581, 2020.
Article in English | MEDLINE | ID: covidwho-688990

ABSTRACT

To ultimately combat the emerging COVID-19 pandemic, it is desired to develop an effective and safe vaccine against this highly contagious disease caused by the SARS-CoV-2 coronavirus. Our literature and clinical trial survey showed that the whole virus, as well as the spike (S) protein, nucleocapsid (N) protein, and membrane (M) protein, have been tested for vaccine development against SARS and MERS. However, these vaccine candidates might lack the induction of complete protection and have safety concerns. We then applied the Vaxign and the newly developed machine learning-based Vaxign-ML reverse vaccinology tools to predict COVID-19 vaccine candidates. Our Vaxign analysis found that the SARS-CoV-2 N protein sequence is conserved with SARS-CoV and MERS-CoV but not from the other four human coronaviruses causing mild symptoms. By investigating the entire proteome of SARS-CoV-2, six proteins, including the S protein and five non-structural proteins (nsp3, 3CL-pro, and nsp8-10), were predicted to be adhesins, which are crucial to the viral adhering and host invasion. The S, nsp3, and nsp8 proteins were also predicted by Vaxign-ML to induce high protective antigenicity. Besides the commonly used S protein, the nsp3 protein has not been tested in any coronavirus vaccine studies and was selected for further investigation. The nsp3 was found to be more conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV than among 15 coronaviruses infecting human and other animals. The protein was also predicted to contain promiscuous MHC-I and MHC-II T-cell epitopes, and the predicted linear B-cell epitopes were found to be localized on the surface of the protein. Our predicted vaccine targets have the potential for effective and safe COVID-19 vaccine development. We also propose that an "Sp/Nsp cocktail vaccine" containing a structural protein(s) (Sp) and a non-structural protein(s) (Nsp) would stimulate effective complementary immune responses.


Subject(s)
Betacoronavirus , Coronavirus Infections , Machine Learning , Pandemics , Pneumonia, Viral , Viral Vaccines , Animals , Betacoronavirus/genetics , Betacoronavirus/immunology , Coronavirus Infections/epidemiology , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Epitopes, B-Lymphocyte/genetics , Epitopes, B-Lymphocyte/immunology , Humans , Immunogenicity, Vaccine , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Pandemics/prevention & control , Pneumonia, Viral/epidemiology , Pneumonia, Viral/genetics , Pneumonia, Viral/immunology , Pneumonia, Viral/prevention & control , Viral Proteins/genetics , Viral Proteins/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology
7.
Indian J Med Microbiol ; 38(1): 9-17, 2020.
Article in English | MEDLINE | ID: covidwho-688963

ABSTRACT

High-throughput, accurate, cost-effective and rapid testing for severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is the need of the hour in face of the global coronavirus disease pandemic. This target is achievable, within a relatively short time through capacity building of reverse transcription polymerase chain reaction (RT-PCR) tests by utilising the strengths of intra and inter institutional networks. These networks act as force multiplier for vital resources which are required for capacity building, namely, leadership, expertise, equipment, space, infection control inputs and human resources. In this article, we report the experience of capacity building for delivery of RT-PCR tests for SARS CoV-2 from a cancer hospital in Eastern India. The relevance, mode of operation and value addition of this essential public health service are discussed in the context of inter departmental collaboration and interaction with other institutes through the existing diagnostic, surveillance and infection control networks. This networking model for service development and delivery could be used by other centres.


Subject(s)
Betacoronavirus/isolation & purification , Capacity Building/organization & administration , Clinical Laboratory Techniques/methods , Community Networks/organization & administration , Coronavirus Infections/diagnosis , Diagnostic Services/organization & administration , Pneumonia, Viral/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Humans , India , Pandemics
8.
Indian J Med Microbiol ; 38(1): 18-23, 2020.
Article in English | MEDLINE | ID: covidwho-688890

ABSTRACT

Background and Objectives: Timely diagnosis is essential for the containment of the disease and breaks in the chain of transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The present situation demands the countries to scale up their testing and design innovative strategies to conserve diagnostic kits and reagents. The pooling of samples saves time, workforce and most importantly diagnostic kits and reagents. In the present study, we tried to define the pool size that could be applied with acceptable confidence for testing. Materials and Methods: We used repeatedly tested positive clinical sample elutes having different levels of SARS CoV 2 RNA and negative sample elutes to prepare seven series of 11 pools each, having pool sizes ranging from 2 to 48 samples to estimate the optimal pool size. Each pool had one positive sample elute in different compositions. All the pools were tested by SARS CoV 2 reverse transcriptase quantitative polymerase chain reaction. Results: Out of the 77 pools, only 53 (68.8%) were found positive. The sensitivity of pools of 2-48 samples was decreased from 100% (95% confidence interval [CL]; 98.4-100) to 41.41% (95% CL; 34.9-48.1). The maximum size of the pool with acceptable sensitivity (>95%) was found to be of six samples. For the pool size of six samples, the sensitivity was 97.8% and the efficiency of pooling was 0.38. Conclusions: The pooling of samples is a practical way for scaling up testing and ultimately containing the further spread of the CoV disease 2019 pandemic.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , Specimen Handling/methods , Betacoronavirus/genetics , Humans , Pandemics , Sensitivity and Specificity
9.
J Proteome Res ; 19(4): 1351-1360, 2020 04 03.
Article in English | MEDLINE | ID: covidwho-688546

ABSTRACT

As the infection of 2019-nCoV coronavirus is quickly developing into a global pneumonia epidemic, the careful analysis of its transmission and cellular mechanisms is sorely needed. In this Communication, we first analyzed two recent studies that concluded that snakes are the intermediate hosts of 2019-nCoV and that the 2019-nCoV spike protein insertions share a unique similarity to HIV-1. However, the reimplementation of the analyses, built on larger scale data sets using state-of-the-art bioinformatics methods and databases, presents clear evidence that rebuts these conclusions. Next, using metagenomic samples from Manis javanica, we assembled a draft genome of the 2019-nCoV-like coronavirus, which shows 73% coverage and 91% sequence identity to the 2019-nCoV genome. In particular, the alignments of the spike surface glycoprotein receptor binding domain revealed four times more variations in the bat coronavirus RaTG13 than in the Manis coronavirus compared with 2019-nCoV, suggesting the pangolin as a missing link in the transmission of 2019-nCoV from bats to human.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Genome, Viral/genetics , Host-Pathogen Interactions , Models, Molecular , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Animals , Betacoronavirus/classification , Eutheria/virology , HIV-1/genetics , Humans , Metagenome , Pandemics , Protein Structure, Tertiary , Sequence Alignment , Sequence Analysis, Protein , Snakes/virology
10.
BMC Bioinformatics ; 21(1): 211, 2020 May 24.
Article in English | MEDLINE | ID: covidwho-687768

ABSTRACT

BACKGROUND: GenBank contains over 3 million viral sequences. The National Center for Biotechnology Information (NCBI) previously made available a tool for validating and annotating influenza virus sequences that is used to check submissions to GenBank. Before this project, there was no analogous tool in use for non-influenza viral sequence submissions. RESULTS: We developed a system called VADR (Viral Annotation DefineR) that validates and annotates viral sequences in GenBank submissions. The annotation system is based on the analysis of the input nucleotide sequence using models built from curated RefSeqs. Hidden Markov models are used to classify sequences by determining the RefSeq they are most similar to, and feature annotation from the RefSeq is mapped based on a nucleotide alignment of the full sequence to a covariance model. Predicted proteins encoded by the sequence are validated with nucleotide-to-protein alignments using BLAST. The system identifies 43 types of "alerts" that (unlike the previous BLAST-based system) provide deterministic and rigorous feedback to researchers who submit sequences with unexpected characteristics. VADR has been integrated into GenBank's submission processing pipeline allowing for viral submissions passing all tests to be accepted and annotated automatically, without the need for any human (GenBank indexer) intervention. Unlike the previous submission-checking system, VADR is freely available (https://github.com/nawrockie/vadr) for local installation and use. VADR has been used for Norovirus submissions since May 2018 and for Dengue virus submissions since January 2019. Since March 2020, VADR has also been used to check SARS-CoV-2 sequence submissions. Other viruses with high numbers of submissions will be added incrementally. CONCLUSION: VADR improves the speed with which non-flu virus submissions to GenBank can be checked and improves the content and quality of the GenBank annotations. The availability and portability of the software allow researchers to run the GenBank checks prior to submitting their viral sequences, and thereby gain confidence that their submissions will be accepted immediately without the need to correspond with GenBank staff. Reciprocally, the adoption of VADR frees GenBank staff to spend more time on services other than checking routine viral sequence submissions.


Subject(s)
Betacoronavirus , Coronavirus Infections , Databases, Nucleic Acid , Molecular Sequence Annotation , Pandemics , Pneumonia, Viral , Software , Betacoronavirus/genetics , Coronavirus Infections/genetics , DNA Viruses , Genomics , Humans , Molecular Sequence Annotation/standards , Pneumonia, Viral/genetics , Viruses
11.
J Med Virol ; 92(6): 584-588, 2020 06.
Article in English | MEDLINE | ID: covidwho-685102

ABSTRACT

Last December 2019, a new virus, named novel Coronavirus (COVID-2019) causing many cases of severe pneumonia was reported in Wuhan, China. The virus knowledge is limited and especially about COVID-2019 pathogenesis. The Open Reading Frame 1ab (ORF1ab) of COVID-2019 has been analyzed to evidence the presence of mutation caused by selective pressure on the virus. For selective pressure analysis fast-unconstrained Bayesian approximation (FUBAR) was used. Homology modelling has been performed by SwissModel and HHPred servers. The presence of transmembrane helical segments in Coronavirus ORF1ab non structural protein 2 (nsp2) and nsp3 was tested by TMHMM, MEMSAT, and MEMPACK tools. Three-dimensional structures have been analyzed and displayed using PyMOL. FUBAR analysis revealed the presence of potential sites under positive selective pressure (P < .05). Position 723 in the COVID-2019 has a serine instead a glycine residue, while at aminoacidic position 1010 a proline instead an isoleucine. Significant (P < .05) pervasive negative selection in 2416 sites (55%) was found. The positive selective pressure could account for some clinical features of this virus compared with severe acute respiratory syndrome (SARS) and Bat SARS-like CoV. The stabilizing mutation falling in the endosome-associated-protein-like domain of the nsp2 protein could account for COVID-2019 high ability of contagious, while the destabilizing mutation in nsp3 proteins could suggest a potential mechanism differentiating COVID-2019 from SARS. These data could be helpful for further investigation aimed to identify potential therapeutic targets or vaccine strategy, especially in the actual moment when the epidemic is ongoing and the scientific community is trying to enrich knowledge about this new viral pathogen.


Subject(s)
Betacoronavirus/genetics , SARS Virus/genetics , Viral Nonstructural Proteins/chemistry , Viral Proteins/chemistry , Betacoronavirus/pathogenicity , Coronavirus Infections/virology , Female , Gene Expression , Humans , Male , Models, Molecular , Mutation , Pandemics , Pneumonia, Viral/virology , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS Virus/pathogenicity , Selection, Genetic , Structural Homology, Protein , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
12.
Cell ; 181(4): 865-876.e12, 2020 05 14.
Article in English | MEDLINE | ID: covidwho-684968

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , CRISPR-Cas Systems , Influenza A Virus, H1N1 Subtype/drug effects , RNA, Viral/antagonists & inhibitors , A549 Cells , Antibiotic Prophylaxis/methods , Base Sequence , Betacoronavirus/genetics , Betacoronavirus/growth & development , Clustered Regularly Interspaced Short Palindromic Repeats , Computer Simulation , Conserved Sequence , Coronavirus/drug effects , Coronavirus/genetics , Coronavirus/growth & development , Coronavirus Infections/drug therapy , Epithelial Cells/virology , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/growth & development , Lung/pathology , Lung/virology , Nucleocapsid Proteins/genetics , Pandemics , Phylogeny , Pneumonia, Viral/drug therapy , RNA Replicase/genetics , Viral Nonstructural Proteins/genetics
13.
J Clin Microbiol ; 58(8)2020 07 23.
Article in English | MEDLINE | ID: covidwho-684521

ABSTRACT

We compared the ability of 2 commercial molecular amplification assays (RealTime SARS-CoV-2 on the m2000 [abbreviated ACOV; Abbott] and ID Now COVID-19 [abbreviated IDNOW; Abbott]) and a laboratory-developed test (modified CDC 2019-nCoV reverse transcriptase PCR [RT-PCR] assay with RNA extraction by eMag [bioMérieux] and amplification on QuantStudio 6 or ABI 7500 real-time PCR system [abbreviated CDC COV]) to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in upper respiratory tract specimens. Discrepant results were adjudicated by medical record review. A total of 200 nasopharyngeal swab specimens in viral transport medium (VTM) were collected from symptomatic patients between 27 March and 9 April 2020. Results were concordant for 167 specimens (83.5% overall agreement), including 94 positive and 73 negative specimens. The ACOV assay yielded 33 additional positive results, 25 of which were also positive by the CDC COV assay but not by the IDNOW assay. In a follow-up evaluation, 97 patients for whom a dry nasal swab specimen yielded negative results by IDNOW had a paired nasopharyngeal swab specimen collected in VTM and tested by the ACOV assay; SARS-CoV-2 RNA was detected in 13 (13.4%) of these specimens. Medical record review deemed all discrepant results to be true positives. The IDNOW test was the easiest to perform and provided a result in the shortest time but detected fewer cases of COVID-19. The ACOV assay detected more cases of COVID-19 than the CDC COV or IDNOW assays.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Adult , Aged , Betacoronavirus/genetics , Centers for Disease Control and Prevention, U.S. , Coronavirus Infections/virology , Female , Humans , Male , Middle Aged , Nasopharynx/virology , Nucleic Acid Amplification Techniques/methods , Pandemics , Pneumonia, Viral/virology , Sensitivity and Specificity , Specimen Handling/methods , Time Factors , United States
14.
J Clin Microbiol ; 58(8)2020 Jul 23.
Article in English | MEDLINE | ID: covidwho-684350

ABSTRACT

Molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the gold standard for diagnosis of coronavirus disease 2019 (COVID-19), but the clinical performance of these tests is still poorly understood, particularly with regard to disease course, patient-specific factors, and viral shedding. From 10 March to 1 May 2020, NewYork-Presbyterian laboratories performed 27,377 SARS-CoV-2 molecular assays from 22,338 patients. Repeat testing was performed for 3,432 patients, of which 2,413 had initial negative and 802 had initial positive results. Repeat-tested patients were more likely to have severe disease and low viral loads. The negative predictive value of the first-day result among repeat-tested patients was 81.3% The clinical sensitivity of SARS-CoV-2 molecular assays was estimated between 58% and 96%, depending on the unknown number of false-negative results in single-tested patients. Conversion to negative was unlikely to occur before 15 to 20 days after initial testing or 20 to 30 days after the onset of symptoms, with 50% conversion occurring at 28 days after initial testing. Conversion from first-day negative to positive results increased linearly with each day of testing, reaching 25% probability in 20 days. Sixty patients fluctuated between positive and negative results over several weeks, suggesting that caution is needed when single-test results are acted upon. In summary, our study provides estimates of the clinical performance of SARS-CoV-2 molecular assays and suggests time frames for appropriate repeat testing, namely, 15 to 20 days after a positive test and the same day or next 2 days after a negative test for patients with high suspicion for COVID-19.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Diagnostic Tests, Routine/methods , Pneumonia, Viral/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Betacoronavirus/genetics , Child , Child, Preschool , Coronavirus Infections/pathology , Coronavirus Infections/virology , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , New York , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Predictive Value of Tests , Sensitivity and Specificity , Viral Load , Young Adult
16.
Genome Med ; 12(1): 68, 2020 07 28.
Article in English | MEDLINE | ID: covidwho-680048

ABSTRACT

BACKGROUND: SARS-CoV-2 is a recently emerged respiratory pathogen that has significantly impacted global human health. We wanted to rapidly characterise the transcriptomic, proteomic and phosphoproteomic landscape of this novel coronavirus to provide a fundamental description of the virus's genomic and proteomic potential. METHODS: We used direct RNA sequencing to determine the transcriptome of SARS-CoV-2 grown in Vero E6 cells which is widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. Allied to this, we used tandem mass spectrometry to investigate the proteome and phosphoproteome of the same virally infected cells. RESULTS: Our integrated analysis revealed that the viral transcripts (i.e. subgenomic mRNAs) generally fitted the expected transcription model for coronaviruses. Importantly, a 24 nt in-frame deletion was detected in over half of the subgenomic mRNAs encoding the spike (S) glycoprotein and was predicted to remove a proposed furin cleavage site from the S glycoprotein. Tandem mass spectrometry identified over 500 viral peptides and 44 phosphopeptides in virus-infected cells, covering almost all proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein. CONCLUSIONS: Detection of an apparently viable deletion in the furin cleavage site of the S glycoprotein, a leading vaccine target, shows that this and other regions of SARS-CoV-2 proteins may readily mutate. The furin site directs cleavage of the S glycoprotein into functional subunits during virus entry or exit and likely contributes strongly to the pathogenesis and zoonosis of this virus. Our data emphasises that the viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.


Subject(s)
Betacoronavirus/growth & development , Gene Expression Profiling/methods , Proteomics/methods , Sequence Deletion , Spike Glycoprotein, Coronavirus/genetics , Animals , Betacoronavirus/genetics , Betacoronavirus/metabolism , Chlorocebus aethiops , Phosphorylation , Sequence Analysis, RNA , Serial Passage , Tandem Mass Spectrometry , Vero Cells
17.
Jpn J Infect Dis ; 73(4)2020 Jul 22.
Article in English | MEDLINE | ID: covidwho-678395

ABSTRACT

During the emergence of novel coronavirus 2019 (nCoV) outbreak in Wuhan city, China at the end of 2019, there was movement of many airline travelers between Wuhan and Japan, suggesting that the Japanese population was at high risk of infection by the virus. Hence, we urgently developed diagnostic systems for detection of 2019 nCoV. Two nested RT-PCR and two real-time RT-PCR assays were adapted for use in Japan. As of February 8, 2020, these assays have successfully detected 25 positive cases of infection in Japan.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Humans , Japan , Pandemics , Reverse Transcriptase Polymerase Chain Reaction/methods , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics
18.
Am J Pathol ; 190(8): 1680-1690, 2020 08.
Article in English | MEDLINE | ID: covidwho-677458

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for >100 years. Patients (n = 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28, 2020, to April 14, 2020. Patients were transfused with convalescent plasma, obtained from donors with confirmed severe acute respiratory syndrome coronavirus 2 infection who had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 after transfusion. Clinical improvement was assessed on the basis of a modified World Health Organization six-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 after transfusion with convalescent plasma, nine patients had at least a one-point improvement in clinical scale, and seven of those were discharged. By day 14 after transfusion, 19 (76%) patients had at least a one-point improvement in clinical status, and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.


Subject(s)
Coronavirus Infections/therapy , Pneumonia, Viral/therapy , Adult , Aged , Betacoronavirus/genetics , Female , Humans , Immunization, Passive , Investigational New Drug Application , Male , Middle Aged , Pandemics , Texas , Whole Genome Sequencing , Young Adult
19.
Medicine (Baltimore) ; 99(29): e21334, 2020 Jul 17.
Article in English | MEDLINE | ID: covidwho-676910

ABSTRACT

RATIONALE: The outbreak of coronavirus disease 2019 (COVID-19) in 2019 has become a global pandemic. It is not known whether the disease is associated with a higher risk of infection in pregnant women or whether intrauterine vertical transmission can occur. We report 2 cases of pregnant women diagnosed with COVID-19. PATIENT CONCERNS: In all of Yichang city from January 20, 2020, to April 9, 2020, only 2 pregnant women, who were in the late stage of pregnancy, were diagnosed with COVID-19; one patient was admitted for fever with limb asthenia, and the other patient was admitted for abnormal chest computed tomography results. DIAGNOSES: Both pregnant women were diagnosed with COVID-19. INTERVENTIONS: After the medical staff prepared for isolation and protection, the 2 pregnant women quickly underwent cesarean sections. A series of tests, such as laboratory, imaging, and SARS-CoV-2 nucleic acid examinations, were performed on the 2 women with COVID-19 and their newborns. OUTCOMES: One of the 2 infected pregnant women had severe COVID-19, and the other had mild disease. Both babies were delivered by cesarean section. Both of the women with COVID-19 worsened 3 to 6 days after delivery. Chest computed tomography suggested that the lesions due to SARS-CoV-2 infection increased. These women began to exhibit fever or reduced blood oxygen saturation again. One of the 2 newborns was born prematurely, and the other was born at full term. Neither infant was infected with COVID-19, but both had increased prothrombin time and fibrinogen, lactate dehydrogenase, phosphocreatine kinase, and creatine kinase isoenzyme contents. LESSONS: SARS-CoV-2 infection was not found in the newborns born to the 2 pregnant women with COVID-19, but transient coagulation dysfunction and myocardial damage occurred in the 2 newborns. Effective management strategies for pregnant women with COVID-19 will help to control the outbreak of COVID-19 among pregnant women.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Adult , Asthenia/etiology , Betacoronavirus/isolation & purification , Cesarean Section/methods , China/epidemiology , Coronavirus Infections/drug therapy , Disease Outbreaks/prevention & control , Female , Fever/etiology , Humans , Infant, Newborn/blood , Infant, Newborn/metabolism , Infectious Disease Transmission, Vertical/prevention & control , Pandemics , Pneumonia, Viral/drug therapy , Pregnancy , Pregnancy Complications, Infectious/epidemiology , Pregnancy Complications, Infectious/virology , Pregnancy Outcome/epidemiology , Premature Birth/epidemiology , Thorax/diagnostic imaging , Tomography, X-Ray Computed/methods
20.
J Clin Microbiol ; 58(8)2020 Jul 23.
Article in English | MEDLINE | ID: covidwho-676653

ABSTRACT

In this commentary, we provide a broad overview of how the rapidly evolving coronavirus disease 2019 (COVID-19) diagnostic landscape has impacted clinical care during the COVID-19 pandemic. We review aspects of both molecular and serologic testing and discuss the logistical challenges faced with each. We also highlight the progress that has been made in the development and implementation of these assays as well as the need for ongoing improvement in diagnostic testing capabilities.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Betacoronavirus/genetics , Betacoronavirus/immunology , Clinical Laboratory Techniques/trends , Diagnostic Tests, Routine/methods , Diagnostic Tests, Routine/trends , Humans , Pandemics
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