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1.
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
2.
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
3.
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
4.
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
5.
Int J Mol Med ; 46(3): 957-964, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-676117

ABSTRACT

Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) is the gold standard method for the diagnosis of COVID­19 infection. Due to pre­analytical and technical limitations, samples with low viral load are often misdiagnosed as false­negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT­qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID­19 were analyzed by droplet digital PCR (ddPCR) and RT­qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)­approved probe for the SARS­CoV­2 N gene were used. SYBR­Green RT­qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT­qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT­qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10­fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT­qPCR for the diagnosis of COVID­19 infection in false­negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID­19 and the follow­up of positive patients until complete remission.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Humans , Nucleocapsid Proteins/genetics , Pandemics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics
6.
Internist (Berl) ; 61(8): 789-792, 2020 Aug.
Article in German | MEDLINE | ID: covidwho-670575

ABSTRACT

The new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has far reaching effects on society, the economy and medical treatment. It is all the more important to understand the characteristics of the virus and to utilize them diagnostically, therapeutically and epidemiologically. This article firstly elucidates the medical importance of coronaviruses in general. Then angiotensin-converting enzyme 2 (ACE2) as the binding site of SARS-CoV­2 and the possible influence on the disease susceptibility are explained. The gold standard for detection of an active SARS-CoV­2 infection is the direct detection of the pathogen with nucleic acid amplification techniques. At the onset of symptoms, a swab of the upper airway is especially suitable due to the high viral burden. At a later stage direct detection can be achieved in samples from the lower airway or a stool or anal swab. Antigen or antibody tests cannot replace the direct detection of the pathogen; however, the detection of immunoglobulin G antibodies are of special interest for epidemiological questions (seroconversion time of sometimes several weeks). The plaque reduction neutralization test exclusively detects antibodies which neutralize viruses but the procedure is complicated. In addition, the importance of these antibodies with respect to immunity against a second infection is uncertain. Thanks to modern techniques thousands of SARS-CoV­2 sequences are already available, which show a genomic variability. The D614G mutation in the S spikes seems to cause a higher infectiosity. Mutations can impair the diagnostics and treatment, which makes monitoring necessary.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Pneumonia, Viral/virology , Antibodies, Viral/blood , Coronavirus Infections/diagnosis , Genome, Viral , Humans , Immunoglobulin G/blood , Nucleic Acid Amplification Techniques , Pandemics , Peptidyl-Dipeptidase A , Pneumonia, Viral/diagnosis , Receptors, Virus , Spike Glycoprotein, Coronavirus/genetics
7.
J Infect Dis ; 222(2): 223-233, 2020 06 29.
Article in English | MEDLINE | ID: covidwho-656287

ABSTRACT

Severe acute respiratory syndrome coronavirus (SARS-CoV) was discovered as a novel pathogen in the 2002-2003 SARS epidemic. The emergence and disappearance of this pathogen have brought questions regarding its source and evolution. Within the genome sequences of 281 SARS-CoVs, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and SARS-related CoVs (SARSr-CoVs), a ~430 bp genomic region (from 27 701 bp to 28 131 bp in AY390556.1) with regular variations was investigated. This ~430 bp region overlaps with the ORF8 gene and is prone to deletions and nucleotide substitutions. Its complexity suggested the need for a new genotyping method for coronaviruses related to SARS-similar coronaviruses (SARS-CoV, SARSr-CoV, and SARS-CoV-2). Bat SARSr-CoV presented 3 genotypes, of which type 0 is only seen in bat SARSr-CoV, type I is present in SARS in the early phase, and type II is found in all SARS-CoV-2. This genotyping also shows potential usage in distinguishing the SARS-similar coronaviruses from different hosts and geographic areas. This genomic region has important implications for predicting the epidemic trend and studying the evolution of coronavirus.


Subject(s)
Betacoronavirus/genetics , Genome, Viral , SARS Virus/genetics , Viral Matrix Proteins/genetics , Animals , Base Sequence , Chiroptera/virology , Eutheria/virology , Evolution, Molecular , Genes, Viral , Genetic Variation , Humans , Open Reading Frames , Phylogeny , Sequence Alignment , Sequence Deletion , Spike Glycoprotein, Coronavirus/genetics , Viverridae/virology
8.
Clin Immunol ; 216: 108459, 2020 07.
Article in English | MEDLINE | ID: covidwho-642982

ABSTRACT

The COVID-19 pandemic is one of the greatest infectious challenges in recent history. Presently, few treatment options exist and the availability of effective vaccines is at least one year away. There is an urgent need to find currently available, effective therapies in the treatment of patients with COVID-19 infection. In this review, we compare and contrast the use of intravenous immunoglobulin and hyperimmune globulin in the treatment of COVID-19 infection.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Cytokine Release Syndrome/pathology , Immunoglobulins, Intravenous/administration & dosage , Immunologic Factors/administration & dosage , Pandemics , Pneumonia, Viral/drug therapy , Adaptive Immunity/drug effects , Antibody-Dependent Enhancement/drug effects , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Coronavirus Infections/therapy , Coronavirus Infections/virology , Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/prevention & control , Gene Expression , Humans , Immunity, Innate/drug effects , Immunization, Passive/adverse effects , Immunization, Passive/methods , Immunoglobulins, Intravenous/adverse effects , Immunologic Factors/adverse effects , Molecular Targeted Therapy , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/immunology , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/virology
9.
Nat Commun ; 11(1): 3523, 2020 07 09.
Article in English | MEDLINE | ID: covidwho-640262

ABSTRACT

The spread of the SARS-CoV-2 into a global pandemic within a few months of onset motivates the development of a rapidly scalable vaccine. Here, we present a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle (LNP) as a vaccine. We observe remarkably high and dose-dependent SARS-CoV-2 specific antibody titers in mouse sera, as well as robust neutralization of both a pseudo-virus and wild-type virus. Upon further characterization we find that the neutralization is proportional to the quantity of specific IgG and of higher magnitude than recovered COVID-19 patients. saRNA LNP immunizations induce a Th1-biased response in mice, and there is no antibody-dependent enhancement (ADE) observed. Finally, we observe high cellular responses, as characterized by IFN-γ production, upon re-stimulation with SARS-CoV-2 peptides. These data provide insight into the vaccine design and evaluation of immunogenicity to enable rapid translation to the clinic.


Subject(s)
Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Nanoparticles/chemistry , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , Animals , Antibodies, Viral/blood , Antibodies, Viral/metabolism , Antibody-Dependent Enhancement/immunology , Betacoronavirus/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cytokines/immunology , Disease Models, Animal , Humans , Immunity, Cellular , Immunoglobulin G/blood , Mice , Mice, Inbred BALB C , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , RNA, Viral/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunology , Viral Vaccines/chemistry
10.
Theranostics ; 10(16): 7448-7464, 2020.
Article in English | MEDLINE | ID: covidwho-640241

ABSTRACT

The COVID-19 pandemic is an emerging threat to global public health. While our current understanding of COVID-19 pathogenesis is limited, a better understanding will help us develop efficacious treatment and prevention strategies for COVID-19. One potential therapeutic target is angiotensin converting enzyme 2 (ACE2). ACE2 primarily catalyzes the conversion of angiotensin I (Ang I) to a nonapeptide angiotensin or the conversion of angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7) and has direct effects on cardiac function and multiple organs via counter-regulation of the renin-angiotensin system (RAS). Significant to COVID-19, ACE2 is postulated to serve as a major entry receptor for SARS-CoV-2 in human cells, as it does for SARS-CoV. Many infected individuals develop COVID-19 with fever, cough, and shortness of breath that can progress to pneumonia. Disease progression promotes the activation of immune cells, platelets, and coagulation pathways that can lead to multiple organ failure and death. ACE2 is expressed by epithelial cells of the lungs at high level, a major target of the disease, as seen in post-mortem lung tissue of patients who died with COVID-19, which reveals diffuse alveolar damage with cellular fibromyxoid exudates bilaterally. Comparatively, ACE2 is expressed at low level by vascular endothelial cells of the heart and kidney but may also be targeted by the virus in severe COVID-19 cases. Interestingly, SARS-CoV-2 infection downregulates ACE2 expression, which may also play a critical pathogenic role in COVID-19. Importantly, targeting ACE2/Ang 1-7 axis and blocking ACE2 interaction with the S protein of SARS-CoV-2 to curtail SARS-CoV-2 infection are becoming very attractive therapeutics potential for treatment and prevention of COVID-19. Here, we will discuss the following subtopics: 1) ACE2 as a receptor of SARS-CoV-2; 2) clinical and pathological features of COVID-19; 3) role of ACE2 in the infection and pathogenesis of SARS; 4) potential pathogenic role of ACE2 in COVID-19; 5) animal models for pathological studies and therapeutics; and 6) therapeutics development for COVID-19.


Subject(s)
Betacoronavirus , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , Receptors, Virus/metabolism , Angiotensin II Type 1 Receptor Blockers/therapeutic use , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , Antiviral Agents/therapeutic use , Betacoronavirus/chemistry , Betacoronavirus/pathogenicity , Betacoronavirus/physiology , Coronavirus Infections/prevention & control , Coronavirus Infections/therapy , Disease Models, Animal , Host Microbial Interactions/physiology , Humans , Mice , Models, Biological , Pandemics , Pneumonia, Viral/therapy , Renin-Angiotensin System/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Theranostic Nanomedicine , Viral Vaccines/isolation & purification , Virus Internalization
11.
Genes (Basel) ; 11(7)2020 07 07.
Article in English | MEDLINE | ID: covidwho-639723

ABSTRACT

The pandemic caused by the spread of SARS-CoV-2 has led to considerable interest in its evolutionary origin and genome structure. Here, we analyzed mutation patterns in 34 human SARS-CoV-2 isolates and a closely related RaTG13 isolated from Rhinolophus affinis (a horseshoe bat). We also evaluated the CpG dinucleotide contents in SARS-CoV-2 and other human and animal coronavirus genomes. Out of 1136 single nucleotide variations (~4% divergence) between human SARS-CoV-2 and bat RaTG13, 682 (60%) can be attributed to C>U and U>C substitutions, far exceeding other types of substitutions. An accumulation of C>U mutations was also observed in SARS-CoV2 variants that arose within the human population. Globally, the C>U substitutions increased the frequency of codons for hydrophobic amino acids in SARS-CoV-2 peptides, while U>C substitutions decreased it. In contrast to most other coronaviruses, both SARS-CoV-2 and RaTG13 exhibited CpG depletion in their genomes. The data suggest that C-to-U conversion mediated by C deamination played a significant role in the evolution of the SARS-CoV-2 coronavirus. We hypothesize that the high frequency C>U transitions reflect virus adaptation processes in their hosts, and that SARS-CoV-2 could have been evolving for a relatively long period in humans following the transfer from animals before spreading worldwide.


Subject(s)
Betacoronavirus/genetics , Cytosine/metabolism , Evolution, Molecular , SARS Virus/genetics , Uracil/metabolism , Animals , Base Sequence , Betacoronavirus/classification , Betacoronavirus/isolation & purification , Chiroptera/virology , CpG Islands , Humans , Phylogeny , Polymorphism, Single Nucleotide , SARS Virus/classification , SARS Virus/isolation & purification , Spike Glycoprotein, Coronavirus/genetics
12.
Emerg Infect Dis ; 26(7): 1610-1612, 2020 07.
Article in English | MEDLINE | ID: covidwho-610766

ABSTRACT

We characterized novel coronaviruses detected in US bottlenose dolphins (BdCoVs) with diarrhea. These viruses are closely related to the other 2 known cetacean coronaviruses, Hong Kong BdCoV and beluga whale CoV. A deletion in the spike gene and insertions in the membrane gene and untranslated regions were found in US BdCoVs (unrelated to severe acute respiratory syndrome coronavirus 2).


Subject(s)
Bottle-Nosed Dolphin/virology , Coronavirus Infections/veterinary , Diarrhea/veterinary , Gammacoronavirus/classification , Gammacoronavirus/genetics , Animals , Coronavirus Infections/virology , Diarrhea/virology , Gammacoronavirus/isolation & purification , Gammacoronavirus/physiology , Genes, Viral , Genome, Viral , Mutation , Phylogeny , Sequence Deletion , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/genetics
13.
J Chin Med Assoc ; 83(6): 537-543, 2020 06.
Article in English | MEDLINE | ID: covidwho-601886

ABSTRACT

BACKGROUND: China announced an outbreak of new coronavirus in the city of Wuhan on December 31, 2019; lash to now, the virus transmission has become pandemic worldwide. Severe cases from the Huanan Seafood Wholesale market in Wuhan were confirmed pneumonia with a novel coronavirus (2019-nCoV). Understanding the molecular mechanisms of genome selection and packaging is critical for developing antiviral strategies. Thus, we defined the correlation in 10 severe acute respiratory syndrome coronavirus (SARS-CoV2) sequences from different countries to analyze the genomic patterns of disease origin and evolution aiming for developing new control pandemic processes. METHODS: We apply genomic analysis to observe SARS-CoV2 sequences from GenBank (http://www.ncbi.nim.nih.gov/genebank/): MN 908947 (China, C1), MN985325 (USA: WA, UW), MN996527 (China, C2), MT007544 (Australia: Victoria, A1), MT027064 (USA: CA, UC), MT039890 (South Korea, K1), MT066175 (Taiwan, T1), MT066176 (Taiwan, T2), LC528232 (Japan, J1), and LC528233 (Japan, J2) for genomic sequence alignment analysis. Multiple Sequence Alignment by Clustalw (https://www.genome.jp/tools-bin/clustalw) web service is applied as our alignment tool. RESULTS: We analyzed 10 sequences from the National Center for Biotechnology Information (NCBI) database by genome alignment and found no difference in amino acid sequences within M and N proteins. There are two amino acid variances in the spike (S) protein region. One mutation found from the South Korea sequence is verified. Two possible "L" and "S" SNPs found in ORF1ab and ORF8 regions are detected. CONCLUSION: We performed genomic analysis and comparative multiple sequences of SARS-CoV2. Studies about the biological symptoms of SARS-CoV2 in clinic animals and humans will manipulate an understanding on the origin of pandemic crisis.


Subject(s)
Betacoronavirus/genetics , Genome, Viral , Amino Acid Sequence , Polymorphism, Single Nucleotide , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
14.
Exp Mol Med ; 52(6): 963-977, 2020 06.
Article in English | MEDLINE | ID: covidwho-601243

ABSTRACT

SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , DNA Primers , Pneumonia, Viral/virology , Polymerase Chain Reaction/methods , Coronavirus Infections/diagnosis , HEK293 Cells , Humans , Multiplex Polymerase Chain Reaction/methods , Pandemics , Pharynx/virology , Pneumonia, Viral/diagnosis , RNA Replicase/genetics , Real-Time Polymerase Chain Reaction/methods , SARS Virus/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics , Viral Proteins/genetics
15.
J Phys Chem Lett ; 11(14): 5661-5667, 2020 Jul 16.
Article in English | MEDLINE | ID: covidwho-598380

ABSTRACT

Coronaviruses may produce severe acute respiratory syndrome (SARS). As a matter of fact, a new SARS-type virus, SARS-CoV-2, is responsible for the global pandemic in 2020 with unprecedented sanitary and economic consequences for most countries. In the present contribution we study, by all-atom equilibrium and enhanced sampling molecular dynamics simulations, the interaction between the SARS Unique Domain and RNA guanine quadruplexes, a process involved in eluding the defensive response of the host thus favoring viral infection of human cells. Our results evidence two stable binding modes involving an interaction site spanning either the protein dimer interface or only one monomer. The free energy profile unequivocally points to the dimer mode as the thermodynamically favored one. The effect of these binding modes in stabilizing the protein dimer was also assessed, being related to its biological role in assisting the SARS viruses to bypass the host protective response. This work also constitutes a first step in the possible rational design of efficient therapeutic agents aiming at perturbing the interaction between SARS Unique Domain and guanine quadruplexes, hence enhancing the host defenses against the virus.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/genetics , Coronavirus Infections/virology , G-Quadruplexes/drug effects , Pneumonia, Viral/virology , RNA, Viral/chemistry , RNA, Viral/genetics , Betacoronavirus/drug effects , Dimerization , Humans , Models, Molecular , Molecular Dynamics Simulation , Pandemics , Protein Binding , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
16.
Proc Natl Acad Sci U S A ; 117(26): 15193-15199, 2020 06 30.
Article in English | MEDLINE | ID: covidwho-595720

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an immediate, major threat to public health across the globe. Here we report an in-depth molecular analysis to reconstruct the evolutionary origins of the enhanced pathogenicity of SARS-CoV-2 and other coronaviruses that are severe human pathogens. Using integrated comparative genomics and machine learning techniques, we identify key genomic features that differentiate SARS-CoV-2 and the viruses behind the two previous deadly coronavirus outbreaks, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), from less pathogenic coronaviruses. These features include enhancement of the nuclear localization signals in the nucleocapsid protein and distinct inserts in the spike glycoprotein that appear to be associated with high case fatality rate of these coronaviruses as well as the host switch from animals to humans. The identified features could be crucial contributors to coronavirus pathogenicity and possible targets for diagnostics, prognostication, and interventions.


Subject(s)
Betacoronavirus/genetics , Evolution, Molecular , Genome, Viral , Nucleocapsid Proteins/genetics , Spike Glycoprotein, Coronavirus/genetics , Animals , Betacoronavirus/classification , Betacoronavirus/pathogenicity , Host Specificity , Humans , Machine Learning , Middle East Respiratory Syndrome Coronavirus/classification , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Mutagenesis, Insertional , Nuclear Localization Signals/genetics , Nucleocapsid Proteins/chemistry , Phylogeny , Sequence Homology , Spike Glycoprotein, Coronavirus/chemistry , Virulence/genetics
17.
Signal Transduct Target Ther ; 5(1): 89, 2020 06 10.
Article in English | MEDLINE | ID: covidwho-595441

ABSTRACT

Coronavirus infections of multiple origins have spread to date worldwide, causing severe respiratory diseases. Seven coronaviruses that infect humans have been identified: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. Among them, SARS-CoV and MERS-CoV caused outbreaks in 2002 and 2012, respectively. SARS-CoV-2 (COVID-19) is the most recently discovered. It has created a severe worldwide outbreak beginning in late 2019, leading to date to over 4 million cases globally. Viruses are genetically simple, yet highly diverse. However, the recent outbreaks of SARS-CoV and MERS-CoV, and the ongoing outbreak of SARS-CoV-2, indicate that there remains a long way to go to identify and develop specific therapeutic treatments. Only after gaining a better understanding of their pathogenic mechanisms can we minimize viral pandemics. This paper mainly focuses on SARS-CoV, MERS-CoV, and SARS-CoV-2. Here, recent studies are summarized and reviewed, with a focus on virus-host interactions, vaccine-based and drug-targeted therapies, and the development of new approaches for clinical diagnosis and treatment.


Subject(s)
Antiviral Agents/therapeutic use , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Host-Pathogen Interactions/drug effects , Pandemics , Pneumonia, Viral/drug therapy , Signal Transduction/drug effects , Betacoronavirus/genetics , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cytokines/antagonists & inhibitors , Cytokines/genetics , Cytokines/immunology , Gene Expression Regulation , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Molecular Targeted Therapy/methods , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/immunology , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , SARS Virus/drug effects , SARS Virus/genetics , SARS Virus/immunology , SARS Virus/pathogenicity , Severe Acute Respiratory Syndrome/drug therapy , Severe Acute Respiratory Syndrome/epidemiology , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Signal Transduction/genetics , Signal Transduction/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/virology
19.
Molecules ; 25(11)2020 Jun 11.
Article in English | MEDLINE | ID: covidwho-593255

ABSTRACT

Flavonoids are widely used as phytomedicines. Here, we report on flavonoid phytomedicines with potential for development into prophylactics or therapeutics against coronavirus disease 2019 (COVID-19). These flavonoid-based phytomedicines include: caflanone, Equivir, hesperetin, myricetin, and Linebacker. Our in silico studies show that these flavonoid-based molecules can bind with high affinity to the spike protein, helicase, and protease sites on the ACE2 receptor used by the severe acute respiratory syndrome coronavirus 2 to infect cells and cause COVID-19. Meanwhile, in vitro studies show potential of caflanone to inhibit virus entry factors including, ABL-2, cathepsin L, cytokines (IL-1ß, IL-6, IL-8, Mip-1α, TNF-α), and PI4Kiiiß as well as AXL-2, which facilitates mother-to-fetus transmission of coronavirus. The potential for the use of smart drug delivery technologies like nanoparticle drones loaded with these phytomedicines to overcome bioavailability limitations and improve therapeutic efficacy are discussed.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Coronavirus OC43, Human/drug effects , Flavonoids/pharmacology , Peptidyl-Dipeptidase A/chemistry , Pneumonia, Viral/drug therapy , Spike Glycoprotein, Coronavirus/chemistry , Animals , Antiviral Agents/chemistry , Betacoronavirus/chemistry , Betacoronavirus/growth & development , Binding Sites , Chloroquine/chemistry , Chloroquine/pharmacology , Coronavirus Infections/genetics , Coronavirus OC43, Human/chemistry , Coronavirus OC43, Human/growth & development , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Flavonoids/chemistry , Humans , Interleukins/antagonists & inhibitors , Interleukins/chemistry , Interleukins/genetics , Interleukins/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/virology , Lung/drug effects , Lung/pathology , Lung/virology , Mice , Molecular Docking Simulation , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Pandemics , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Phytotherapy/methods , Pneumonia, Viral/genetics , Primary Cell Culture , Protein Binding , Protein Interaction Domains and Motifs , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Thermodynamics , Virus Internalization/drug effects
20.
Vopr Virusol ; 65(2): 62-70, 2020.
Article in Russian | MEDLINE | ID: covidwho-593172

ABSTRACT

Since the early 2000s, three novel zooanthroponous coronaviruses (Betacoronavirus) have emerged. The first outbreak of infection (SARS) caused by SARS-CoV virus occurred in the fall of 2002 in China (Guangdong Province). A second outbreak (MERS) associated with the new MERS-CoV virus appeared in Saudi Arabia in autumn 2012. The third epidemic, which turned into a COVID-19 pandemic caused by SARS-CoV-2 virus, emerged in China (Hubei Province) in the autumn 2019. This review focuses on ecological and genetic aspects that lead to the emergence of new human zoanthroponous coronaviruses. The main mechanism of adaptation of zoonotic betacoronaviruses to humans is to changes in the receptor-binding domain of surface protein (S), as a result of which it gains the ability to bind human cellular receptors of epithelial cells in respiratory and gastrointestinal tract. This process is caused by the high genetic diversity and variability combined with frequent recombination, during virus circulation in their natural reservoir - bats (Microchiroptera, Chiroptera). Appearance of SARS-CoV, SARS-CoV-2 (subgenus Sarbecovirus), and MERS (subgenus Merbecovirus) viruses is a result of evolutionary events occurring in bat populations with further transfer of viruses to the human directly or through the intermediate vertebrate hosts, ecologically connected with bats. This review is based on the report at the meeting «Coronavirus - a global challenge to science¼ of the Scientific Council «Life Science¼ of the Russian Academy of Science: Lvov D.K., Alkhovsky S.V., Burtseva E.I. COVID-19 pandemic sources: origin, biology and genetics of coronaviruses of SARS-CoV, SARS-CoV-2, MERS-CoV (Conference hall of Presidium of RAS, 14 Leninsky Prospect, Moscow, Russia. April 16, 2020).


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Pandemics , Pneumonia, Viral/epidemiology , Spike Glycoprotein, Coronavirus/genetics , Zoonoses/epidemiology , Animals , Betacoronavirus/classification , Betacoronavirus/pathogenicity , Chiroptera/virology , Coronavirus Infections/transmission , Coronavirus Infections/virology , Ecology , Evolution, Molecular , Gene Expression , Mutation , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Phylogeny , Phylogeography , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Reassortant Viruses/classification , Reassortant Viruses/genetics , Reassortant Viruses/pathogenicity , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombination, Genetic , Spike Glycoprotein, Coronavirus/metabolism , Zoonoses/transmission , Zoonoses/virology
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