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1.
Bull Exp Biol Med ; 171(2): 230-233, 2021 May.
Article in English | MEDLINE | ID: covidwho-1525548

ABSTRACT

The presence of IgG and IgM antibodies in the venous blood of 76 patients with confirmed COVID-19 infection was determined by ELISA using Russian test systems. Different levels of IgM antibodies to N-protein and receptor binding domain of the Spike protein (RBD) were revealed. The dynamics of IgG antibodies to the whole virion antigen and recombinant antigens showed high values on weeks 4-5 of the disease. The level of IgG antibodies to Nprotein remained low throughout the observation period. The characteristic dynamics of IgG measured using test systems with sorbed whole virion or recombinant spike proteins reflects the duration of the disease.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/immunology , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , Immunoglobulin G/blood , Immunoglobulin M/blood , Spike Glycoprotein, Coronavirus/immunology , Antigens, Viral/genetics , COVID-19/blood , COVID-19/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Enzyme-Linked Immunosorbent Assay , Humans , Immunity, Humoral , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Time Factors , Virion/genetics , Virion/immunology
2.
Sci Rep ; 11(1): 21284, 2021 10 28.
Article in English | MEDLINE | ID: covidwho-1493215

ABSTRACT

We quantified the presence of SARS-CoV-2 RNA in the air of different hospital settings and the autopsy room of the largest medical centre in Sao Paulo, Brazil. Real-time reverse-transcription PCR was used to determine the presence of the envelope protein of SARS-CoV-2 and the nucleocapsid protein genes. The E-gene was detected in 5 out of 6 samples at the ICU-COVID-19 ward and in 5 out of 7 samples at the ward-COVID-19. Similarly, in the non-dedicated facilities, the E-gene was detected in 5 out of 6 samples collected in the ICU and 4 out of 7 samples in the ward. In the necropsy room, 6 out of 7 samples were positive for the E-gene. When both wards were compared, the non-COVID ward presented a significantly higher concentration of the E-gene than in the COVID-19 ward (p = 0.003). There was no significant difference in E-gene concentration between the ICU-COVID-19 and the ICU (p = 0.548). Likewise, there was no significant difference among E-gene concentrations found in the autopsy room versus the ICUs and wards (dedicated or not) (p = 0.245). Our results show the widespread presence of aerosol contamination in different hospital units.


Subject(s)
Air Microbiology , COVID-19/virology , Hospitals , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Aerosols , Autopsy , Brazil/epidemiology , COVID-19/epidemiology , COVID-19/transmission , COVID-19 Nucleic Acid Testing , Genome, Viral , Hospital Units , Humans , Intensive Care Units , Pandemics , Pathology Department, Hospital , RNA, Viral/analysis , RNA, Viral/genetics , Virion/genetics , Virion/isolation & purification
3.
Int J Mol Sci ; 22(16)2021 Aug 23.
Article in English | MEDLINE | ID: covidwho-1367852

ABSTRACT

The SARS-CoV-2 pseudovirus is a commonly used strategy that mimics certain biological functions of the authentic virus by relying on biological legitimacy at the molecular level. Despite the fact that spike (S), envelope (E), and membrane (M) proteins together wrap up the SARS-CoV-2 virion, most of the reported pseudotype viruses consist of only the S protein. Here, we report that the presence of E and M increased the virion infectivity by promoting the S protein priming. The S, E, and M (SEM)-coated pseudovirion is spherical, containing crown-like spikes on the surface. Both S and SEM pseudoviruses packaged the same amounts of viral RNA, but the SEM virus bound more efficiently to cells stably expressing the viral receptor human angiotensin-converting enzyme II (hACE2) and became more infectious. Using this SEM pseudovirus, we examined the infectivity and antigenic properties of the natural SARS-CoV-2 variants. We showed that some variants have higher infectivity than the original virus and that some render the neutralizing plasma with lower potency. These studies thus revealed possible mechanisms of the dissemination advantage of these variants. Hence, the SEM pseudovirion provides a useful tool to evaluate the viral infectivity and capability of convalescent sera in neutralizing specific SARS-CoV-2 S dominant variants.


Subject(s)
Antibodies, Viral/metabolism , COVID-19/immunology , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/pathogenicity , Viral Matrix Proteins/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/virology , Cell Line , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/immunology , Coronavirus Envelope Proteins/ultrastructure , Cricetinae , Humans , Microscopy, Electron, Transmission , Mutation , Neutralization Tests , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Viral Matrix Proteins/ultrastructure , Virion/genetics , Virion/immunology , Virion/metabolism , Virion/ultrastructure
4.
Front Immunol ; 12: 669103, 2021.
Article in English | MEDLINE | ID: covidwho-1348487

ABSTRACT

Targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), especially severe cases, are currently lacking. As macrophages have unique effector functions as a first-line defense against invading pathogens, we genetically armed human macrophages with chimeric antigen receptors (CARs) to reprogram their phagocytic activity against SARS-CoV-2. After investigation of CAR constructs with different intracellular receptor domains, we found that although cytosolic domains from MERTK (CARMERTK) did not trigger antigen-specific cellular phagocytosis or killing effects, unlike those from MEGF10, FcRγ and CD3ζ did, these CARs all mediated similar SARS-CoV-2 clearance in vitro. Notably, we showed that CARMERTK macrophages reduced the virion load without upregulation of proinflammatory cytokine expression. These results suggest that CARMERTK drives an 'immunologically silent' scavenger effect in macrophages and pave the way for further investigation of CARs for the treatment of individuals with COVID-19, particularly those with severe cases at a high risk of hyperinflammation.


Subject(s)
COVID-19/drug therapy , COVID-19/immunology , Immunotherapy, Adoptive , Macrophages/immunology , SARS-CoV-2/immunology , Virion/immunology , Animals , COVID-19/genetics , Chlorocebus aethiops , Humans , Phagocytosis , SARS-CoV-2/genetics , THP-1 Cells , Vero Cells , Virion/genetics
5.
Nat Commun ; 12(1): 4317, 2021 07 14.
Article in English | MEDLINE | ID: covidwho-1310803

ABSTRACT

The COVID-19 pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include lengthy multi-step processes for nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report an Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, and requires minimal supplies and infrastructure to perform. We validated the performance of OIL-TAS using contrived SARS-CoV-2 viral particle samples and clinical nasopharyngeal swab samples. OIL-TAS showed a 93% positive predictive agreement (n = 57) and 100% negative predictive agreement (n = 10) with clinical SARS-CoV-2 qPCR assays in testing clinical samples, highlighting its potential to be a faster, cheaper, and easier-to-deploy alternative for infectious disease testing.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/instrumentation , Equipment Design , Humans , Molecular Diagnostic Techniques , Nasopharynx/virology , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reproducibility of Results , SARS-CoV-2/genetics , Sensitivity and Specificity , Time Factors , Virion/genetics , Virion/isolation & purification
6.
Viruses ; 13(7)2021 06 30.
Article in English | MEDLINE | ID: covidwho-1287278

ABSTRACT

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.


Subject(s)
Host-Pathogen Interactions , Membrane Proteins/genetics , Viral Envelope Proteins , Virion/metabolism , Viruses/metabolism , HEK293 Cells , HIV-1/metabolism , Humans , Leukemia Virus, Murine/metabolism , Membrane Proteins/immunology , Retroviridae/classification , Retroviridae/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Virion/genetics , Virus Internalization , Viruses/chemistry , Viruses/classification , Viruses/genetics
7.
Nat Commun ; 12(1): 3917, 2021 06 24.
Article in English | MEDLINE | ID: covidwho-1281717

ABSTRACT

SARS-CoV-2 carries the largest single-stranded RNA genome and is the causal pathogen of the ongoing COVID-19 pandemic. How the SARS-CoV-2 RNA genome is folded in the virion remains unknown. To fill the knowledge gap and facilitate structure-based drug development, we develop a virion RNA in situ conformation sequencing technology, named vRIC-seq, for probing viral RNA genome structure unbiasedly. Using vRIC-seq data, we reconstruct the tertiary structure of the SARS-CoV-2 genome and reveal a surprisingly "unentangled globule" conformation. We uncover many long-range duplexes and higher-order junctions, both of which are under purifying selections and contribute to the sequential package of the SARS-CoV-2 genome. Unexpectedly, the D614G and the other two accompanying mutations may remodel duplexes into more stable forms. Lastly, the structure-guided design of potent small interfering RNAs can obliterate the SARS-CoV-2 in Vero cells. Overall, our work provides a framework for studying the genome structure, function, and dynamics of emerging deadly RNA viruses.


Subject(s)
COVID-19/pathology , RNA, Viral/chemistry , SARS-CoV-2/genetics , Sequence Analysis, RNA/methods , Virion/genetics , Animals , COVID-19/genetics , COVID-19/virology , Cells, Cultured , Chlorocebus aethiops , Genome, Viral , Humans , Nucleic Acid Conformation , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/pathogenicity , Virion/chemistry , Virion/metabolism
8.
Biotechnol Bioeng ; 118(7): 2660-2675, 2021 07.
Article in English | MEDLINE | ID: covidwho-1176262

ABSTRACT

The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Virus-like particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV-1 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV-1 VLPs depending on the cell-based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, mass-spectrometry-based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617 ± 17 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.


Subject(s)
Vaccines, Virus-Like Particle , Virion , gag Gene Products, Human Immunodeficiency Virus , COVID-19 Vaccines , HEK293 Cells , HIV-1/genetics , Humans , Virion/chemistry , Virion/genetics , gag Gene Products, Human Immunodeficiency Virus/analysis , gag Gene Products, Human Immunodeficiency Virus/chemistry , gag Gene Products, Human Immunodeficiency Virus/genetics
9.
Viruses ; 13(4)2021 03 26.
Article in English | MEDLINE | ID: covidwho-1154536

ABSTRACT

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting the destructive effects of inactivation. We observed that 15 min of incubation at 65 °C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 µJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody detection assays. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the quality of resulting viral proteins and virions were differentially impacted depending on inactivation method and time. Here, we outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.


Subject(s)
COVID-19/virology , Disinfection/methods , SARS-CoV-2/radiation effects , Virion/radiation effects , Virus Inactivation/radiation effects , Disinfection/instrumentation , Hot Temperature , Humans , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Ultraviolet Rays , Viral Proteins/genetics , Viral Proteins/metabolism , Virion/chemistry , Virion/genetics , Virion/physiology
10.
Virus Res ; 295: 198305, 2021 04 02.
Article in English | MEDLINE | ID: covidwho-1065651

ABSTRACT

In this study, we showed that a codon optimized version of the spike (S) protein of SARS-CoV-2 can migrate to the cell membrane. However, efficient production of Moloney murine leukemia (MLV) infectious viral particles was only achieved with stable expression of a shorter S version in C-terminal (ΔS) in MLV Gag-pol expressing cells. As compared to transient transfections, this platform generated viruses with a 1000-fold higher titer. ΔS was 15-times more efficiently incorporated into VLPs as compared to S, and that was not due to steric interference between the cytoplasmic tail and the MLV capsid, as similar differences were also observed with extracellular vesicles. The amount of ΔS incorporated into VLPs released from producer cells was high and estimated at 1.25 µg/mL S2 equivalent (S is comprised of S1 and S2). The resulting VLPs could potentially be used alone or as a boost of other immunization strategies for COVID-19.


Subject(s)
COVID-19 Vaccines/immunology , Spike Glycoprotein, Coronavirus/biosynthesis , Virion/genetics , Cell Line , Humans , Moloney murine leukemia virus/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Synthetic/immunology , Virion/immunology
11.
J Biol Chem ; 296: 100111, 2021.
Article in English | MEDLINE | ID: covidwho-1066049

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.


Subject(s)
Coronavirus Envelope Proteins/genetics , Nucleocapsid Proteins/genetics , SARS-CoV-2/growth & development , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/genetics , Virion/growth & development , Virus Assembly/physiology , Animals , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Envelope Proteins/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Gene Expression , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Golgi Apparatus/virology , HEK293 Cells , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Hepatocytes/virology , Host-Pathogen Interactions/genetics , Humans , Nucleocapsid Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Matrix Proteins/metabolism , Virion/genetics , Virion/metabolism , Virus Internalization , Virus Release/physiology
12.
Viruses ; 12(12)2020 12 21.
Article in English | MEDLINE | ID: covidwho-1000348

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3' untranslated region (UTR) of this ß-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3' UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3' UTRs in the infectious virions and the minigene-transfected cells are almost identical.


Subject(s)
3' Untranslated Regions/genetics , COVID-19/virology , Nucleic Acid Conformation , Pandemics , RNA, Viral/genetics , SARS-CoV-2/genetics , Animals , Base Sequence , Cell Line , Conserved Sequence , Cricetinae , High-Throughput Nucleotide Sequencing , Humans , Mesocricetus , Models, Molecular , Plasmids , Point Mutation , Reverse Genetics/methods , SARS-CoV-2/physiology , Sequence Alignment , Sequence Homology, Nucleic Acid , Sulfuric Acid Esters , Transcription, Genetic , Virion/genetics , Virion/physiology
13.
Anal Chem ; 93(2): 715-721, 2021 01 19.
Article in English | MEDLINE | ID: covidwho-962725

ABSTRACT

The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. To meet the urgent and massive demand for the screening and diagnosis of infected individuals, many in vitro diagnostic assays using nucleic acid tests (NATs) have been urgently authorized by regulators worldwide. A reference standard with a well-characterized concentration or titer is of the utmost importance for the study of limit of detection (LoD), which is a crucial feature for a diagnostic assay. Although several reference standards of plasmids or synthetic RNA have already been announced, a reference standard for inactivated virus particles with an accurate concentration is still needed to evaluate the complete procedure. Here, we performed a collaborative study to estimate the NAT-detectable units as a viral genomic equivalent quantity (GEQ) of an inactivated whole-virus SARS-CoV-2 reference standard candidate using digital PCR (dPCR) on multiple commercialized platforms. The median of the quantification results (4.6 × 105 ± 6.5 × 104 GEQ/mL) was treated as the consensus true value of GEQ of virus particles in the reference standard. This reference standard was then used to challenge the LoDs of six officially approved diagnostic assays. Our study demonstrates that an inactivated whole virus quantified by dPCR can serve as a reference standard and provides a unified solution for assay development, quality control, and regulatory surveillance.


Subject(s)
COVID-19/diagnosis , Polymerase Chain Reaction/methods , RNA, Viral/analysis , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/methods , COVID-19 Nucleic Acid Testing/standards , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus Nucleocapsid Proteins/standards , Humans , Limit of Detection , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphoproteins/standards , Polymerase Chain Reaction/standards , Polyproteins/genetics , Polyproteins/metabolism , Polyproteins/standards , Quality Control , RNA, Viral/metabolism , RNA, Viral/standards , Reagent Kits, Diagnostic , Reference Standards , SARS-CoV-2/isolation & purification , Viral Proteins/genetics , Viral Proteins/metabolism , Viral Proteins/standards , Virion/genetics , Virion/isolation & purification
14.
Nat Commun ; 11(1): 5920, 2020 11 20.
Article in English | MEDLINE | ID: covidwho-939437

ABSTRACT

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Nucleic Acid Amplification Techniques/methods , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Humans , RNA/metabolism , RNA, Viral/genetics , RNA, Viral/isolation & purification , RNA-Directed DNA Polymerase/metabolism , Real-Time Polymerase Chain Reaction , Recombinases/metabolism , SARS-CoV-2 , Saliva/virology , Virion/genetics
15.
J Biol Chem ; 296: 100103, 2021.
Article in English | MEDLINE | ID: covidwho-936211

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China, and expeditiously spread across the globe causing a global pandemic. Research on SARS-CoV-2, as well as the closely related SARS-CoV-1 and MERS coronaviruses, is restricted to BSL-3 facilities. Such BSL-3 classification makes SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the United States; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus' genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form virus-like particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions. These systems should be useful to those looking to circumvent BSL-3 work with SARS-CoV-2 yet study the mechanisms by which SARS-CoV-2 enters and exits human cells.


Subject(s)
Coronavirus Envelope Proteins/genetics , Nucleocapsid Proteins/genetics , SARS-CoV-2/growth & development , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/genetics , Virion/growth & development , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Containment of Biohazards/classification , Coronavirus Envelope Proteins/metabolism , Gene Expression , Genes, Reporter , Government Regulation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Electron , Nucleocapsid Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/metabolism , Viral Matrix Proteins/metabolism , Virion/genetics , Virion/metabolism , Virion/ultrastructure , Virus Assembly/physiology , Virus Internalization , Virus Release/physiology
16.
Cell Host Microbe ; 28(6): 880-891.e8, 2020 12 09.
Article in English | MEDLINE | ID: covidwho-921850

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) mediates viral entry into cells and is critical for vaccine development against coronavirus disease 2019 (COVID-19). Structural studies have revealed distinct conformations of S, but real-time information that connects these structures is lacking. Here we apply single-molecule fluorescence (Förster) resonance energy transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to human receptor angiotensin-converting enzyme 2 (hACE2), S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences observed upon exposure to convalescent plasma or antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to the receptor-binding domain (RBD) or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.


Subject(s)
COVID-19/genetics , Protein Conformation , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/ultrastructure , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , Epitopes/immunology , Humans , Membrane Glycoproteins/genetics , Membrane Glycoproteins/ultrastructure , Protein Binding/immunology , Receptors, Virus/genetics , Receptors, Virus/immunology , Receptors, Virus/ultrastructure , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Virion/genetics , Virion/ultrastructure , Virus Internalization
17.
Proc Natl Acad Sci U S A ; 117(41): 25759-25770, 2020 10 13.
Article in English | MEDLINE | ID: covidwho-807358

ABSTRACT

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.


Subject(s)
Coronavirus/physiology , Hemagglutinins, Viral/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Fusion Proteins/genetics , Virion/metabolism , Animals , Biological Evolution , Cell Line , Coronavirus/genetics , Coronavirus/metabolism , Coronavirus Infections/virology , Coronavirus OC43, Human/genetics , Coronavirus OC43, Human/metabolism , Coronavirus OC43, Human/physiology , Coronavirus, Bovine/genetics , Coronavirus, Bovine/metabolism , Coronavirus, Bovine/physiology , Hemagglutinins, Viral/chemistry , Hemagglutinins, Viral/metabolism , Humans , Lectins/genetics , Lectins/metabolism , Mice , Mutation , Protein Binding , Protein Domains , Receptors, Virus/metabolism , Selection, Genetic , Sialic Acids/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/metabolism , Virion/genetics , Virus Attachment , Virus Release
18.
J Virol ; 94(21)2020 10 14.
Article in English | MEDLINE | ID: covidwho-709870

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals.IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus/physiology , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/genetics , Betacoronavirus/immunology , Betacoronavirus/metabolism , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/metabolism , HEK293 Cells , HIV-1/genetics , HIV-1/metabolism , Humans , Leukemia Virus, Murine , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Pneumonia, Viral/metabolism , SARS-CoV-2 , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/metabolism , Virion/genetics , Virion/immunology , Virion/metabolism , Virus Internalization
19.
Infect Genet Evol ; 84: 104451, 2020 10.
Article in English | MEDLINE | ID: covidwho-630854

ABSTRACT

WHO has declared the outbreak of COVID-19 as a public health emergency of international concern. The ever-growing new cases have called for an urgent emergency for specific anti-COVID-19 drugs. Three structural proteins (Membrane, Envelope and Nucleocapsid protein) play an essential role in the assembly and formation of the infectious virion particles. Thus, the present study was designed to identify potential drug candidates from the unique collection of 548 anti-viral compounds (natural and synthetic anti-viral), which target SARS-CoV-2 structural proteins. High-end molecular docking analysis was performed to characterize the binding affinity of the selected drugs-the ligand, with the SARS-CoV-2 structural proteins, while high-level Simulation studies analyzed the stability of drug-protein interactions. The present study identified rutin, a bioflavonoid and the antibiotic, doxycycline, as the most potent inhibitor of SARS-CoV-2 envelope protein. Caffeic acid and ferulic acid were found to inhibit SARS-CoV-2 membrane protein while the anti-viral agent's simeprevir and grazoprevir showed a high binding affinity for nucleocapsid protein. All these compounds not only showed excellent pharmacokinetic properties, absorption, metabolism, minimal toxicity and bioavailability but were also remain stabilized at the active site of proteins during the MD simulation. Thus, the identified lead compounds may act as potential molecules for the development of effective drugs against SARS-CoV-2 by inhibiting the envelope formation, virion assembly and viral pathogenesis.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Nucleocapsid Proteins/chemistry , Viral Envelope Proteins/chemistry , Viral Matrix Proteins/chemistry , Virion/drug effects , Amides , Amino Acid Sequence , Antiviral Agents/chemistry , Betacoronavirus/genetics , Betacoronavirus/metabolism , Binding Sites , COVID-19 , Caffeic Acids/chemistry , Caffeic Acids/pharmacology , Carbamates , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Coumaric Acids/chemistry , Coumaric Acids/pharmacology , Cyclopropanes , Doxycycline/chemistry , Doxycycline/pharmacology , Gene Expression , Humans , Kinetics , Molecular Docking Simulation , Molecular Dynamics Simulation , Nucleocapsid Proteins/antagonists & inhibitors , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Quinoxalines/chemistry , Quinoxalines/pharmacology , Rutin/chemistry , Rutin/pharmacology , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Amino Acid , Simeprevir/chemistry , Simeprevir/pharmacology , Sulfonamides , Thermodynamics , Viral Envelope Proteins/antagonists & inhibitors , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Matrix Proteins/antagonists & inhibitors , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Virion/genetics
20.
J Pak Med Assoc ; 70(Suppl 3)(5): S38-S43, 2020 May.
Article in English | MEDLINE | ID: covidwho-609354

ABSTRACT

COVID-19 has taken the world by storm in the ongoing pandemic. The virus responsible for COVID-19 disease is 'severe acute respiratory syndrome coronavirus-2' SARS-CoV-2, an enveloped RNA beta-coronavirus from the family Coronaviridae. There have been similar beta-coronavirus disease outbreaks previously: Severe acute respiratory syndrome (SARS - 2002) and Middle East respiratory syndrome (MERS - 2012) epidemics. SARS-CoV-2 origins have been traced to bat reservoirs. A virus with a high capacity for mutation, SARS-CoV-2 poses unique challenges both in the current form of disease control and management, while also leaving the door open for future novel diseases and pandemics. An understanding of the virion structure and genomic organisation will help us in understanding their origins and likely course of future evolution. Moreover, novel cost-effective methodologies for genetic surveillance may help in mitigating the emergence of these viral infections in future. In this manuscript, the authors have detailed the unique aspects of the SARS-CoV-2 virus genome and its clinical implications.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Pneumonia, Viral/virology , Animals , Asia , COVID-19 , Chiroptera/virology , Coronavirus Infections/transmission , Genome, Viral/genetics , High-Throughput Nucleotide Sequencing , Humans , Mutation/genetics , Mutation Rate , Pandemics , Pneumonia, Viral/transmission , RNA, Viral/genetics , SARS-CoV-2 , Virion/genetics
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