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2.
Nucleic Acids Res ; 50(14): 8080-8092, 2022 Aug 12.
Article in English | MEDLINE | ID: covidwho-1948397

ABSTRACT

Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represses translation, and induces mRNA degradation, while the host elicits an anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-mediated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5'UTR of SARS-CoV-2 drives cap-independent translation and promotes expression of NSP1 in an eIF4E-independent and Torin1-resistant manner. Upon expression, NSP1 further enhances cap-independent translation. However, the sub-genomic 5'UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin1. We conclude that the combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5'UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.


Subject(s)
COVID-19 , SARS-CoV-2 , 5' Untranslated Regions , Base Sequence , COVID-19/genetics , Eukaryotic Initiation Factor-4E/genetics , Humans , Protein Biosynthesis , RNA Caps/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism
3.
Int J Infect Dis ; 118: 211-213, 2022 May.
Article in English | MEDLINE | ID: covidwho-1838859

ABSTRACT

SARS-CoV-2 Omicron strain emergence raised concerns that its enhanced infectivity is partly due to altered spread/contamination modalities. We therefore sampled high-contact surfaces and air in close proximity to patients who were verified as infected with the Omicron strain, using identical protocols applied to sample patients positive to the original or Alpha strains. Cumulatively, for all 3 strains, viral RNA was detected in 90 of 168 surfaces and 6 of 49 air samples (mean cycle threshold [Ct]=35.2±2.5). No infective virus was identified. No significant differences in prevalence were found between strains.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , RNA, Viral/genetics , Specimen Handling
4.
Anal Chem ; 94(10): 4380-4389, 2022 03 15.
Article in English | MEDLINE | ID: covidwho-1713090

ABSTRACT

A multi-component microarray, applying a novel analysis algorithm, was developed for quantitative evaluation of the SARS-CoV-2 vaccines' immunogenicity. The array enables simultaneous quantitation of IgG, IgM, and IgA, specific to the SARS-CoV-2 spike, receptor binding domain, and nucleocapsid proteins. The developed methodology is based on calculating an apparent immunoglobulin signal from the linear range of the fluorescent read-outs generated by scanning the microarray slides at different exposure times. A dedicated algorithm, employing a rigorous set of embedded conditions, then generates a normalized signal for each of the unique assays. Qualification of the multi-component array performance (evaluating linearity, extended dynamic-range, specificity, precision, and accuracy) was carried out with an in-house COVID-19, qRT-PCR positive serum, as well as pre-pandemic commercial negative sera. Results were compared to the WHO international standard for anti-SARS-CoV-2 immunoglobulins. Specific IgG, IgM, and IgA signals obtained by this array enabled successful discrimination between SARS-CoV-2 q-RT-PCR positive (seroconverted SARS-CoV-2 patients) and negative (naïve) samples. This array is currently used for evaluation of the humoral response to BriLife, the VSV-based Israeli vaccine during phase I/II clinical trials.


Subject(s)
COVID-19 Vaccines , COVID-19 , Antibodies, Viral , COVID-19/diagnosis , COVID-19/prevention & control , Humans , Immunoglobulin G , Immunoglobulin M , SARS-CoV-2/genetics , Sensitivity and Specificity
5.
Arch Virol ; 167(4): 1041-1049, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1709039

ABSTRACT

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis in 2019. Currently, the main method for identification of SARS-CoV-2 is a reverse transcription (RT)-PCR assay designed to detect viral RNA in oropharyngeal (OP) or nasopharyngeal (NP) samples. While the PCR assay is considered highly specific and sensitive, this method cannot determine the infectivity of the sample, which may assist in evaluation of virus transmissibility from patients and breaking transmission chains. Thus, cell-culture-based approaches such as cytopathic effect (CPE) assays are routinely employed for the identification of infectious viruses in NP/OP samples. Despite their high sensitivity, CPE assays take several days and require additional diagnostic tests in order to verify the identity of the pathogen. We have therefore developed a rapid immunofluorescence assay (IFA) for the specific detection of SARS-CoV-2 in NP/OP samples following cell culture infection. Initially, IFA was carried out on Vero E6 cultures infected with SARS-CoV-2 at defined concentrations, and infection was monitored at different time points. This test was able to yield positive signals in cultures infected with 10 pfu/ml at 12 hours postinfection (PI). Increasing the incubation time to 24 hours reduced the detectable infective dose to 1 pfu/ml. These IFA signals occur before the development of CPE. When compared to the CPE test, IFA has the advantages of specificity, rapid detection, and sensitivity, as demonstrated in this work.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Fluorescent Antibody Technique , Humans , Nasopharynx , Pandemics , RNA, Viral/genetics , Sensitivity and Specificity
6.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-320971

ABSTRACT

This study describes the development of a novel assay for SARS-CoV-2 identification using LC-MS/MS analysis. A multi-step procedure for the rational down-selection of a set of markers has leaded to the discovery of six SARS-CoV-2 specific and sensitive markers, enabling the reliable identification of the virus. A rapid and simple assay was developed, successfully applied to clinical nasopharyngeal samples. The assay may potentially serve as a complementary approach for SARS-CoV-2 identification.

7.
Arch Toxicol ; 96(3): 859-875, 2022 03.
Article in English | MEDLINE | ID: covidwho-1634984

ABSTRACT

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 108 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2.


Subject(s)
COVID-19 Vaccines/toxicity , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Vaccines/immunology , Cricetinae , Female , Membrane Glycoproteins/genetics , Mesocricetus , Mice , Mice, Inbred C57BL , Rabbits , Swine , Vaccination , Vaccines, Synthetic/toxicity , Viral Envelope Proteins/genetics
8.
Anal Bioanal Chem ; 414(5): 1949-1962, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1607761

ABSTRACT

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Chromatography, Liquid/methods , Immunomagnetic Separation/methods , SARS-CoV-2/genetics , Tandem Mass Spectrometry/methods , Amino Acid Sequence , Antibodies, Viral/chemistry , Biomarkers/chemistry , COVID-19/immunology , COVID-19/virology , COVID-19 Testing/instrumentation , COVID-19 Testing/standards , Chromatography, Liquid/instrumentation , Chromatography, Liquid/standards , Humans , Immunomagnetic Separation/instrumentation , Immunomagnetic Separation/standards , Nasopharynx/virology , Peptides/chemistry , Peptides/immunology , SARS-CoV-2/immunology , Sensitivity and Specificity , Tandem Mass Spectrometry/instrumentation , Tandem Mass Spectrometry/standards
9.
Microbiol Spectr ; 9(2): e0087021, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1455682

ABSTRACT

The first case of SARS-CoV-2 was discovered in Israel in late February 2020. Three major outbreaks followed, resulting in over 800,000 cases and over 6,000 deaths by April 2021. Our aim was characterization of a serological snapshot of Israeli patients and healthy adults in the early months of the COVID-19 pandemic. Sera from 55 symptomatic COVID-19 patients and 146 healthy subjects (early-pandemic, reverse transcription-quantitative PCR [qRT-PCR]-negative), collected in Israel between March and April 2020, were screened for SARS-CoV-2-specific IgG, IgM, and IgA antibodies, using a 6-plex antigen microarray presenting the whole inactivated virus and five viral antigens: a stabilized version of the spike ectodomain (S2P), spike subunit 1 (S1), receptor-binding-domain (RBD), N-terminal-domain (NTD), and nucleocapsid (NC). COVID-19 patients, 4 to 40 days post symptom onset, presented specific IgG to all of the viral antigens (6/6) in 54 of the 55 samples (98% sensitivity). Specific IgM and IgA antibodies for all six antigens were detected in only 10% (5/55) and 4% (2/55) of the patients, respectively, suggesting that specific IgG is a superior serological marker for COVID-19. None of the qRT-PCR-negative sera reacted with all six viral antigens (100% specificity), and 48% (70/146) were negative throughout the panel. Our findings confirm a low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population prior to the COVID-19 outbreak. We further suggest that the presence of low-level cross-reacting antibodies in naive individuals calls for a combined, multiantigen analysis for accurate discrimination between naive and exposed individuals. IMPORTANCE A 6-plex protein array presenting the whole inactivated virus and five nucleocapsid and spike-derived SARS-CoV-2 antigens was used to generate a serological snapshot of SARS-CoV-2 seroprevalence and seroconversion in Israel in the early months of the pandemic. Our findings confirm a very low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population. We further propose that the presence of low-level nonspecific antibodies in naive individuals calls for a combined, multiantigen analysis for accurate discrimination between naive and exposed individuals enabling accurate determination of seroconversion. The developed assay is currently applied to evaluate immune responses to the Israeli vaccine during human phase I/II trials.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/epidemiology , Microarray Analysis/methods , SARS-CoV-2/immunology , Adult , Aged , Antigens, Viral/immunology , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Immunoassay/methods , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Israel/epidemiology , Male , Middle Aged , Phosphoproteins/immunology , Sensitivity and Specificity , Seroepidemiologic Studies , Spike Glycoprotein, Coronavirus/immunology , Young Adult
10.
Nat Biotechnol ; 39(12): 1556-1562, 2021 12.
Article in English | MEDLINE | ID: covidwho-1287813

ABSTRACT

Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24 h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.


Subject(s)
COVID-19 Testing/methods , COVID-19 , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , COVID-19/diagnosis , Humans
11.
JCI Insight ; 6(12)2021 06 22.
Article in English | MEDLINE | ID: covidwho-1223641

ABSTRACT

Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary morbidities rendered SARS-CoV-2-refractive CD-1 mice susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low doses of the acute lung injury stimulants bleomycin or ricin caused severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates greater than 50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart, and serum of low-dose ricin-pretreated mice compared with non-pretreated mice. Furthermore, lung extracts prepared 2-3 days after viral infection contained subgenomic mRNA and virus particles capable of replication only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against the SARS-CoV-2 receptor binding domain (RBD). Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of coronavirus disease 2019 (COVID-19) pathologies in patients with preexisting morbidities.


Subject(s)
Bleomycin/toxicity , COVID-19/pathology , Lung Injury , Ricin/toxicity , Animals , Chlorocebus aethiops , Comorbidity , Disease Models, Animal , Female , Lung Injury/chemically induced , Lung Injury/virology , Mice , Vero Cells , Virus Attachment , Virus Internalization/drug effects
12.
Anal Bioanal Chem ; 413(13): 3501-3510, 2021 May.
Article in English | MEDLINE | ID: covidwho-1151992

ABSTRACT

Public health experts emphasize the need for quick, point-of-care SARS-CoV-2 detection as an effective strategy for controlling virus spread. To this end, many "antigen" detection devices were developed and commercialized. These devices are mostly based on detecting SARS-CoV-2's nucleocapsid protein. Recently, alerts issued by both the FDA and the CDC raised concerns regarding the devices' tendency to exhibit false positive results. In this work, we developed a novel alternative spike-based antigen assay, comprising four high-affinity, specific monoclonal antibodies, directed against different epitopes on the spike's S1 subunit. The assay's performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an in-house nucleocapsid-based assay, composed of novel antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR positive and negative nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct < 25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct < 25) while less specific (87% specificity). Despite being outperformed by qRT-PCR, we suggest that there is room for such tests in the clinical setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results. Schematic representation of sample collection and analysis. The figure was created using BioRender.com.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/analysis , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/analysis , Enzyme-Linked Immunosorbent Assay/methods , Humans , Phosphoproteins/analysis , Sensitivity and Specificity , Specimen Handling
13.
ACS Omega ; 6(5): 3525-3534, 2021 Feb 09.
Article in English | MEDLINE | ID: covidwho-1083828

ABSTRACT

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis. Rapid and reliable clinical diagnosis is essential for effectively controlling transmission. The gold standard assay for SARS-CoV-2 identification is the highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR); however, this assay depends on specialized reagents and may suffer from false results. Thus, additional assays based on different approaches could be beneficial. Here, we present a novel method for SARS-CoV-2 identification based on mass spectrometry. The approach we implemented combines a multistep procedure for the rational down-selection of a set of reliable markers out of all optional in silico derived tryptic peptides in viral proteins, followed by monitoring of peptides derived from tryptic digests of purified proteins, cell-cultured SARS-CoV-2, and nasopharyngeal (NP) swab matrix spiked with the virus. The marker selection was based on specificity to SARS-CoV-2 and on analytical parameters including sensitivity, linearity, and reproducibility. The final assay is based on six unique and specific peptide markers for SARS-CoV-2 identification. The simple and rapid (2.5 h) protocol we developed consists of virus heat inactivation and denaturation, tryptic digestion, and identification of the selected markers by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The developed assay enabled the identification of 104 PFU/mL SARS-CoV-2 spiked into buffer. Finally, the assay was successfully applied to 16 clinical samples diagnosed by RT-qPCR, achieving 94% concordance with the current gold standard assay. To conclude, the novel MS-based assay described here is specific, rapid, simple, and is believed to provide a complementary assay to the RT-qPCR method.

14.
Nat Commun ; 11(1): 6402, 2020 12 16.
Article in English | MEDLINE | ID: covidwho-983658

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 imposes an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we show the development of a replication competent recombinant VSV-∆G-spike vaccine, in which the glycoprotein of VSV is replaced by the spike protein of SARS-CoV-2. In-vitro characterization of this vaccine indicates the expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in-vivo model for COVID-19 is implemented. We show that a single-dose vaccination results in a rapid and potent induction of SARS-CoV-2 neutralizing antibodies. Importantly, vaccination protects hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss, and  alleviation of the extensive tissue damage and viral loads in lungs and nasal turbinates. Taken together, we suggest the recombinant VSV-∆G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Synthetic/immunology , Vesicular stomatitis Indiana virus/immunology , Animals , Antibodies, Viral/immunology , Antigens, Viral/immunology , Body Weight , COVID-19/virology , Cell Line , Cricetinae , Disease Models, Animal , Dose-Response Relationship, Immunologic , Genome, Viral , Lung/pathology , Lung/virology , Mice, Inbred C57BL , Mutation/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/ultrastructure , Vaccination , Viral Load
15.
Clin Microbiol Infect ; 26(12): 1658-1662, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-753742

ABSTRACT

OBJECTIVES: Environmental surfaces have been suggested as likely contributors in the transmission of COVID-19. This study assessed the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contaminating surfaces and objects in two hospital isolation units and a quarantine hotel. METHODS: SARS-CoV-2 virus stability and infectivity on non-porous surfaces was tested under controlled laboratory conditions. Surface and air sampling were conducted at two COVID-19 isolation units and in a quarantine hotel. Viral RNA was detected by RT-PCR and infectivity was assessed by VERO E6 CPE test. RESULTS: In laboratory-controlled conditions, SARS-CoV-2 gradually lost its infectivity completely by day 4 at ambient temperature, and the decay rate of viral viability on surfaces directly correlated with increase in temperature. Viral RNA was detected in 29/55 surface samples (52.7%) and 16/42 surface samples (38%) from the surroundings of symptomatic COVID-19 patients in isolation units of two hospitals and in a quarantine hotel for asymptomatic and very mild COVID-19 patients. None of the surface and air samples from the three sites (0/97) were found to contain infectious titres of SARS-Cov-2 on tissue culture assay. CONCLUSIONS: Despite prolonged viability of SARS-CoV-2 under laboratory-controlled conditions, uncultivable viral contamination of inanimate surfaces might suggest low feasibility for indirect fomite transmission.


Subject(s)
COVID-19/transmission , Fomites/virology , Hospitals, Isolation/statistics & numerical data , Housing/statistics & numerical data , Microbial Viability , SARS-CoV-2/isolation & purification , COVID-19/virology , Humans , RNA, Viral/isolation & purification , Surface Properties , Temperature
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