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1.
Front Immunol ; 13: 831849, 2022.
Article in English | MEDLINE | ID: covidwho-1785339

ABSTRACT

COVID-19 is accompanied by a myriad of both transient and long-lasting autoimmune responses. Dermatan sulfate (DS), a glycosaminoglycan crucial for wound healing, has unique affinity for autoantigens (autoAgs) from apoptotic cells. DS-autoAg complexes are capable of stimulating autoreactive B cells and autoantibody production. We used DS-affinity proteomics to define the autoantigen-ome of lung fibroblasts and bioinformatics analyses to study the relationship between autoantigenic proteins and COVID-induced alterations. Using DS-affinity, we identified an autoantigen-ome of 408 proteins from human HFL1 cells, at least 231 of which are known autoAgs. Comparing with available COVID data, 352 proteins of the autoantigen-ome have thus far been found to be altered at protein or RNA levels in SARS-CoV-2 infection, 210 of which are known autoAgs. The COVID-altered proteins are significantly associated with RNA metabolism, translation, vesicles and vesicle transport, cell death, supramolecular fibrils, cytoskeleton, extracellular matrix, and interleukin signaling. They offer clues to neurological problems, fibrosis, smooth muscle dysfunction, and thrombosis. In particular, 150 altered proteins are related to the nervous system, including axon, myelin sheath, neuron projection, neuronal cell body, and olfactory bulb. An association with the melanosome is also identified. The findings from our study illustrate a connection between COVID infection and autoimmunity. The vast number of COVID-altered proteins with high intrinsic propensity to become autoAgs offers an explanation for the diverse autoimmune complications in COVID patients. The variety of autoAgs related to mRNA metabolism, translation, and vesicles suggests a need for long-term monitoring of autoimmunity in COVID. The COVID autoantigen atlas we are establishing provides a detailed molecular map for further investigation of autoimmune sequelae of the pandemic, such as "long COVID" syndrome. Summary Sentence: An autoantigen-ome by dermatan sulfate affinity from human lung HFL1 cells may explain neurological and autoimmune manifestations of COVID-19.


Subject(s)
COVID-19 , Autoantigens , Dermatan Sulfate , Humans , Lung/metabolism , Proteins , RNA , SARS-CoV-2
2.
PLoS One ; 17(4): e0266419, 2022.
Article in English | MEDLINE | ID: covidwho-1779768

ABSTRACT

The pandemic caused by the SARS-CoV-2 virus (COVID-19) is still a major health issue. The COVID-19 pandemic has forced the university teaching to consider in high priority the switch from in-presence teaching to remote teaching, including laboratory teaching. While excellent virtual-laboratory teaching has been proposed and turned out to be very useful, the need of a real-laboratory in-presence teaching is still a major need. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. SARS-CoV-2 diagnostics, and (b) on technologies that are playing a central role in applied biochemistry and molecular biology, i.e. PCR and RT-PCR. The aims of the practical laboratory were to determine: (a) the possibility to identify SARS-CoV-2 sequences starting from a recombinant plasmid and (b) the possibility to discriminate cells with respect to the expression of SARS-CoV-2 Spike protein. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). We suggest that this laboratory practical exercises should be considered for face-to-face teaching especially if the emergency related to the COVID-19 pandemic is maintained. The teaching protocol here described might be considered in order to perform fast but meaningful in-presence teaching, making feasible the division of crowded classes in low-number cohorts of students, allowing the maintenance of the required social distance.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/epidemiology , Humans , Pandemics , RNA , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
3.
Nat Commun ; 13(1): 1722, 2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1773975

ABSTRACT

The rapidly growing popularity of RNA structure probing methods is leading to increasingly large amounts of available RNA structure information. This demands the development of efficient tools for the identification of RNAs sharing regions of structural similarity by direct comparison of their reactivity profiles, hence enabling the discovery of conserved structural features. We here introduce SHAPEwarp, a largely sequence-agnostic SHAPE-guided algorithm for the identification of structurally-similar regions in RNA molecules. Analysis of Dengue, Zika and coronavirus genomes recapitulates known regulatory RNA structures and identifies novel highly-conserved structural elements. This work represents a preliminary step towards the model-free search and identification of shared and conserved RNA structural features within transcriptomes.


Subject(s)
Zika Virus Infection , Zika Virus , Algorithms , Humans , Nucleic Acid Conformation , RNA/chemistry , RNA/genetics , RNA, Guide , Sequence Analysis, RNA/methods , Zika Virus/genetics
4.
PLoS Genet ; 18(3): e1010130, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1770640

ABSTRACT

SARS-CoV-2 is a positive-sense, single-stranded RNA virus responsible for the COVID-19 pandemic. It remains unclear whether and to what extent the virus in human host cells undergoes RNA editing, a major RNA modification mechanism. Here we perform a robust bioinformatic analysis of metatranscriptomic data from multiple bronchoalveolar lavage fluid samples of COVID-19 patients, revealing an appreciable number of A-to-I RNA editing candidate sites in SARS-CoV-2. We confirm the enrichment of A-to-I RNA editing signals at these candidate sites through evaluating four characteristics specific to RNA editing: the inferred RNA editing sites exhibit (i) stronger ADAR1 binding affinity predicted by a deep-learning model built from ADAR1 CLIP-seq data, (ii) decreased editing levels in ADAR1-inhibited human lung cells, (iii) local clustering patterns, and (iv) higher RNA secondary structure propensity. Our results have critical implications in understanding the evolution of SARS-CoV-2 as well as in COVID-19 research, such as phylogenetic analysis and vaccine development.


Subject(s)
COVID-19 , SARS-CoV-2 , Adenosine Deaminase/metabolism , COVID-19/genetics , Humans , Nucleotides/metabolism , Pandemics , Phylogeny , RNA/metabolism , RNA Editing/genetics , SARS-CoV-2/genetics
5.
Nat Immunol ; 23(4): 532-542, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1764192

ABSTRACT

The use of lipid-formulated RNA vaccines for cancer or COVID-19 is associated with dose-limiting systemic inflammatory responses in humans that were not predicted from preclinical studies. Here, we show that the 'interleukin 1 (IL-1)-interleukin 1 receptor antagonist (IL-1ra)' axis regulates vaccine-mediated systemic inflammation in a host-specific manner. In human immune cells, RNA vaccines induce production of IL-1 cytokines, predominantly IL-1ß, which is dependent on both the RNA and lipid formulation. IL-1 in turn triggers the induction of the broad spectrum of pro-inflammatory cytokines (including IL-6). Unlike humans, murine leukocytes respond to RNA vaccines by upregulating anti-inflammatory IL-1ra relative to IL-1 (predominantly IL-1α), protecting mice from cytokine-mediated toxicities at >1,000-fold higher vaccine doses. Thus, the IL-1 pathway plays a key role in triggering RNA vaccine-associated innate signaling, an effect that was unexpectedly amplified by certain lipids used in vaccine formulations incorporating N1-methyl-pseudouridine-modified RNA to reduce activation of Toll-like receptor signaling.


Subject(s)
COVID-19 , Interleukin 1 Receptor Antagonist Protein , Animals , Interleukin 1 Receptor Antagonist Protein/genetics , Lipids , Mice , RNA , Vaccines, Synthetic
6.
Toxicol Appl Pharmacol ; 442: 116003, 2022 May 01.
Article in English | MEDLINE | ID: covidwho-1763987

ABSTRACT

Molnupiravir is an orally active nucleoside analog antiviral drug that recently was approved by the U.S. FDA for emergency treatment of adult patients infected with the SARS-CoV-2 (COVID-19) virus and at risk for severe progression. The active form of the drug, N-hydroxycytidine (NHC) triphosphate competes for incorporation by RNA-dependent RNA-polymerase (RdRp) into the replicating viral genome resulting in mutations and arrest of the replicating virus. Historically, some nucleoside analog antiviral drugs have been found to lack specificity for the virus and also inhibit replication and/or expression of the mitochondrial genome. The objective of the present study was to test whether molnupiravir and/or NHC also target mitochondrial DNA polymerase gamma (PolG) or RNA polymerase (POLRMT) activity to inhibit the replication and/or expression of the mitochondrial genome leading to impaired mitochondrial function. Human-derived HepG2 cells were exposed for 48 h in culture to increasing concentrations of either molnupiravir or NHC after which cytotoxicity, mtDNA copy number and mitochondrial gene expression were determined. The phenotypic endpoint, mitochondrial respiration, was measured with the Seahorse® XF96 Extracellular Flux Analyzer. Both molnupiravir and NHC were cytotoxic at concentrations of ≥10 µM. However, at non-cytotoxic concentrations, neither significantly altered mitochondrial gene dose or transcription, or mitochondrial respiration. From this we conclude that mitochondrial toxicity is not a primary off target in the mechanism of cytotoxicity for either molnupiravir or its active metabolite NHC in the HepG2 cell line.


Subject(s)
COVID-19 , Nucleosides , Antiviral Agents/toxicity , COVID-19/drug therapy , Cytidine/analogs & derivatives , Humans , Hydroxylamines , Mitochondria/metabolism , RNA , SARS-CoV-2
7.
Cells ; 11(6)2022 03 17.
Article in English | MEDLINE | ID: covidwho-1760409

ABSTRACT

Cancer therapy is an emergent application for mRNA therapeutics. While in tumor immunotherapy, mRNA encoding for tumor-associated antigens is delivered to antigen-presenting cells in spleen and lymph nodes, other therapeutic options benefit from immediate delivery of mRNA nanomedicines directly to the tumor. However, tumor targeting of mRNA therapeutics is still a challenge, since, in addition to delivery of the cargo to the tumor, specifics of the targeted cell type as well as its interplay with the tumor microenvironment are crucial for successful intervention. This study investigated lipoplex nanoparticle-mediated mRNA delivery to spheroid cell culture models of melanoma. Insights into cell-type specific targeting, non-cell-autonomous effects, and penetration capacity in tumor and stroma cells of the mRNA lipoplex nanoparticles were obtained. It was shown that both coculture of different cell types as well as three-dimensional cell growth characteristics can modulate distribution and transfection efficiency of mRNA lipoplex formulations. The results demonstrate that three-dimensional coculture spheroids can provide a valuable surplus of information in comparison to adherent cells. Thus, they may represent in vitro models with enhanced predictivity for the in vivo activity of cancer nanotherapeutics.


Subject(s)
Melanoma , Nanoparticles , Coculture Techniques , Humans , Melanoma/therapy , Nanoparticles/therapeutic use , RNA , RNA, Messenger/genetics , Tumor Microenvironment
8.
Nat Commun ; 13(1): 1536, 2022 03 22.
Article in English | MEDLINE | ID: covidwho-1758235

ABSTRACT

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured "superfolder" mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.


Subject(s)
COVID-19 , RNA , COVID-19/therapy , Humans , Pseudouridine/metabolism , RNA Stability/genetics , RNA, Messenger/metabolism
9.
Nat Commun ; 13(1): 1444, 2022 03 17.
Article in English | MEDLINE | ID: covidwho-1751716

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to have devastating consequences worldwide. Recently, great efforts have been made to identify SARS-CoV-2 host factors, but the regulatory mechanisms of these host molecules, as well as the virus per se, remain elusive. Here we report a role of RNA G-quadruplex (RG4) in SARS-CoV-2 infection. Combining bioinformatics, biochemical and biophysical assays, we demonstrate the presence of RG4s in both SARS-CoV-2 genome and host factors. The biological and pathological importance of these RG4s is then exemplified by a canonical 3-quartet RG4 within Tmprss2, which can inhibit Tmprss2 translation and prevent SARS-CoV-2 entry. Intriguingly, G-quadruplex (G4)-specific stabilizers attenuate SARS-CoV-2 infection in pseudovirus cell systems and mouse models. Consistently, the protein level of TMPRSS2 is increased in lungs of COVID-19 patients. Our findings reveal a previously unknown mechanism underlying SARS-CoV-2 infection and suggest RG4 as a potential target for COVID-19 prevention and treatment.


Subject(s)
COVID-19 , Virus Internalization , Animals , Humans , Mice , RNA , SARS-CoV-2 , Serine Endopeptidases/genetics
10.
Am J Physiol Cell Physiol ; 322(4): C787-C793, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1745645

ABSTRACT

Similar to epigenetic DNA modification, RNA can be methylated and altered for stability and processing. RNA modifications, namely, epitranscriptomes, involve the following three functions: writing, erasing, and reading of marks. Methods for measurement and position detection are useful for the assessment of cellular function and human disease biomarkers. After pyrimidine 5-methylcytosine was reported for the first time a hundred years ago, numerous techniques have been developed for studying nucleotide modifications, including RNAs. Recent studies have focused on high-throughput and direct measurements for investigating the precise function of epitranscriptomes, including the characterization of severe acute respiratory syndrome coronavirus 2. The current study presents an overview of the development of detection techniques for epitranscriptomic marks and briefs about the recent progress in this field.


Subject(s)
COVID-19 , Transcriptome , Epigenesis, Genetic , Humans , RNA/genetics , RNA/metabolism , RNA Processing, Post-Transcriptional , Transcriptome/genetics
11.
Turk J Med Sci ; 51(SI-1): 3246-3252, 2021 12 17.
Article in English | MEDLINE | ID: covidwho-1726140

ABSTRACT

Due to the COVID-19 infection, which was recognized as a global pandemic by the WHO on March 11, 2020, the number of cases and disease-related deaths increases day by day globally. For this reason, antiviral agents used in treatment and vaccines, the most effective weapon in prevention, continue to be the most popular topic of the plan. Several situations are expected to affect the course of the pandemic. The loss of the ability of the virus to mutate and cause disease, the fact that those who become immunized by having the disease in the society reach a critical rate and create social immunity (herd immunity), and the provision of social immunity with effective vaccination can be counted as some of these situations. Candidate vaccines in the clinical phase among RNA-based vaccines: This review aimed to examine COVID-19 vaccine candidates using RNA technology and compile its current data. We used PubMed, Google Scholar, and World Health Organization (WHO) databases. Also, we followed up on the latest news and developments on vaccine companies' websites. Conclusion: Vaccination trials, which started due to the seriousness and urgency of the situation that we are in, continue exceptionally quickly and effectively. As per the WHO>s data on July 9, 2021, there have been 291 vaccine trials, 107 of which are in the clinical phase, and 18 (16%) of the vaccine candidates in the clinical phase are RNA-based vaccines. Also, the number of RNA-based vaccines with ongoing preclinical trials is 2


Subject(s)
COVID-19 Vaccines , COVID-19/prevention & control , COVID-19/epidemiology , Clinical Trials as Topic , Humans , Pandemics , RNA , SARS-CoV-2 , Vaccination
12.
Biosensors (Basel) ; 12(2)2022 Jan 19.
Article in English | MEDLINE | ID: covidwho-1715104

ABSTRACT

CRISPR-Cas systems have a great and still largely untapped potential for in vitro applications, in particular, for RNA biosensing. However, there is currently no systematic guide on selecting the most appropriate RNA-targeting CRISPR-Cas system for a given application among thousands of potential candidates. We provide an overview of the currently described Cas effector systems and review existing Cas-based RNA detection methods. We then propose a set of systematic selection criteria for selecting CRISPR-Cas candidates for new applications. Using this approach, we identify four candidates for in vitro RNA.


Subject(s)
CRISPR-Cas Systems , RNA
13.
Elife ; 112022 01 21.
Article in English | MEDLINE | ID: covidwho-1716085

ABSTRACT

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.


Subject(s)
DNA Methylation/physiology , Methyltransferases/metabolism , DNA, Single-Stranded/metabolism , Deoxyadenosines/metabolism , Humans , RNA/chemistry , RNA/metabolism
14.
Immunity ; 54(12): 2673-2675, 2021 Dec 14.
Article in English | MEDLINE | ID: covidwho-1709840

ABSTRACT

We talk to first and last authors Katalin Karikó and Drew Weissman about their seminal 2005 paper ''Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA", about how they see the work in retrospect, the current progress in the field, and their inspiration-then and now.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , RNA/immunology , SARS-CoV-2/physiology , Toll-Like Receptors/metabolism , /immunology , Access to Information , Animals , Humans , Information Dissemination , Mice , Nucleosides/immunology
15.
Biochem Biophys Res Commun ; 601: 129-136, 2022 04 23.
Article in English | MEDLINE | ID: covidwho-1699331

ABSTRACT

COVID-19, caused by SARS-CoV-2, has been spreading worldwide for more than two years and has led to immense challenges to human health. Despite the great efforts that have been made, our understanding of SARS-CoV-2 is still limited. The viral helicase, NSP13 is an important enzyme involved in SARS-CoV-2 replication and transcription. Here we highlight the important role of the stalk domain in the enzymatic activity of NSP13. Without the stalk domain, NSP13 loses its dsRNA unwinding ability due to the lack of ATPase activity. The stalk domain of NSP13 also provides a rigid connection between the ZBD and helicase domain. We found that the tight connection between the stalk and helicase is necessary for NSP13-mediated dsRNA unwinding. When a short flexible linker was inserted between the stalk and helicase domains, the helicase activity of NSP13 was impaired, although its ATPase activity remained intact. Further study demonstrated that linker insertion between the stalk and helicase domains attenuated the RNA binding ability and affected the thermal stability of NSP13. In summary, our results suggest the crucial role of the stalk domain in NSP13 enzymatic activity and provide mechanistic insight into dsRNA unwinding by SARS-CoV-2 NSP13.


Subject(s)
COVID-19/prevention & control , Methyltransferases/metabolism , RNA Helicases/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Binding Sites/genetics , COVID-19/virology , Enzyme Stability , Humans , Methyltransferases/chemistry , Methyltransferases/genetics , Models, Molecular , Mutation , Protein Conformation , RNA/chemistry , RNA/genetics , RNA/metabolism , RNA Helicases/chemistry , RNA Helicases/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Temperature , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
16.
Front Immunol ; 12: 797390, 2021.
Article in English | MEDLINE | ID: covidwho-1686476

ABSTRACT

Phosphodiesterase 4 (PDE4) inhibitors are immunomodulatory drugs approved to treat diseases associated with chronic inflammatory conditions, such as COPD, psoriasis and atopic dermatitis. Tanimilast (international non-proprietary name of CHF6001) is a novel, potent and selective inhaled PDE4 inhibitor in advanced clinical development for the treatment of COPD. To begin testing its potential in limiting hyperinflammation and immune dysregulation associated to SARS-CoV-2 infection, we took advantage of an in vitro model of dendritic cell (DC) activation by SARS-CoV-2 genomic ssRNA (SCV2-RNA). In this context, Tanimilast decreased the release of pro-inflammatory cytokines (TNF-α and IL-6), chemokines (CCL3, CXCL9, and CXCL10) and of Th1-polarizing cytokines (IL-12, type I IFNs). In contrast to ß-methasone, a reference steroid anti-inflammatory drug, Tanimilast did not impair the acquisition of the maturation markers CD83, CD86 and MHC-II, nor that of the lymph node homing receptor CCR7. Consistent with this, Tanimilast did not reduce the capability of SCV2-RNA-stimulated DCs to activate CD4+ T cells but skewed their polarization towards a Th2 phenotype. Both Tanimilast and ß-methasone blocked the increase of MHC-I molecules in SCV2-RNA-activated DCs and restrained the proliferation and activation of cytotoxic CD8+ T cells. Our results indicate that Tanimilast can modulate the SCV2-RNA-induced pro-inflammatory and Th1-polarizing potential of DCs, crucial regulators of both the inflammatory and immune response. Given also the remarkable safety demonstrated by Tanimilast, up to now, in clinical studies, we propose this inhaled PDE4 inhibitor as a promising immunomodulatory drug in the scenario of COVID-19.


Subject(s)
COVID-19/immunology , Dendritic Cells , Phosphodiesterase 4 Inhibitors/pharmacology , RNA/pharmacology , SARS-CoV-2/physiology , Virus Activation/drug effects , CD8-Positive T-Lymphocytes/immunology , COVID-19/drug therapy , Cytokines/immunology , Dendritic Cells/immunology , Dendritic Cells/virology , Humans , Th1 Cells/immunology , Th2 Cells/immunology , Virus Activation/immunology
17.
Lancet Microbe ; 2(12): e704-e714, 2021 12.
Article in English | MEDLINE | ID: covidwho-1683809

ABSTRACT

BACKGROUND: An optimised standard experimental setup across different hospitals is urgently needed to ensure consistency in nucleic acid test results for SARS-CoV-2 detection. A standard comparison across different nucleic acid tests and their optimal experimental setups is not present. We assessed the performance of three common nucleic acid tests, namely digital PCR (dPCR), quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP), to detect SARS-CoV-2 in clinical settings. METHODS: In this systematic review and meta-analysis we compared sensitivity and specificity of qPCR, dPCR, and LAMP and their performances when different experimental setups (namely specimen type used, use of RNA extraction, primer-probe sets, and RNA extraction methods) are applied. We searched PubMed, BioRxiv, MedRxiv, SciFinder, and ScienceDirect for studies and preprints published between Feb 29 and Dec 15, 2020. Included dPCR, qPCR, and LAMP studies using any type of human specimens should report the number of true-positive, true-negative, false-positive, and false-negative cases with Emergency Use Authorization (EUA)-approved PCR assays as the comparator. Studies with a sample size of less than ten, descriptive studies, case studies, reviews, and duplicated studies were excluded. Pooled sensitivity and specificity were computed from the true and false positive and negative cases using Reitsma's bivariate random-effects and bivariate latent class models. Test performance reported in area under the curve (AUC) of the three nucleic acid tests was further compared by pooling studies with similar experimental setups (eg, tests that used RNA extracted pharyngeal swabs but with either the open reading frame 1ab or the N primer). Heterogeneity was assessed and reported in I 2 and τ2. FINDINGS: Our search identified 1277 studies of which we included 66 studies (11 dPCR, 32 qPCR, and 23 LAMP) with 15 017 clinical samples in total in our systematic review and 52 studies in our meta-analysis. dPCR had the highest pooled diagnostic sensitivity (94·1%, 95% CI 88·9-96·6, by Reitsma's model and 95·8%, 54·9-100·0, by latent class model), followed by qPCR (92·7%, 88·3-95·6, and 93·4%, 60·9-99·9) and LAMP (83·3%, 76·9-88·2, and 86·2%, 20·7-99·9), using EUA-approved PCR kits as the reference standard. LAMP was the most specific with a pooled estimate of 96·3% (93·8-97·8) by Reitsma's model and 94·3% (49·1-100·0) by latent class model, followed by qPCR (92·9%, 87·2-96·2, and 93·1%, 47·1-100·0) and dPCR (78·5%, 57·4-90·8, and 73·8%, 0·9-100·0). The overall heterogeneity was I 2 0·5% (τ2 2·79) for dPCR studies, 0% (4·60) for qPCR studies, and 0% (3·96) for LAMP studies. AUCs of the three nucleic acid tests were the highest and differed the least between tests (ie, AUC>0·98 for all tests) when performed with RNA extracted pharyngeal swabs using SARS-CoV-2 open reading frame 1ab primer. INTERPRETATION: All three nucleic acid tests consistently perform better with pharyngeal swabs using SARS-CoV-2 open reading frame 1ab primer with RNA extraction. dPCR was shown to be the most sensitive, followed by qPCR and LAMP. However, their accuracy does not differ significantly. Instead, accuracy depends on specific experimental conditions, implying that more efforts should be directed to optimising the experimental setups for the nucleic acid tests. Hence, our results could be a reference for optimising and establishing a standard nucleic acid test protocol that is applicable in laboratories worldwide. FUNDING: University Grants Committee and The Chinese University of Hong Kong.


Subject(s)
COVID-19 , Nucleic Acids , COVID-19/diagnosis , Hospitals , Humans , RNA , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics
18.
Cells ; 11(3)2022 01 30.
Article in English | MEDLINE | ID: covidwho-1667057

ABSTRACT

The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still ongoing, as is research on the molecular mechanisms underlying cellular infection by coronaviruses, with the hope of developing therapeutic agents against this pandemic. Other important respiratory viruses such as 2009 pandemic H1N1 and H7N9 avian influenza virus (AIV), influenza A viruses, are also responsible for a possible outbreak due to their respiratory susceptibility. However, the interaction of these viruses with host cells and the regulation of post-transcriptional genes remains unclear. In this study, we detected and analyzed the comparative transcriptome profiling of SARS-CoV-2, panH1N1 (A/California/07/2009), and H7N9 (A/Shanghai/1/2013) infected cells. The results showed that the commonly upregulated genes among the three groups were mainly involved in autophagy, pertussis, and tuberculosis, which indicated that autophagy plays an important role in viral pathogenicity. There are three groups of commonly downregulated genes involved in metabolic pathways. Notably, unlike panH1N1 and H7N9, SARS-CoV-2 infection can inhibit the m-TOR pathway and activate the p53 signaling pathway, which may be responsible for unique autophagy induction and cell apoptosis. Particularly, upregulated expression of IRF1 was found in SARS-CoV-2, panH1N1, and H7N9 infection. Further analysis showed SARS-CoV-2, panH1N1, and H7N9 infection-induced upregulation of lncRNA-34087.27 could serve as a competitive endogenous RNA to stabilize IRF1 mRNA by competitively binding with miR-302b-3p. This study provides new insights into the molecular mechanisms of influenza A virus and SARS-CoV-2 infection.


Subject(s)
COVID-19/immunology , Immunity/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H7N9 Subtype/immunology , Influenza, Human/immunology , RNA/immunology , Transcriptome/immunology , A549 Cells , Animals , COVID-19/genetics , COVID-19/virology , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Immunity/genetics , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H7N9 Subtype/physiology , Influenza, Human/genetics , Influenza, Human/virology , Interferon Regulatory Factor-1/genetics , Interferon Regulatory Factor-1/immunology , Interferon Regulatory Factor-1/metabolism , MicroRNAs/genetics , MicroRNAs/immunology , MicroRNAs/metabolism , Pandemics/prevention & control , RNA/genetics , RNA/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/immunology , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Messenger/metabolism , RNA-Seq/methods , SARS-CoV-2/physiology , Signal Transduction/genetics , Signal Transduction/immunology , Transcriptome/genetics
19.
Int J Cardiol ; 353: 131-134, 2022 04 15.
Article in English | MEDLINE | ID: covidwho-1652578

ABSTRACT

BACKGROUND: The incidence of myocarditis after RNA-based vaccines for coronavirus has gained social and medical interest. METHODS: We performed an intention-to-treat meta-analysis, following the PRISMA statement. After a systematic search, without language restriction, 9 publications were selected. Two were excluded (one was only in subjects with age 12-17 and other might had included subjects from a larger publication). We followed the PRISMA guidelines for abstracting data and assessing data quality and validity. Data was verified by 2 investigators. RESULTS: We analyzed 17,704,413 subjects, from 7 studies, that included 627 cases of confirmed myocarditis). The incidence of myocarditis was 0.0035% (95% CI 0.0034-0.0035). Mean incidence rate was 10.69 per 100.000 persons-year. Cases reported from Israel represented 45.14% from total (283 out of the 627). Only 1 case of fatal myocarditis or death was reported. There was significant heterogeneity between results. The meta-regression analysis excluded mean age, region, number of cases or number of people included as sources of heterogeneity. No small-study effect was observed (p = 0.19). CONCLUSIONS AND RELEVANCE: Myocarditis incidence after RNA vaccines is very rare (0.0035%) and has a very favorable clinical course.


Subject(s)
COVID-19 , Myocarditis , Adolescent , Child , Humans , Myocarditis/epidemiology , Myocarditis/etiology , RNA
20.
Mol Diagn Ther ; 26(2): 229-238, 2022 03.
Article in English | MEDLINE | ID: covidwho-1649215

ABSTRACT

BACKGROUND AND AIMS: Rapid and accurate detection of COVID-19 is crucial for mitigation of the pandemic. We evaluated the performance of six molecular kits and the effect of several factors on the performance of the kits. MATERIALS AND METHODS: Two hundred and four nasopharyngeal samples were collected from participants aged ≥18 years at the Baruch Padeh Medical Center Poriya, Israel, between June and August 2020. Samples were tested by: Allplex 2019-nCOV Assay (Seegene), Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV (BGI Genomics), Xpert® Xpress SARS-CoV-2 test (Cepheid), Simplexa® COVID-19 Direct Kit (Focus Diagnostics), BD SARS-CoV-2 Reagents for BD MAX™ System (BD), and Logix Smart™ Coronavirus Disease 2019 (COVID-19) Test kit (CO-DIAGNOSTICS). RESULTS: Xpert® Xpress SARS-CoV-2 test and Logix Smart™ COVID-19 Kit had the highest (91.2%) and the lowest (74.5%) sensitivity, respectively. Symptoms were a predictor of a positive result. Traditional assays had a higher minimum cycle threshold (min Ct), i.e. detected lower viral load, compared to rapid assays (p = 0.012). Samples of symptomatic participants had lower min Ct, than samples of asymptomatic participants (p < 0.001). Additionally, the more genes were detected, the lower the min Ct (p < 0.001), indicating that a greater percentage of the viral genome was amplified. CONCLUSIONS: Taken together, most assays had overall good performance. Since several factors affect the performance of kits, each laboratory must be familiar with its kit's limitations in order to produce the most reliable results.


Subject(s)
COVID-19 , Adolescent , Adult , COVID-19/diagnosis , Humans , Pandemics , RNA , SARS-CoV-2/genetics , Sensitivity and Specificity
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