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1.
Anal Bioanal Chem ; 413(22): 5619-5632, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-2174032

ABSTRACT

In the face of the COVID-19 pandemic, the need for rapid serological tests that allow multiplexing emerged, as antibody seropositivity can instruct about individual immunity after an infection with SARS-CoV-2 or after vaccination. As many commercial antibody tests are either time-consuming or tend to produce false negative or false positive results when only one antigen is considered, we developed an automated, flow-based chemiluminescence microarray immunoassay (CL-MIA) that allows for the detection of IgG antibodies to SARS-CoV-2 receptor-binding domain (RBD), spike protein (S1 fragment), and nucleocapsid protein (N) in human serum and plasma in less than 8 min. The CoVRapid CL-MIA was tested with a set of 65 SARS-CoV-2 serology positive or negative samples, resulting in 100% diagnostic specificity and 100% diagnostic sensitivity, thus even outcompeting commercial tests run on the same sample set. Additionally, the prospect of future quantitative assessments (i.e., quantifying the level of antibodies) was demonstrated. Due to the fully automated process, the test can easily be operated in hospitals, medical practices, or vaccination centers, offering a valuable tool for COVID-19 serosurveillance. Graphical abstract.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , Immunoassay/methods , Immunoglobulin G/blood , SARS-CoV-2/immunology , Antigens, Viral/chemistry , Antigens, Viral/immunology , Automation, Laboratory , Coronavirus Nucleocapsid Proteins/immunology , Humans , Immobilized Proteins/chemistry , Immobilized Proteins/immunology , Immune Sera , Immunoassay/instrumentation , Lab-On-A-Chip Devices , Luminescent Measurements , Phosphoproteins/immunology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Time Factors
2.
Intern Med ; 61(20): 3053-3062, 2022 Oct 15.
Article in English | MEDLINE | ID: covidwho-2079926

ABSTRACT

Objective To examine the continuation of antibody prevalence status after 12 months and background factors in antibody-positive subjects following asymptomatic infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods We initially determined the SARS-CoV-2 anti-nucleocapsid protein immunoglobulin G (anti-N IgG) antibody prevalence in 1,603 patients, doctors, and nurses at 65 medical institutions in Kanagawa Prefecture, Japan. We then obtained consent from 33 of the 39 subjects who tested positive and performed follow-up for 12 months. Results Follow-up for up to 12 months showed that a long-term response of the anti-N IgG antibody could be detected in 6 of the 33 participants (18.2%). The proportions with hypertension, using an angiotensin-receptor blocker, and without a drinking habit were higher among the participants with a long-term anti-N IgG antibody response for up to 12 months than among those without a long-term antibody response. Conclusions The proportion of individuals with subclinical COVID-19 who continuously had a positive result for the anti-N IgG antibody at 12 months was low.


Subject(s)
COVID-19 , Immunoglobulin G , Angiotensin Receptor Antagonists , Antibodies, Viral/blood , Asymptomatic Infections/epidemiology , COVID-19/epidemiology , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Humans , Immunoglobulin G/blood , Phosphoproteins/immunology , SARS-CoV-2
3.
Cells ; 11(19)2022 Sep 23.
Article in English | MEDLINE | ID: covidwho-2043599

ABSTRACT

Circulating cell-free mitochondrial DNA (cf-mtDNA) has been found in the plasma of severely ill COVID-19 patients and is now known as a strong predictor of mortality. However, the underlying mechanism of mtDNA release is unexplored. Here, we show a novel mechanism of SARS-CoV-2-mediated pro-inflammatory/pro-apoptotic mtDNA release and a rational therapeutic stem cell-based approach to mitigate these effects. We systematically screened the effects of 29 SARS-CoV-2 proteins on mitochondrial damage and cell death and found that NSP4 and ORF9b caused extensive mitochondrial structural changes, outer membrane macropore formation, and the release of inner membrane vesicles loaded with mtDNA. The macropore-forming ability of NSP4 was mediated through its interaction with BCL2 antagonist/killer (BAK), whereas ORF9b was found to inhibit the anti-apoptotic member of the BCL2 family protein myeloid cell leukemia-1 (MCL1) and induce inner membrane vesicle formation containing mtDNA. Knockdown of BAK and/or overexpression of MCL1 significantly reversed SARS-CoV-2-mediated mitochondrial damage. Therapeutically, we engineered human mesenchymal stem cells (MSCs) with a simultaneous knockdown of BAK and overexpression of MCL1 (MSCshBAK+MCL1) and named these cells IMAT-MSCs (intercellular mitochondrial transfer-assisted therapeutic MSCs). Upon co-culture with SARS-CoV-2-infected or NSP4/ORF9b-transduced airway epithelial cells, IMAT-MSCs displayed functional intercellular mitochondrial transfer (IMT) via tunneling nanotubes (TNTs). The mitochondrial donation by IMAT-MSCs attenuated the pro-inflammatory and pro-apoptotic mtDNA release from co-cultured epithelial cells. Our findings thus provide a new mechanistic basis for SARS-CoV-2-induced cell death and a novel therapeutic approach to engineering MSCs for the treatment of COVID-19.


Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins/metabolism , DNA, Mitochondrial , Viral Nonstructural Proteins/metabolism , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Humans , Mitochondria/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Phosphoproteins/metabolism , SARS-CoV-2
4.
J Infect Dis ; 226(5): 812-821, 2022 09 13.
Article in English | MEDLINE | ID: covidwho-2029035

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antigen (N-Ag) can be detected in the blood of patients with coronavirus disease 2019 (COVID-19). We used a highly sensitive and specific assay to explore the presence of N-Ag in urine during the course of COVID-19 and its relationship with the severity of disease. METHODS: We studied urinary and plasma N-Ag using a highly sensitive immunoassay in 82 patients with SARS-CoV-2 infection proved by polymerase chain reaction. RESULTS: In the first and second weeks of COVID-19, hospitalized patients tested positive for urinary N-Ag (81.25% and 71.79%, respectively) and plasma N-Ag (93.75% and 94.87%, respectively). High urinary N-Ag levels were associated with the absence of SARS-CoV-2 nucleocapsid antibodies, admission in intensive care units, high C-reactive protein levels, lymphopenia, eosinopenia, and high lactate dehydrogenase levels. Higher accuracy was observed for urinary N-Ag as a predictor of severe COVID-19 than for plasma N-Ag. CONCLUSIONS: Our study demonstrates that N-Ag is present in the urine of patients hospitalized in the early phase of COVID-19. As a direct marker of SARS-CoV-2, urinary N-Ag reflects the dissemination of viral compounds in the body. Urinary N-Ag may be a useful marker for disease severity in SARS-CoV-2 infections.


Subject(s)
COVID-19 , Antibodies, Viral , Antigens, Viral , Coronavirus Nucleocapsid Proteins , Humans , Nucleocapsid/analysis , SARS-CoV-2 , Sensitivity and Specificity
5.
Signal Transduct Target Ther ; 7(1): 318, 2022 09 14.
Article in English | MEDLINE | ID: covidwho-2028663

ABSTRACT

Excessive inflammatory responses contribute to the pathogenesis and lethality of highly pathogenic human coronaviruses, but the underlying mechanism remains unclear. In this study, the N proteins of highly pathogenic human coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were found to bind MASP-2, a key serine protease in the lectin pathway of complement activation, resulting in excessive complement activation by potentiating MBL-dependent MASP-2 activation, and the deposition of MASP-2, C4b, activated C3 and C5b-9. Aggravated inflammatory lung injury was observed in mice infected with adenovirus expressing the N protein. Complement hyperactivation was also observed in SARS-CoV-2-infected patients. Either blocking the N protein:MASP-2 interaction, MASP-2 depletion or suppressing complement activation can significantly alleviate N protein-induced complement hyperactivation and lung injury in vitro and in vivo. Altogether, these data suggested that complement suppression may represent a novel therapeutic approach for pneumonia induced by these highly pathogenic coronaviruses.


Subject(s)
COVID-19 , Lung Injury , Animals , COVID-19/genetics , Complement Pathway, Mannose-Binding Lectin/genetics , Coronavirus Nucleocapsid Proteins , Humans , Inflammation/genetics , Mannose-Binding Protein-Associated Serine Proteases/genetics , Mannose-Binding Protein-Associated Serine Proteases/metabolism , Mice , SARS-CoV-2
6.
Nature ; 609(7928): 785-792, 2022 09.
Article in English | MEDLINE | ID: covidwho-1972633

ABSTRACT

Highly pathogenic coronaviruses, including severe acute respiratory syndrome coronavirus 2 (refs. 1,2) (SARS-CoV-2), Middle East respiratory syndrome coronavirus3 (MERS-CoV) and SARS-CoV-1 (ref. 4), vary in their transmissibility and pathogenicity. However, infection by all three viruses results in substantial apoptosis in cell culture5-7 and in patient tissues8-10, suggesting a potential link between apoptosis and pathogenesis of coronaviruses. Here we show that caspase-6, a cysteine-aspartic protease of the apoptosis cascade, serves as an important host factor for efficient coronavirus replication. We demonstrate that caspase-6 cleaves coronavirus nucleocapsid proteins, generating fragments that serve as interferon antagonists, thus facilitating virus replication. Inhibition of caspase-6 substantially attenuates lung pathology and body weight loss in golden Syrian hamsters infected with SARS-CoV-2 and improves the survival of mice expressing human DPP4 that are infected with mouse-adapted MERS-CoV. Our study reveals how coronaviruses exploit a component of the host apoptosis cascade to facilitate virus replication.


Subject(s)
Aspartic Acid , Caspase 6 , Coronavirus Infections , Coronavirus , Cysteine , Host-Pathogen Interactions , Virus Replication , Animals , Apoptosis , Aspartic Acid/metabolism , Caspase 6/metabolism , Coronavirus/growth & development , Coronavirus/pathogenicity , Coronavirus Infections/enzymology , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/metabolism , Cricetinae , Cysteine/metabolism , Dipeptidyl Peptidase 4/genetics , Dipeptidyl Peptidase 4/metabolism , Humans , Interferons/antagonists & inhibitors , Interferons/immunology , Lung/pathology , Mesocricetus , Mice , Middle East Respiratory Syndrome Coronavirus , SARS Virus , SARS-CoV-2 , Survival Rate , Weight Loss
7.
PLoS One ; 17(2): e0262591, 2022.
Article in English | MEDLINE | ID: covidwho-1968842

ABSTRACT

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/chemistry , COVID-19/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Immunoglobulin G/blood , Inclusion Bodies/chemistry , Protein Refolding , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Hydrogen-Ion Concentration , Hydrostatic Pressure , Immunoglobulin G/immunology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Solubility
8.
Emerg Infect Dis ; 28(9): 1859-1862, 2022 09.
Article in English | MEDLINE | ID: covidwho-1963356

ABSTRACT

Given widespread use of spike antibody in generating coronavirus disease vaccines, SARS-CoV-2 nucleocapsid antibodies are increasingly used to indicate previous infection in serologic surveys. However, longitudinal kinetics and seroreversion are poorly defined. We found substantial seroreversion of nucleocapsid total immunoglobulin, underscoring the need to account for seroreversion in seroepidemiologic studies.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/epidemiology , Coronavirus Nucleocapsid Proteins/immunology , Humans , Kinetics , Nucleocapsid , Phosphoproteins/immunology , Seroepidemiologic Studies
9.
Nucleic Acids Res ; 50(14): 8168-8192, 2022 08 12.
Article in English | MEDLINE | ID: covidwho-1961119

ABSTRACT

Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.


Subject(s)
Coronavirus Nucleocapsid Proteins , RNA, Double-Stranded , SARS-CoV-2 , Binding Sites , Coronavirus Nucleocapsid Proteins/chemistry , Phosphoproteins/chemistry , RNA, Double-Stranded/genetics , RNA, Viral/genetics , RNA-Binding Proteins/metabolism , SARS-CoV-2/genetics , Temperature
10.
Microbiol Spectr ; 10(4): e0078122, 2022 08 31.
Article in English | MEDLINE | ID: covidwho-1938014

ABSTRACT

The emergence and evolution of SARS-CoV-2 is characterized by the occurrence of diverse sets of mutations that affect virus characteristics, including transmissibility and antigenicity. Recent studies have focused mostly on spike protein mutations; however, SARS-CoV-2 variants of interest (VoI) or concern (VoC) contain significant mutations in the nucleocapsid protein as well. To study the relevance of mutations at the virion level, recombinant baculovirus expression system-based virus-like particles (VLPs) were generated for the prototype Wuhan sequence along with spike protein mutants like D614G and G1124V and the significant RG203KR mutation in nucleocapsid. All four structural proteins were assembled in a particle for which the morphology and size, confirmed by transmission electron microscopy, closely resembled that of the native virion. The VLP harboring RG203KR mutations in nucleocapsid exhibited augmentation of humoral immune responses and enhanced neutralization by immunized mouse sera. Results demonstrate a noninfectious platform to quickly assess the implication of mutations in structural proteins of the emerging variant. IMPORTANCE Since its origin in late 2019, the SARS-CoV-2 virus has been constantly mutating and evolving. Current studies mostly employ spike protein (S) pseudovirus systems to determine the effects of mutations on the infectivity and immunogenicity of variants. Despite its functional importance and emergence as a mutational hot spot, the nucleocapsid (N) protein has not been widely studied. The generation of SARS-CoV-2 VLPs in a baculoviral system in this study, with mutations in the S and N proteins, allowed examination of the involvement of all the structural proteins involved in viral entry and eliciting an immune response. This approach provides a platform to study the effect of mutations in structural proteins of SARS-CoV-2 that potentially contribute to cell infectivity, immune response, and immune evasion, bypassing the use of infectious virus for the same analyses.


Subject(s)
Coronavirus Nucleocapsid Proteins , SARS-CoV-2 , Animals , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Mice , Mutation , Phosphoproteins/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus , Virion/genetics
11.
Biosci Rep ; 41(9)2021 09 30.
Article in English | MEDLINE | ID: covidwho-1915305

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic of the Coronavirus disease in late 2019 (COVID-19). Vaccine development efforts have predominantly been aimed at 'Extra-viral' Spike (S) protein as vaccine vehicles, but there are concerns regarding 'viral immune escape' since multiple mutations may enable the mutated virus strains to escape from immunity against S protein. The 'Intra-viral' Nucleocapsid (N-protein) is relatively conserved among mutant strains of coronaviruses during spread and evolution. Herein, we demonstrate novel vaccine candidates against SARS-CoV-2 by using the whole conserved N-protein or its fragment/peptides. Using ELISA assay, we showed that high titers of specific anti-N antibodies (IgG, IgG1, IgG2a, IgM) were maintained for a reasonably long duration (> 5 months), suggesting that N-protein is an excellent immunogen to stimulate host immune system and robust B-cell activation. We synthesized three peptides located at the conserved regions of N-protein among CoVs. One peptide showed as a good immunogen for vaccination as well. Cytokine arrays on post-vaccination mouse sera showed progressive up-regulation of various cytokines such as IFN-γ and CCL5, suggesting that TH1 associated responses are also stimulated. Furthermore, vaccinated mice exhibited an elevated memory T cells population. Here, we propose an unconventional vaccine strategy targeting the conserved N-protein as an alternative vaccine target for coronaviruses. Moreover, we generated a mouse monoclonal antibody specifically against an epitope shared between SARS-CoV and SARS-CoV-2, and we are currently developing the First-in-Class humanized anti-N-protein antibody to potentially treat patients infected by various CoVs in the future.


Subject(s)
Antibodies, Viral/blood , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Coronavirus Nucleocapsid Proteins/immunology , Animals , Antibodies, Monoclonal, Murine-Derived , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Coronavirus Nucleocapsid Proteins/genetics , Epitopes/immunology , Humans , Immune Evasion , Immunogenicity, Vaccine , Mice , Models, Animal , Pandemics/prevention & control , SARS Virus/genetics , SARS Virus/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/immunology , Th1 Cells/immunology , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology
12.
J Struct Biol ; 214(3): 107879, 2022 09.
Article in English | MEDLINE | ID: covidwho-1914725

ABSTRACT

14-3-3 proteins are important dimeric scaffolds that regulate the function of hundreds of proteins in a phosphorylation-dependent manner. The SARS-CoV-2 nucleocapsid (N) protein forms a complex with human 14-3-3 proteins upon phosphorylation, which has also been described for other coronaviruses. Here, we report a high-resolution crystal structure of 14-3-3 bound to an N phosphopeptide bearing the phosphoserine 197 in the middle. The structure revealed two copies of the N phosphopeptide bound, each in the central binding groove of each 14-3-3 monomer. A complex network of hydrogen bonds and water bridges between the peptide and 14-3-3 was observed explaining the high affinity of the N protein for 14-3-3 proteins.


Subject(s)
14-3-3 Proteins , Coronavirus Nucleocapsid Proteins , SARS-CoV-2 , 14-3-3 Proteins/chemistry , COVID-19 , Coronavirus Nucleocapsid Proteins/chemistry , Humans , Phosphopeptides/chemistry , Phosphoproteins/chemistry , Protein Binding
13.
Sci Rep ; 12(1): 10366, 2022 06 20.
Article in English | MEDLINE | ID: covidwho-1900657

ABSTRACT

The Covid-19 pandemic, caused by SARS-CoV-2, has resulted in over 6 million reported deaths worldwide being one of the biggest challenges the world faces today. Here we present optimizations of all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay to a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals and 103 individuals without a confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals from our cohort before and after receiving ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It is also a valuable tool for elucidating the immunological consequences of new viral strains and monitoring vaccination coverage and duration of response to different immunization regimens.


Subject(s)
COVID-19 , Seroconversion , Antibodies, Viral/analysis , Brazil , COVID-19/diagnosis , COVID-19/prevention & control , COVID-19 Vaccines/administration & dosage , ChAdOx1 nCoV-19/administration & dosage , Coronavirus Nucleocapsid Proteins/immunology , Humans , Immunoglobulin A , Immunoglobulin G , Immunoglobulin M , Pandemics , Phosphoproteins/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Vaccines, Inactivated/administration & dosage
14.
Clin Chim Acta ; 533: 42-47, 2022 Aug 01.
Article in English | MEDLINE | ID: covidwho-1885664

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID19) caused by the new severe acute respiratory syndrome coronavirus 2 (SARSCoV2) is a global public health emergency. Age and gender are two important factors related to the risk and outcome of various diseases. Cycle threshold (Ct) value is believed to have relation with age and gender. OBJECTIVE: This study has been conducted to investigates the association between SARS-CoV-2 cycle threshold to age and gender of COVID-19 patients, to investigate whether the population-wide change of SARSCoV2 RTPCR Ct value over time is corelated to the number of new COVID19 cases and to investigate the dynamic of RdRp and N genes. METHODS: 72,811 individuals from second wave of COVID19, were observed in current study at Pure Health Lab, Mafraq Hospital, Abu Dhabi, UAE. RESULTS: 15,201/72,811 (21 %) positivity was observed. COVID-19 were more prevalent in males (59.35%) as compared to female (40.65%). The Positivity rate were significantly higher in Male than in Female cases (p-Value = 0.04). The Ct values for both targets of all the samples were ranged from 4.57 to 29.73. Longitudinal analysis showed significant increased during the study period from starting to end as were hypothesized. Interestingly, both the targets (RdRp and N) were present in age < 1 year. Which may indicate that mutated strains are not prevalent in children's < 1 year. CONCLUSION: There was no statistically significant difference in viral loads in between age-groups. Males were tending to higher viral load compared to females. The findings have implications for preventive strategies.


Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2 , Age Distribution , Child , Female , Humans , Male , RNA, Viral , RNA-Dependent RNA Polymerase , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sex Characteristics
15.
J Infect Dis ; 226(5): 766-777, 2022 09 13.
Article in English | MEDLINE | ID: covidwho-1883015

ABSTRACT

BACKGROUND: Excessive complement activation has been implicated in the pathogenesis of coronavirus disease 2019 (COVID-19), but the mechanisms leading to this response remain unclear. METHODS: We measured plasma levels of key complement markers, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and antibodies against SARS-CoV-2 and seasonal human common cold coronaviruses (CCCs) in hospitalized patients with COVID-19 of moderate (n = 18) and critical severity (n = 37) and in healthy controls (n = 10). RESULTS: We confirmed that complement activation is systemically increased in patients with COVID-19 and is associated with a worse disease outcome. We showed that plasma levels of C1q and circulating immune complexes were markedly increased in patients with severe COVID-19 and correlated with higher immunoglobulin (Ig) G titers, greater complement activation, and higher disease severity score. Additional analyses showed that the classical pathway was the main arm responsible for augmented complement activation in severe patients. In addition, we demonstrated that a rapid IgG response to SARS-CoV-2 and an anamnestic IgG response to the nucleoprotein of the CCCs were strongly correlated with circulating immune complex levels, complement activation, and disease severity. CONCLUSIONS: These findings indicate that early, nonneutralizing IgG responses may play a key role in complement overactivation in severe COVID-19. Our work underscores the urgent need to develop therapeutic strategies to modify complement overactivation in patients with COVID-19.


Subject(s)
COVID-19 , Antibodies, Viral , Coronavirus Nucleocapsid Proteins , Humans , Immunoglobulin G , SARS-CoV-2
16.
Viruses ; 14(5)2022 05 09.
Article in English | MEDLINE | ID: covidwho-1875802

ABSTRACT

SARS-CoV-2 is constantly evolving with lineages emerging and others eclipsing. Some lineages have an important epidemiological impact and are known as variants of interest (VOIs), variants under monitoring (VUMs) or variants of concern (VOCs). Lineage A.27 was first defined as a VUM since it holds mutations of concern. Here, we report additional lineage A.27 data and sequences from five African countries and describe the molecular characteristics, and the genetic history of this lineage worldwide. Based on the new sequences investigated, the most recent ancestor (tMRCA) of lineage A.27 was estimated to be from April 2020 from Niger. It then spread to Europe and other parts of the world with a peak observed between February and April 2021. The detection rate of A.27 then decreased with only a few cases reported during summer 2021. The phylogenetic analysis revealed many sub-lineages. Among them, one was defined by the substitution Q677H in the spike (S) gene, one was defined by the substitution D358N in the nucleoprotein (N) gene and one was defined by the substitution A2143V in the ORF1b gene. This work highlights the importance of molecular characterization and the timely submission of sequences to correctly describe the circulation of particular strains in order to be proactive in monitoring the pandemic.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Humans , Pandemics , Phosphoproteins/genetics , Phylogeny , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
17.
Sci Rep ; 12(1): 9045, 2022 05 31.
Article in English | MEDLINE | ID: covidwho-1873542

ABSTRACT

Long-term antibody responses to SARS-CoV-2 have focused on responses to full-length spike protein, specific domains within spike, or nucleoprotein. In this study, we used high-density peptide microarrays representing the complete proteome of SARS-CoV-2 to identify binding sites (epitopes) targeted by antibodies present in the blood of COVID-19 resolved cases at 5 months post-diagnosis. Compared to previous studies that evaluated epitope-specific responses early post-diagnosis (< 60 days), we found that epitope-specific responses to nucleoprotein and spike protein have contracted, and that responses to membrane protein have expanded. Although antibody titers to full-length spike and nucleoprotein remain steady over months, taken together our data suggest that the population of epitope-specific antibodies that contribute to this reactivity is dynamic and evolves over time. Further, the spike epitopes bound by polyclonal antibodies in COVID-19 convalescent serum samples aligned with known target sites that can neutralize viral activity suggesting that the maintenance of these antibodies might provide rapid serological immunity. Finally, the most dominant epitopes for membrane protein and spike showed high diagnostic accuracy providing novel biomarkers to refine blood-based antibody tests. This study provides new insights into the specific regions of SARS-CoV-2 targeted by serum antibodies long after infection.


Subject(s)
Antibodies, Viral , COVID-19 , Convalescence , Antibodies, Viral/blood , COVID-19/blood , COVID-19/therapy , Coronavirus Nucleocapsid Proteins , Epitopes , Humans , Immunization, Passive , Phosphoproteins , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
18.
mBio ; 13(3): e0130022, 2022 06 28.
Article in English | MEDLINE | ID: covidwho-1874506

ABSTRACT

Ubiquitin signaling is essential for immunity to restrict pathogen proliferation. Due to its enormous impact on human health and the global economy, intensive efforts have been invested in studying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its interactions with hosts. However, the role of the ubiquitin network in pathogenicity has not yet been explored. Here, we found that ORF9b of SARS-CoV-2 is ubiquitinated on Lys-4 and Lys-40 by unknown E3 ubiquitin ligases and is degraded by the ubiquitin proteasomal system. Importantly, we identified USP29 as a host factor that prevents ORF9b ubiquitination and subsequent degradation. USP29 interacts with the carboxyl end of ORF9b and removes ubiquitin chains from the protein, thereby inhibiting type I interferon (IFN) induction and NF-κB activation. We also found that ORF9b stabilization by USP29 enhanced the virulence of VSV-eGFP and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). Moreover, we observed that the mRNA level of USP29 in SARS-CoV-2 patients was higher than that in healthy people. Our findings provide important evidence indicating that targeting USP29 may effectively combat SARS-CoV-2 infection. IMPORTANCE Coronavirus disease 2019 (COVID-19) is a current global health threat caused by SARS-CoV-2. The innate immune response such as type I IFN (IFN-I) is the first line of host defense against viral infections, whereas SARS-CoV-2 proteins antagonize IFN-I production through distinct mechanisms. Among them, ORF9b inhibits the canonical IκB kinase alpha (IKKɑ)/ß/γ-NF-κB signaling and subsequent IFN production; therefore, discovering the regulation of ORF9b by the host might help develop a novel antiviral strategy. Posttranslational modification of proteins by ubiquitination regulates many biological processes, including viral infections. Here, we report that ORF9b is ubiquitinated and degraded through the proteasome pathway, whereas deubiquitinase USP29 deubiquitinates ORF9b and prevents its degradation, resulting in the enhancement of ORF9b-mediated inhibition of IFN-I and NF-κB activation and the enhancement of virulence of VSV-eGFP and SARS-CoV-2 trVLP.


Subject(s)
Biological Phenomena , COVID-19 , Coronavirus Nucleocapsid Proteins/metabolism , Deubiquitinating Enzymes , Humans , Immunity, Innate , NF-kappa B , Phosphoproteins/metabolism , Proteasome Endopeptidase Complex , SARS-CoV-2/genetics , Ubiquitin-Specific Proteases , Ubiquitins , Virulence
19.
J Virol ; 96(12): e0041222, 2022 06 22.
Article in English | MEDLINE | ID: covidwho-1874504

ABSTRACT

SARS-CoV-2 is the causative agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19) and poses a significant threat to global health. N protein (NP), which is a major pathogenic protein among betacoronaviruses, binds to the viral RNA genome to allow viral genome packaging and viral particle release. Recent studies showed that NP antagonizes interferon (IFN) induction and mediates phase separation. Using live SARS-CoV-2 viruses, this study provides solid evidence showing that SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming G3BP1-mediated antiviral innate immunity. G3BP1 conditional knockout mice (g3bp1fl/fL, Sftpc-Cre) exhibit significantly higher lung viral loads after SARS-CoV-2 infection than wild-type mice. Our findings contribute to the growing body of knowledge regarding the pathogenicity of NPSARS-CoV-2 and provide insight into new therapeutics targeting NPSARS-CoV-2. IMPORTANCE In this study, by in vitro assay and live SARS-CoV-2 virus infection, we provide solid evidence that the SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming antiviral innate immunity mediated by G3BP1 in A549 cell lines and G3BP1 conditional knockout mice (g3bp1-cKO) mice, which provide in-depth evidence showing the mechanism underlying NP-related SARS-CoV-2 pathogenesis through G3BPs.


Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins , Poly-ADP-Ribose Binding Proteins , SARS-CoV-2 , Virus Replication , Adaptor Proteins, Signal Transducing/metabolism , Animals , COVID-19/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , DNA Helicases/metabolism , Host Microbial Interactions/immunology , Mice , Phosphoproteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , RNA-Binding Proteins/metabolism , Stress Granules , Virus Replication/genetics
20.
Pharm Biol ; 60(1): 862-878, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1860677

ABSTRACT

CONTEXT: Coronavirus disease 2019 is a global pandemic. Studies suggest that folic acid has antiviral effects. Molecular docking shown that folic acid can act on SARS-CoV-2 Nucleocapsid Phosphoprotein (SARS-CoV-2 N). OBJECTIVE: To identify novel molecular therapeutic targets for SARS-CoV-2. MATERIALS AND METHODS: Traditional Chinese medicine targets and virus-related genes were identified with network pharmacology and big data analysis. Folic acid was singled out by molecular docking, and its potential target SARS-CoV-2 N was identified. Inhibition of SARS-CoV-2 N of folic acid was verified at the cellular level. RESULTS: In total, 8355 drug targets were potentially involved in the inhibition of SARS-CoV-2. 113 hub genes were screened by further association analysis between targets and virus-related genes. The hub genes related compounds were analysed and folic acid was screened as a potential new drug. Moreover, molecular docking showed folic acid could target on SARS-CoV-2 N which inhibits host RNA interference (RNAi). Therefore, this study was based on RNAi to verify whether folic acid antagonises SARS-CoV-2 N. Cell-based experiments shown that RNAi decreased mCherry expression by 81.7% (p < 0.001). This effect was decreased by 8.0% in the presence of SARS-CoV-2 N, indicating that SARS-CoV-2 N inhibits RNAi. With increasing of folic acid concentration, mCherry expression decreased, indicating that folic acid antagonises the regulatory effect of SARS-CoV-2 N on host RNAi. DISCUSSION AND CONCLUSIONS: Folic acid may be an antagonist of SARS-CoV-2 N, but its effect on viruses unclear. In future, the mechanisms of action of folic acid against SARS-CoV-2 N should be studied.


Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins , Folic Acid , SARS-CoV-2 , COVID-19/drug therapy , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Folic Acid/pharmacology , Humans , Molecular Docking Simulation , Phosphoproteins/antagonists & inhibitors
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