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2.
Journal of Light Industry ; 37(4):34-40, 2022.
Article in Chinese | Academic Search Complete | ID: covidwho-2025551

ABSTRACT

Bioinformatics methods were used to predict the hydrophilicity, hydrophobicity, antigen epitopes and analyse multiple sequence alignment of the nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The recombinant plasmid pET28a/ N was constructed. In the prokaryotic expression system of Escherichia coli, the solubility and expression level of the protein were improved by adjusting the change of induction temperature and time, and the expressed recombinant N protein was purified and identified. The results showed that SARS-CoV-2 N encoded 419 amino acids, with an isoelectric point (PI) of 10.10, no transmembrane region, no signal peptide sequence, and strong local hydrophilicity. The full-length protein had a high antigenic index and was highly conserved, and its homology with SARS-CoV N protein was 90.5%. After fermentation with Escherichia coli prokaryotic expression system, the engineering strain BL21 (DE3)/pET28a/N was induced at 16 °C for 20 h with the final IPTG concentration of 0.2 mmol/L, and the protein was soluble and most pressed at this time, accounting for 70% of the total protein expression. The target protein purified by Ni-NTA affinity chromatography and gel filtration chromatography had a purity of 90% and a molecular weight of 55 kDa, which was specific. [ FROM AUTHOR] Copyright of Journal of Light Industry is the property of Journal of Zhengzhou University of Light Industry, Natural Science Edition and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

3.
Biophys Rev ; 14(3): 709-715, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1982362

ABSTRACT

SARS-CoV-2 is the coronavirus causing the ongoing pandemic with > 460 millions of infections and > 6 millions of deaths. SARS-CoV-2 nucleocapsid (N) is the only structural protein which plays essential roles in almost all key steps of the viral life cycle with its diverse functions depending on liquid-liquid phase separation (LLPS) driven by interacting with various nucleic acids. The 419-residue N protein is highly conserved in all variants including delta and omicron, and composed of both folded N-/C-terminal domains (NTD/CTD) as well as three long intrinsically disordered regions (IDRs). Recent results have suggested that its CTD and IDRs are also cryptic nucleic acid-binding domains. In this context, any small molecules capable of interfering in its interaction with nucleic acids are anticipated to modulate its LLPS and associated functions. Indeed, ATP, the energy currency existing at very high concentrations (2-12 mM) in all living cells but absent in viruses, modulates LLPS of N protein, and consequently appears to be evolutionarily hijacked by SARS-CoV-2 to promote its life cycle. Hydroxychloroquine (HCQ) has been also shown to specifically bind NTD and CTD to inhibit their interactions with nucleic acids, as well as to disrupt LLPS. Particularly, the unique structure of the HCQ-CTD complex offers a promising strategy for further design of anti-SARS-CoV-2 drugs with better affinity and specificity. The finding may indicate that LLPS is indeed druggable by small molecules, thus opening up a promising direction for drug discovery/design by targeting LLPS in general.

4.
Asia-Pacific Journal of Molecular Biology and Biotechnology ; 30:50, 2022.
Article in English | ProQuest Central | ID: covidwho-1981291

ABSTRACT

Introduction: The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARSCoV2) is the causative agent responsible for the COVID-19 pandemic and has resulted in devastating impacts on global public health. The nucleocapsid (N) protein of other coronaviruses, such as SARS-CoV-1, have been previously implicated in the deregulation of the host cell cycle through interactions with cell cycle checkpoint proteins, Cyclin-Dependent Kinases (CDKs) or cyclins. In this study, we highlight the role of SARS-CoV-2 N-protein in modulating CDK expression, thereby, deregulating the host cell cycle. Methods: A549 cells were transfected with pCMV plasmids, harbouring the SARS-CoV-2 N-protein. Protein extracts of control and Nprotein transfected cells were electrophoresed on SDS-PAGE, transferred onto a nitrocellulose membrane and incubated with CDK2 and CDK4 antibodies. The blots were visualized and protein quantification was performed using ImageJ analysis. Results: Transfection of SARS-CoV-2 N resulted in differential expression of CDK2 and CDK4, which are key regulators that drive cell cycle progression through G0 or G1 phase into S phase. Notably, preliminary findings also demonstrate that N protein results in decreased CDK2 and CDK4 expression. Conclusion: The differential expression of CDKs caused by SARS-CoV-2 N-protein suggests its role in inducing cell cycle arrest at the S phase to facilitate SARS-CoV-2 replication. The results from this research may aid in future characterisation of the mechanisms for SARS-CoV-2-mediated cell cycle arrest, and contribute towards the development of novel antiviral strategies and therapies.

5.
Asia-Pacific Journal of Molecular Biology and Biotechnology ; 30:81, 2022.
Article in English | ProQuest Central | ID: covidwho-1981290

ABSTRACT

Introduction: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), responsible for the coronavirus disease 2019 (COVID-19) pandemic, has resulted in significantly disruptive global impacts. Cytokine storm syndrome (CSS) can accompany SARSCoV2 infection, and involves the excessive release of pro-inflammatory cytokines that lead to acute respiratory distress syndrome (ARDS) in infected patients. Given the correlation between ARDS and poor patient prognosis, inflammatory pathways (e.g., interferon-1 (IFN-1)) would be a target area for antiviral development. Our preliminary results have demonstrated a direct correlation between the SARS-CoV-2 nucleocapsid (N) protein and host intracellular IFN-1 pathway components IRF3 and STAT1. Methods: A549 cells were transfected with pCMV-GFP vectors expressing N protein and harvested. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting were performed. The membranes were then incubated with STAT-1, p-STAT1 and IRF3 antibodies and visualised. Protein content was quantified using ImageJ software. Results: Transfection with SARS-CoV-2 N was correlated with a decrease in intracellular IRF3 and reduced phosphorylation of STAT1, suggesting the involvement of N protein in the delayed IFN-1 response commonly observed in SARS-CoV-2 patients. These findings suggest that IRF3 and STAT1 may be part of the innate immune response affected by SARS-CoV-2 infection. Conclusion: Our results show that IRF3 and STAT1 are responsible for stimulating transcription of interferon signalling genes (ISGs). Future studies on SARS-CoV-2 N and its downstream effectors could provide further insight into the IFN-1 response during infection, and assist in future antiviral development strategies.

6.
Med Nov Technol Devices ; 15: 100159, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1966945

ABSTRACT

The mortality rate of the recent global pandemic corona virus disease 2019 (COVID-19) is currently as high as 7%. The SARS-CoV-2 virus is the culprit behind COVID-19. SARS-CoV-2 is an enveloped single-stranded RNA virus, the genome encodes four types of the structural proteins: S protein, E protein (envelope protein), M protein (matrix protein) and N protein (nucleocapsid protein). In COVID-19, monoclonal antibodies have played a significant role in diagnosis and treatment. This article briefly introduced the development of monoclonal antibodies targeting on S protein and N protein, which represents the main direction of monoclonal antibody drugs used in the diagnosis and treatment of COVID-19. Meanwhile, the traditional Chinese medicine also plays important role in the fight against COVID-19 by regulating human immunity. The article introduced the use of traditional Chinese medicine in fighting against COVID-19.

7.
J Virol ; 96(13): e0061822, 2022 07 13.
Article in English | MEDLINE | ID: covidwho-1962091

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) is the globally distributed alphacoronavirus that can cause lethal watery diarrhea in piglets, causing substantial economic damage. However, the current commercial vaccines cannot effectively the existing diseases. Thus, it is of great necessity to identify the host antiviral factors and the mechanism by which the host immune system responds against PEDV infection required to be explored. The current work demonstrated that the host protein, the far upstream element-binding protein 3 (FUBP3), could be controlled by the transcription factor TCFL5, which could suppress PEDV replication through targeting and degrading the nucleocapsid (N) protein of the virus based on selective autophagy. For the ubiquitination of the N protein, FUBP3 was found to recruit the E3 ubiquitin ligase MARCH8/MARCHF8, which was then identified, transported to, and degraded in autolysosomes via NDP52/CALCOCO2 (cargo receptors), resulting in impaired viral proliferation. Additionally, FUBP3 was found to positively regulate type-I interferon (IFN-I) signaling and activate the IFN-I signaling pathway by interacting and increasing the expression of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3). Collectively, this study showed a novel mechanism of FUBP3-mediated virus restriction, where FUBP3 was found to degrade the viral N protein and induce IFN-I production, aiming to hinder the replication of PEDV. IMPORTANCE PEDV refers to the alphacoronavirus that is found globally and has re-emerged recently, causing severe financial losses. In PEDV infection, the host activates various host restriction factors to maintain innate antiviral responses to suppress virus replication. Here, FUBP3 was detected as a new host restriction factor. FUBP3 was found to suppress PEDV replication via the degradation of the PEDV-encoded nucleocapsid (N) protein via E3 ubiquitin ligase MARCH8 as well as the cargo receptor NDP52/CALCOCO2. Additionally, FUBP3 upregulated the IFN-I signaling pathway by interacting with and increasing tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) expression. This study further demonstrated that another layer of complexity could be added to the selective autophagy and innate immune response against PEDV infection are complicated.


Subject(s)
Coronavirus Infections , Interferon Type I , Nucleocapsid Proteins , Porcine epidemic diarrhea virus , Transcription Factors , Animals , Antiviral Agents , Cell Line , Chlorocebus aethiops , Coronavirus Infections/metabolism , Interferon Type I/genetics , Interferon Type I/metabolism , Nucleocapsid Proteins/metabolism , Porcine epidemic diarrhea virus/physiology , Swine , TNF Receptor-Associated Factor 3 , Transcription Factors/metabolism , Ubiquitin-Protein Ligases , Vero Cells
8.
Biosens Bioelectron ; 213: 114436, 2022 Oct 01.
Article in English | MEDLINE | ID: covidwho-1944325

ABSTRACT

The emergence of the COVID-19 epidemic has affected the lives of hundreds of millions of people globally. There is no doubt that the development of fast and sensitive detection methods is crucial while the worldwide effective vaccination programs are miles away from actualization. In this study, we have reported an electrochemical N protein aptamer sensor with complementary oligonucleotide as probe for the specific detection of COVID-19. The electrochemical aptasensor was prepared by fixing the double-stranded DNA hybrid obtained by the hybridization of N protein aptamer and its Fc-labeled complementary strand on the surface of a gold electrode. After incubation with the target, the aptamer dissociated from the labeled complementary DNA oligonucleotide hybrid to preferentially bind with N protein in the solution. The concentration of N protein was measured by detecting the changes in electrochemical current signals induced by the conformational transformation of the complementary DNA oligonucleotide left on the electrode surface. The sensor had a linear relationship between the logarithm of the N protein concentration from 10 fM to 100 nM (ΔIp = 0.098 log CN protein/fM - 0.08433, R2 = 0.99), and the detection limitation was 1 fM (S/N = 3). The electrochemical aptamer sensor was applied to test the spiked concentrations of throat swabs and blood samples from three volunteers, and the obtained results proved that the sensor has great potentials for the early detection of COVID-19 in patients.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , COVID-19 , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , COVID-19/diagnosis , DNA, Complementary , Electrochemical Techniques/methods , Electrodes , Gold/chemistry , Humans , Limit of Detection , Protein Binding
9.
Indian Journal of Chemistry ; 61(4):370-384, 2022.
Article in English | Web of Science | ID: covidwho-1925432

ABSTRACT

Amid the pandemic COVID-19, there is a desperate and urgent need for a therapeutic solution for COVID-19. Our present studies have adapted the SAR-based approach to explore in silico several selected ferrocene-based complexes as the potential inhibitors of the major viral proteins (Spike, RdRp, M-pro, N protein) of the SARS-CoV-2 virus. The SAR-based molecular docking studies have revealed that compound 1 is the strongest inhibitor of the major viral proteins with a binding energy of >9.0 kcal/mol. Compound 1 is also able to inhibit the human Ca2+ channel and thereby potentially able to prevent the strong inflammatory signalling cascades causing severe respiratory distress to the COVID-19 patients. Overall, our computational studies explored ferrocene-based compounds as the emerging multi-targeting therapeutic solution for COVID-19 by inhibiting viral replication as well as modulating the inflammatory signalling cascades.

10.
Biosensors (Basel) ; 12(7)2022 Jun 22.
Article in English | MEDLINE | ID: covidwho-1911186

ABSTRACT

The pandemic of new coronary pneumonia caused by the COVID-19 virus continues to ravage the world. Large-scale population testing is the key to controlling infection and related mortality worldwide. Lateral flow immunochromatographic assay (LFIA) is fast, inexpensive, simple to operate, and easy to carry, very suitable for detection sites. This study developed a COVID-19 N protein detect strip based on p-toluenesulfonyl modified rare earth fluorescent microspheres. The p-toluenesulfonyl-activated nanomaterials provide reactive sulfonyl esters to covalently attach antibodies or other ligands containing primary amino or sulfhydryl groups to the nanomaterial surface. Antibodies are immobilized on these nanomaterials through the Fc region, which ensures optimal orientation of the antibody, thereby increasing the capture rate of the target analyte. The use of buffers with high ionic strength can promote hydrophobic binding; in addition, higher pH could promote the reactivity of the tosyl group. The detection limit of the prepared COVID-19 N protein strips can reach 0.01 ng/mL, so it has great application potential in large-scale population screening.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies , COVID-19/diagnosis , Humans , Immunoassay/methods , Limit of Detection , Microspheres
11.
JPEN J Parenter Enteral Nutr ; 46(6): 1404-1411, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1905907

ABSTRACT

BACKGROUND: Not all patients suffer from a severe course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, demanding a definition of groups at risk. Short bowel syndrome (SBS) has been assumed to be a risk factor, because of the complexity of disease, the need for interdisciplinary care, and frequent contact with caretakers. We aimed to establish data on the course of infection and prevalence of SARS-CoV-2 seropositivity in SBS patients in Germany. METHODS: From January 2021 until January 2022 a total of 119 patients from three different tertiary care centers with SBS were included. All patients received an antibody test against the nucleocapsid (N) antigen and were asked to fill out a questionnaire, which included frequency of contact with medical personnel, risk behavior and worries. RESULTS: Sixty-seven percent of SBS patients received parenteral nutrition with a median of 6 days per week. The seroprevalence of SARS-CoV-2 antibodies was 7.6% (n = 9). Seven patients with positive antibodies had coronavirus disease 2019 (COVID-19) with a mild course. None of the patients were hospitalized or needed further treatment. There was no difference in willingness to take risks in SARS-CoV-2 antibody-positive and -negative patients (P = 0.61). Patients were predominantly worried about the economy (61%) and transmitting COVID-19 (52%), less frequent (26%) about receiving insufficient medical treatment. CONCLUSION: These are the first clinical results concerning SARS-CoV-2 seropositivity and COVID-19 disease in patients with SBS. The seropositivity is comparable to national data, which we attribute to increased risk awareness and avoidance. Further studies are warranted to investigate effects of COVID-19 infection in SBS patients.


Subject(s)
COVID-19 , Short Bowel Syndrome , Adult , Antibodies, Viral , COVID-19/epidemiology , Cross-Sectional Studies , Humans , Prevalence , SARS-CoV-2 , Seroepidemiologic Studies , Short Bowel Syndrome/epidemiology , Short Bowel Syndrome/therapy
12.
Chinese General Practice ; 25(14):1741-1748, 2022.
Article in Chinese | Scopus | ID: covidwho-1863322

ABSTRACT

Background: Based on the current prevalence of Coronavirus Disease 2019 (COVID-19), early diagnosis, isolation, and treatment are important methods to prevent and control infectious diseases. The establishment of convenient and efficient immunochromatographic detection techniques is essential for the prevention and control of COVID-19 epidemic. Objective: To establish a method for the detection of SARS-CoV-2 anti-N protein IgG antibody by immun of luorescence chromatography method based on quantum dots labeling technology in August, 2020. In order to determine whether the detected persons had been infected with COVID-19 or been injected with SARS-CoV-2 inactivated vaccine. Methods The prepared rat anti-human secondary antibody and anti-N protein antibody were immobilized on a Nitrocellulose (NC) membrane as detection line (T) and quality control line (C), respectively. Then the SARS-CoV-2 N protein labeled by quantum dots was evenly sprayed on glass fiber, which was assembled, cut and packaged into test strips after drying. The test strips were used to detect the clinical serum of 35 COVID-19 patients and 50 healthy individuals, the results of the initial screening of the ELISA kit were used as a control to calculate the detection specificity and sensitivity of quantum dots fluorescence immunochromatography. The sensitivity of the test strip was detected by using the N protein antibody standard. Results The specificity and sensitivity of the strip were 100.00%, 94.29%, and the susceptibility was 8.53-17.06 ng/ml antibody concentration. Conclusion: The detection of anti-N protein IgG antibody in serum by quantum dots labeling is simple, fast, with strong sensitivity and specificity. Copyright © 2022 by the Chinese General Practice.

13.
Front Mol Biosci ; 9: 871499, 2022.
Article in English | MEDLINE | ID: covidwho-1834465

ABSTRACT

Epidemics caused by coronaviruses (CoVs), namely the severe acute respiratory syndrome (SARS) (2003), Middle East respiratory syndrome (MERS) (2012), and coronavirus disease 2019 (COVID-19) (2019), have triggered a global public health emergency. Drug development against CoVs is inherently arduous. The nucleocapsid (N) protein forms an oligomer and facilitates binding with the viral RNA genome, which is critical in the life cycle of the virus. In the current study, we found a potential allosteric site (Site 1) using PARS, an online allosteric site predictor, in the CoV N-N-terminal RNA-binding domain (NTD) to modulate the N protein conformation. We identified 5-hydroxyindole as the lead via molecular docking to target Site 1. We designed and synthesized four 5-hydroxyindole derivatives, named P4-1 to P4-4, based on the pose of 5-hydroxyindole in the docking model complex. Small-angle X-ray scattering (SAXS) data indicate that two 5-hydroxyindole compounds with higher hydrophobic R-groups mediate the binding between N-NTD and N-C-terminal dimerization domain (CTD) and elicit high-order oligomerization of the whole N protein. Furthermore, the crystal structures suggested that these two compounds act on this novel cavity and create a flat surface with higher hydrophobicity, which may mediate the interaction between N-NTD and N-CTD. Taken together, we discovered an allosteric binding pocket targeting small molecules that induces abnormal aggregation of the CoV N protein. These novel concepts will facilitate protein-protein interaction (PPI)-based drug design against various CoVs.

14.
Viruses ; 14(4)2022 04 08.
Article in English | MEDLINE | ID: covidwho-1810315

ABSTRACT

Porcine deltacoronavirus (PDCoV) mainly causes severe diarrhea and intestinal pathological damage in piglets and poses a serious threat to pig farms. Currently, no effective reagents or vaccines are available to control PDCoV infection. Single-chain fragment variable (scFv) antibodies can effectively inhibit virus infection and may be a potential therapeutic reagent for PDCoV treatment. In this study, a porcine phage display antibody library from the peripheral blood lymphocytes of piglets infected with PDCoV was constructed and used to select PDCoV-specific scFv. The library was screened with four rounds of biopanning using the PDCoV N protein, and the colony with the highest affinity to the PDCoV N protein was obtained (namely, N53). Then, the N53-scFv gene fragment was cloned into plasmid pFUSE-hIgG-Fc2 and expressed in HEK-293T cells. The scFv-Fc antibody N53 (namely, scFv N53) was purified using Protein A-sepharose. The reactive activity of the purified antibody with the PDCoV N protein was confirmed by indirect enzyme-linked immunosorbent assay (ELISA), western blot and indirect immunofluorescence assay (IFA). Finally, the antigenic epitopes that the scFv N53 recognized were identified by a series of truncated PDCoV N proteins. The amino acid residues 82GELPPNDTPATTRVT96 of the PDCoV N protein were verified as the minimal epitope that can be recognized by the scFv-Fc antibody N53. In addition, the interaction between the scFv-Fc antibody N53 and the PDCoV N protein was further analyzed by molecule docking. In conclusion, our research provides some references for the treatment and prevention of PDCoV.


Subject(s)
Bacteriophages , Coronavirus Infections , Single-Chain Antibodies , Swine Diseases , Animals , Antibodies, Viral , Deltacoronavirus , Epitopes , Nucleocapsid Proteins/genetics , Single-Chain Antibodies/genetics , Swine , Technology
15.
J Med Virol ; 94(5): 2067-2078, 2022 05.
Article in English | MEDLINE | ID: covidwho-1777581

ABSTRACT

Rapid detection of antibodies to SARS-CoV-2 is critical for COVID-19 diagnostics, epidemiological research, and studies related to vaccine evaluation. It is known that the nucleocapsid (N) is the most abundant protein of SARS-CoV-2 and can serve as an excellent biomarker due to its strong immunogenicity. This paper reports a rapid and ultrasensitive 3D biosensor for quantification of COVID-19 antibodies in seconds via electrochemical transduction. This sensor consists of an array of three-dimensional micro-length-scale electrode architecture that is fabricated by aerosol jet 3D printing, which is an additive manufacturing technique. The micropillar array is coated with N proteins via an intermediate layer of nano-graphene and is integrated into a microfluidic channel to complete an electrochemical cell that uses antibody-antigen interaction to detect the antibodies to the N protein. Due to the structural innovation in the electrode geometry, the sensing is achieved in seconds, and the sensor shows an excellent limit of detection of 13 fm and an optimal detection range of 100 fm to 1 nm. Furthermore, the sensor can be regenerated at least 10 times, which reduces the cost per test. This work provides a powerful platform for rapid screening of antibodies to SARS-CoV-2 after infection or vaccination.


Subject(s)
Biosensing Techniques , COVID-19 , Antibodies, Viral , Biosensing Techniques/methods , COVID-19/diagnosis , Electrodes , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
16.
Sens Actuators B Chem ; 362: 131764, 2022 Jul 01.
Article in English | MEDLINE | ID: covidwho-1763980

ABSTRACT

The pandemic of the novel coronavirus disease 2019 (COVID-19) is continuously causing hazards for the world. Effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can relieve the impact, but various toxic chemicals are also released into the environment. Fluorescence sensors offer a facile analytical strategy. During fluorescence sensing, biological samples such as tissues and body fluids have autofluorescence, giving false-positive/negative results because of the interferences. Fluorescence near-infrared (NIR) nanosensors can be designed from low-toxic materials with insignificant background signals. Although this research is still in its infancy, further developments in this field have the potential for sustainable detection of SARS-CoV-2. Herein, we summarize the reported NIR fluorescent nanosensors with the potential to detect SARS-CoV-2. The green synthesis of NIR fluorescent nanomaterials, environmentally compatible sensing strategies, and possible methods to reduce the testing frequencies are discussed. Further optimization strategies for developing NIR fluorescent nanosensors to facilitate greener diagnostics of SARS-CoV-2 for pandemic control are proposed.

17.
Virol J ; 19(1): 50, 2022 03 19.
Article in English | MEDLINE | ID: covidwho-1745445

ABSTRACT

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection. METHODS: We recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background. RESULTS: The optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995-0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative. CONCLUSIONS: We conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.


Subject(s)
COVID-19 , Epstein-Barr Virus Infections , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay , Herpesvirus 4, Human , Humans , Nucleocapsid Proteins , SARS-CoV-2
18.
J Virol ; 94(12)2020 06 01.
Article in English | MEDLINE | ID: covidwho-1723543

ABSTRACT

The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that recently emerged in China is thought to have a bat origin, as its closest known relative (BatCoV RaTG13) was described previously in horseshoe bats. We analyzed the selective events that accompanied the divergence of SARS-CoV-2 from BatCoV RaTG13. To this end, we applied a population genetics-phylogenetics approach, which leverages within-population variation and divergence from an outgroup. Results indicated that most sites in the viral open reading frames (ORFs) evolved under conditions of strong to moderate purifying selection. The most highly constrained sequences corresponded to some nonstructural proteins (nsps) and to the M protein. Conversely, nsp1 and accessory ORFs, particularly ORF8, had a nonnegligible proportion of codons evolving under conditions of very weak purifying selection or close to selective neutrality. Overall, limited evidence of positive selection was detected. The 6 bona fide positively selected sites were located in the N protein, in ORF8, and in nsp1. A signal of positive selection was also detected in the receptor-binding motif (RBM) of the spike protein but most likely resulted from a recombination event that involved the BatCoV RaTG13 sequence. In line with previous data, we suggest that the common ancestor of SARS-CoV-2 and BatCoV RaTG13 encoded/encodes an RBM similar to that observed in SARS-CoV-2 itself and in some pangolin viruses. It is presently unknown whether the common ancestor still exists and, if so, which animals it infects. Our data, however, indicate that divergence of SARS-CoV-2 from BatCoV RaTG13 was accompanied by limited episodes of positive selection, suggesting that the common ancestor of the two viruses was poised for human infection.IMPORTANCE Coronaviruses are dangerous zoonotic pathogens; in the last 2 decades, three coronaviruses have crossed the species barrier and caused human epidemics. One of these is the recently emerged SARS-CoV-2. We investigated how, since its divergence from a closely related bat virus, natural selection shaped the genome of SARS-CoV-2. We found that distinct coding regions in the SARS-CoV-2 genome evolved under conditions of different degrees of constraint and are consequently more or less prone to tolerate amino acid substitutions. In practical terms, the level of constraint provides indications about which proteins/protein regions are better suited as possible targets for the development of antivirals or vaccines. We also detected limited signals of positive selection in three viral ORFs. However, we warn that, in the absence of knowledge about the chain of events that determined the human spillover, these signals should not be necessarily interpreted as evidence of an adaptation to our species.


Subject(s)
Betacoronavirus/genetics , Evolution, Molecular , Selection, Genetic , Amino Acid Sequence , Animals , Betacoronavirus/classification , COVID-19 , Chiroptera/virology , Coronavirus Infections/virology , Genome, Viral/genetics , Humans , Models, Molecular , Open Reading Frames/genetics , Pandemics , Phylogeny , Pneumonia, Viral/virology , SARS-CoV-2 , Viral Proteins/chemistry , Viral Proteins/genetics
19.
Front Immunol ; 13: 776861, 2022.
Article in English | MEDLINE | ID: covidwho-1701723

ABSTRACT

Cardiovascular dysfunction and disease are common and frequently fatal complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Indeed, from early on during the SARS-CoV-2 virus pandemic it was recognized that cardiac complications may occur, even in patients with no underlying cardiac disorders, as part of the acute infection, and that these were associated with more severe disease and increased morbidity and mortality. The most common cardiac complication is acute cardiac injury, defined by significant elevation of cardiac troponins. The potential mechanisms of cardiovascular complications include direct viral myocardial injury, systemic inflammation induced by the virus, sepsis, arrhythmia, myocardial oxygen supply-demand mismatch, electrolyte abnormalities, and hypercoagulability. This review is focused on the prevalence, risk factors and clinical course of COVID-19-related myocardial injury, as well as on current data with regard to disease pathogenesis, specifically the interaction of platelets with the vascular endothelium. The latter section includes consideration of the role of SARS-CoV-2 proteins in triggering development of a generalized endotheliitis that, in turn, drives intense activation of platelets. Most prominently, SARS-CoV-2-induced endotheliitis involves interaction of the viral spike protein with endothelial angiotensin-converting enzyme 2 (ACE2) together with alternative mechanisms that involve the nucleocapsid and viroporin. In addition, the mechanisms by which activated platelets intensify endothelial activation and dysfunction, seemingly driven by release of the platelet-derived calcium-binding proteins, SA100A8 and SA100A9, are described. These events create a SARS-CoV-2-driven cycle of intravascular inflammation and coagulation, which contributes significantly to a poor clinical outcome in patients with severe disease.


Subject(s)
Blood Platelets/metabolism , COVID-19/pathology , Cardiovascular Diseases/pathology , Endothelium, Vascular/metabolism , Platelet Activation/immunology , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/mortality , Cardiovascular Diseases/virology , Coronavirus Nucleocapsid Proteins/immunology , Endothelial Cells/metabolism , Humans , Myocardium/pathology , Phosphoproteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
20.
Inform Med Unlocked ; 29: 100889, 2022.
Article in English | MEDLINE | ID: covidwho-1701459

ABSTRACT

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N-protein) is responsible for viral replication by assisting in viral RNA synthesis and attaching the viral genome to the replicase-transcriptase complex (RTC). Numerous studies suggested the N-protein as a drug target. However, the specific N-protein active sites for SARS-CoV-2 drug treatments are yet to be discovered. The purpose of this study was to determine active sites of the SARS-CoV-2 N-protein by identifying torsion angle classifiers for N-protein structural changes that correlated with the respective angle differences between the active and inactive N-protein. In the study, classifiers with a minimum accuracy of 80% determined from molecular simulation data were analyzed by Principal Component Analysis and cross-validated by Logistic Regression, Support Vector Machine, and Random Forest Classification. The ability of torsion angles ψ252 and φ375 to differentiate between phosphorylated and unphosphorylated structures suggested that residues 252 and 375 in the RNA binding domain might be important in N-protein activation. Furthermore, the φ and ψ angles of residue S189 correlated to a 90.7% structural determination accuracy. The key residues involved in the structural changes identified here might suggest possible important functional sites on the N-protein that could be the focus of further study to understand their potential as drug targets.

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