Deep mutational scanning identifies SARS-CoV-2 Nucleocapsid escape mutations of currently available rapid antigen tests.

The effects of mutations in continuously emerging variants of SARS-CoV-2 are a major concern for the performance of rapid antigen tests. To evaluate the impact of mutations on 17 antibodies used in 11 commercially available antigen tests with emergency use authorization, we measured antibody binding for all possible Nucleocapsid point mutations using a mammalian surface-display platform and deep mutational scanning. The results provide a complete map of the antibodies' epitopes and their susceptibility to mutational escape. Our data predict no vulnerabilities for detection of mutations found in variants of concern. We confirm this using the commercial tests and sequence-confirmed COVID-19 patient samples. The antibody escape mutational profiles generated here serve as a valuable resource for predicting the performance of rapid antigen tests against past, current, as well as any possible future variants of SARS-CoV-2, establishing the direct clinical and public health utility of our system.

Single-Cell Profiling of the Differential In Vivo Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Infection Among Lung Tissue Cell Subtypes at the Protein Level.

The complexity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its variants in lung cells can truly be characterized only at the tissue and protein levels among unique cell subtypes. However, in vivo data are limited due to lack of accessible human tissues. Using a transgenic mouse model of SARS-CoV-2 infection and flow cytometry, we provide in vivo novel insight at the protein level that the differential impact of SARS-CoV-2 (Wuhan strain) and its B.1.617.2 (Delta) and BA.1 (Omicron) variants on lung may be attributed to differential patterns of viral protein levels among ciliated airway cells, alveolar types 1 and 2 cells, immune cells, and endothelial lung cells.

Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

SARS-CoV-2 Nucleocapsid Protein Is Not Responsible for Over-Activation of Complement Lectin Pathway.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral structural protein that is abundant in the circulation of infected individuals. Previous published studies reported controversial data about the role of the N protein in the activation of the complement system. It was suggested that the N protein directly interacts with mannose-binding lectin-associated serine protease-2 (MASP-2) and stimulates lectin pathway overactivation/activity. In order to check these data and to reveal the mechanism of activation, we examined the effect of the N protein on lectin pathway activation. We found that the N protein does not bind to MASP-2 and MASP-1 and it does not stimulate lectin pathway activity in normal human serum. Furthermore, the N protein does not facilitate the activation of zymogen MASP-2, which is MASP-1 dependent. Moreover, the N protein does not boost the enzymatic activity of MASP-2 either on synthetic or on protein substrates. In some of our experiments, we observed that MASP-2 digests the N protein. However, it is questionable, whether this activity is biologically relevant. Although surface-bound N protein did not activate the lectin pathway, it did trigger the alternative pathway in 10% human serum. Additionally, we detected some classical pathway activation by the N protein. Nevertheless, we demonstrated that this activation was induced by the bound nucleic acid, rather than by the N protein itself.

Susceptibility of SARS COV-2 nucleocapsid and spike proteins to reactive oxygen species and role in inflammation.

Chemiluminescence was used to test the susceptibility of the SARS-CoV-2 N and S proteins to oxidation by reactive oxygen species (ROS) at pH 7.4 and pH 8.5. The Fenton's system generates various ROS (H2O2, OH, -OH, OOH). All proteins were found to significantly suppress oxidation (the viral proteins exhibited 25-60% effect compared to albumin). In the second system, H2O2 was used both as a strong oxidant and as a ROS. A similar effect was observed (30-70%); N protein approached the effect of albumin at physiological pH (∼45%). In the O2.--generation system, albumin was most effective in the suppression of generated radicals (75%, pH 7.4). The viral proteins were more susceptible to oxidation (inhibition effect no more than 20%, compared to albumin). The standard antioxidant assay confirmed the strong antioxidant capacity of both viral proteins (1.5-1.7 fold higher than albumin). These results demonstrate the effective and significant inhibition of ROS-induced oxidation by the proteins. Obviously, the viral proteins could not be involved in the oxidative stress reactions during the course of the infection. They even suppress the metabolites involved in its progression. These results can be explained by their structure. Probably, an evolutionary self-defense mechanism of the virus has been developed.

Aptamers dimerization inspired biomimetic clamp assay towards impedimetric SARS-CoV-2 antigen detection.

Antigen-detecting rapid diagnostic testing (Ag-RDT) has contributed to containing the spread of SARS-CoV-2 variants of concern (VOCs). In this study, we proposed a biomimetic clamp assay for impedimetric SARS-CoV-2 nucleocapsid protein (Np) detection. The DNA biomimetic clamp (DNA-BC) is formed by a pair of Np aptamers connected via a T20 spacer. The 5'- terminal of the DNA-BC is phosphate-modified and then anchored on the surface of the screen-printed gold electrode, which has been pre-coated with Au@UiO-66-NH2. The integrated DNA-material sensing biochip is fabricated through the strong Zr-O-P bonds to form a clamp-type impedimetric aptasensor. It is demonstrated that the aptasensor could achieve Np detection in one step within 11 min and shows pronounced sensitivity with a detection limit of 0.31 pg mL-1. Above all, the aptasensor displays great specificity and stability under physiological conditions as well as various water environments. It is a potentially promising strategy to exploit reliable Ag-RDT products to confront the ongoing epidemic.

SARS-CoV-2 infection among healthcare workers whom already received booster vaccination during epidemic outbreak of omicron variant in Taiwan.

BACKGROUND/PURPOSE: Healthcare workers (HCWs) are at risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection due to occupational exposure. We aim to investigate the prevalence and risk factors of SARS-CoV-2 infection among HCWs during epidemic outbreak of omicron variant in Taiwan. METHODS: Sequential reserved serum samples collected from our previous study during December 2021 and July 2022 were tested for antibodies against SARS-CoV-2 nucleocapsid protein (NP). Diagnosis of SARS-CoV-2 infection was defined as positive either of anti-SARS-CoV-2 nucleoprotein, rapid antigen test or polymerase chain reaction. Retrospective chart review and a questionnaire were used to access the symptoms and risk factors for SARS-CoV-2 infection. RESULTS: Totally 300 participants (69.3% female) with a median age of 37.9 years were enrolled. A significant increase incidence of SARS-CoV-2 infection was found before and during community outbreak (11.91 versus 230.93 per 100,000 person-days, P < 0.001), which was a trend paralleling that observed in the general population. For 61 SARS-CoV-2 infected participants, nine (14.8%) were asymptomatic. Multivariate analysis revealed recent contact with a SARS-CoV-2 infected household (odds ratio [OR], 7.01; 95% confidence interval [95% CI], 3.70-13.30; P < 0.001) and co-existed underlying autoimmune diseases (OR, 4.46; 95% CI, 1.28-15.51; P = 0.019) were significant risk factors associated with acquisition of SARS-CoV-2 infection among HCWs. CONCLUSION: Community factors, such as closely contact with SARS-CoV-2 infected individuals and underlying immune suppression status, were significant factors for acquisition of SARS-CoV-2 infection among HCWs. We suggest the application of appropriate infection control measures for HCWs should be maintained to reduce risk of SARS-CoV-2 infection.

Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips.

The coronavirus disease 2019 (COVID-19) is outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in viral replication, assembly, and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drug target. Therefore, by synthesizing the N gene sequence of SARS-CoV-2, constructing the pET-28a (+)-N recombinant plasmid, we expressed the N protein in Escherichia coli and obtained 15 monoclonal antibody (mAbs) against SARS-CoV-2-N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established. In this study, we obtained 14 high-titer and high-specificity monoclonal antibodies, and the test strips exclusively react with the SARS-CoV-2-N protein and no cross-reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS-CoV-2 infection through serological antigen.

[Clinical and morphological characteristics of SARS-CoV-2-related myocarditis proven by the presence of viral RNA and proteins in myocardial tissue]. / Kliniko-morfologicheskaya kharakteristika SARS-CoV-2-assotsiirovannogo miokardita, podtverzhdennogo nalichiem RNK i belkov virusa v tkani miokarda.

OBJECTIVE: To investigate the clinical and morphological features of SARS-CoV-2-related myocarditis, by determining the presence of viral RNA and proteins in myocardial tissue. MATERIAL AND METHODS: The study was conducted to examine the material of 32 autopsies with a confirmed diagnosis of myocarditis. There were data of a morphological study, including a standard histological study, as well as immunohistochemical determination of the surface markers CD45, CD3, CD20, and CD68 cells of an inflammatory infiltrate and virus proteins (SARS-CoV-2 nucleocapsid protein and spike protein). Positive and negative control tests were carried out. In addition, coronavirus RNA was detected in the myocardium using a polymerase chain reaction. RESULTS: Polymerase chain reaction (PCR) revealed viral RNA in myocardial tissue. Viral proteins were identified in the macrophages of an inflammatory infiltrate and cardiomyocytes. CONCLUSION: The findings may suggest that the virus persists in the myocardium and chronic myocarditis develops.

A universal three-dimensional hydrogel electrode for electrochemical detection of SARS-CoV-2 nucleocapsid protein and hydrogen peroxide.

Coronavirus disease 2019 (COVID-19) is a highly contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in a global health crisis. The primary diagnostic method for COVID-19 is quantitative reverse transcription PCR, which is time-consuming and requires expensive instrumentation. Here, we developed an electrochemical biosensor for detecting SARS-CoV-2 biomarkers using a 3D porous polyacrylamide/polyaniline hydrogel (PPG) electrode prepared by UV photopolymerization and in situ polymerization. The electrochemical immunosensor for detecting SARS-CoV-2 N protein via the immune sandwich principle demonstrated a lower detection limit of 42 pg/mL and comparable specificity to a commercial enzyme-linked immunosorbent assay, which was additionally validated in pseudoviruses. The electrochemical sensor for hydrogen peroxide showed a low detection limit of 0.5 µM and excellent selectivity, which was further confirmed in cancer cells under oxidative stress. The biomarkers of SARS-CoV-2 were successfully detected due to the signal amplification capability provided by 3D porous electrodes and the high sensitivity of the antigen-antibody specific binding. This study introduces a novel three-dimensional electrode with great potential for the early detection of SARS-CoV-2.

Dual-Mode Lateral Flow Immunoassay Based on "Pompon Mum"-Like Fe3O4@MoS2@Pt Nanotags for Sensitive Detection of Viral Pathogens.

Lateral flow immunoassay (LFIA) has been widely used for the early diagnosis of diseases. However, conventional colorimetric LFIA possesses limited sensitivity, and the single-mode readout signal is easily affected by the external environment, leading to insufficient accuracy. Herein, multifunctional Fe3O4@MoS2@Pt nanotags with a unique "pompon mum"-like structure were triumphantly prepared, exhibiting excellent peroxidase (POD)-like activity, photothermal properties, and magnetic separation capability. Furthermore, the Fe3O4@MoS2@Pt nanotags were used to establish dual-mode LFIA (dLFIA) for the first time, enabling the catalytic colorimetric and photothermal dual-mode detection of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein (SARS-CoV-2 NP) and influenza A (H1N1). The calculated limits of detection (cLODs) of SARS-CoV-2 NP and H1N1 were 80 and 20 ng/mL in catalytic colorimetric mode and 10 and 8 ng/mL in photothermal mode, respectively, demonstrating about 100 times more sensitive than the commercial colloidal Au-LFIA strips (1 ng/mL for SARS-CoV-2 NP; 1 µg/mL for H1N1). The recovery rates of dLFIA in simulated nose swab samples were 95.2-103.8% with a coefficient of variance of 2.3-10.1%. These results indicated that the proposed dLFIA platform showed great potential for the rapid diagnosis of respiratory viruses.

Effectiveness and duration of additional immune defense provided by SARS-CoV-2 infection before and after receiving the mRNA COVID-19 vaccine BNT162b2.

Background: Our investigation focused whether infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before or after receiving the mRNA COVID-19 vaccine can increase immune protection. And we also investigated relationship of infection acquired. Methods: Three shots of the mRNA coronavirus disease 2019 (COVID-19) vaccine BNT162b2 were administered to 736 healthcare workers at Tokyo Shinagawa Hospital. Serum samples were collected before the first shot (P1), at one month (P2), and at six months (P3) after the second shot and at one month after the third shot (P4). The presence of infection was assessed using IgG against the nucleocapsid (IgG (N) and RBD in the spike protein of SARS-CoV-2. We defined infection before P2 as natural infection (NI) and infection between P2 and P3 as breakthrough infection (BI) and compared susceptibility to further infection between the NI (-) and NI (+) groups and between BI (-) and BI (+) groups. Events in 485 participants who had a complete dataset of IgG (N) and IgG (RBD) from P1 to P4 were analyzed. Results: The presence of SARS-CoV-2 infection before P2 were examined by examining the titers of IgG (N)P1, IgG (N) P2, and IgG (RBD) P1 that exceeded the cutoff values. Consequently, 35 participants (7.22 %) were categorized into the NI (+) group, whereas 450 (92.8 %) were categorized into the NI (-) group. Between P2 and P3, the NI (-) group showed a higher rate of SARS-CoV-2 infection than the NI (+) group; however, there was no significant difference in the infection rate between P3 and P4. The infection rate was significantly lower in the BI (+) group than in the BI (-) group. Pre-primary vaccination infection significantly increased IgG (RBD) levels between P1 and P3. Post-primary vaccination infection significantly increased IgG (RBD) levels between P3 and P4. Conclusions: Infection with SARS-CoV-2 before or after receiving the mRNA COVID-19 vaccine can increase immune protection; however, the duration of this effect may be limited.

A self-enhanced electrochemiluminescence array chip for portable label-free detection of SARS-CoV-2 nucleocapsid protein with smartphone.

SARS-CoV-2 antigen detection plays a key role in the rapid diagnosis of COVID-19. However, current clinically used immunoassays are often limited by assay throughput, sensitivity, accuracy, and field operating conditions. To address these challenges, we constructed a self-enhanced electrochemiluminescence (ECL) array chip (SE2AC) for highly sensitive and label-free detection of SARS-CoV-2 nucleocapsid protein (N protein) with a facile and portable assay setup. Firstly, the self-enhanced ECL nanomaterials with inherent film-forming properties were synthesized by co-doping Ru(bpy)32+ and polyethyleneimine (PEI) in silica nanoparticles (Ru/PEI@SiO2). Secondly, a resistance-induced potential difference-based single-electrode electrochemical system (SEES) was adapted to serve as the electrode array to facilitate one-step assembly without the need for chip alignment. Thirdly, the chip electrode array was functionalized with the synthesized self-enhanced ECL emitters and captured antibodies. In addition, a portable detection box equipped with a smartphone was 3D-printed to serve as the chip holder and "dark room" for imaging acquisition. The SE2AC performance was validated with N protein with a limit of detection (LOD) of 0.47 pg/mL in the range of 1-10,000 pg/mL. Furthermore, the chip successfully detected the viral antigen residue as low as 1.92 pg/mL from diluted rehabilitation patients' serum samples. This is the first study reporting label-free detection of SARS-Cov-2 N protein based on a self-enhanced ECL immunosensor, which provides a novel facile method for highly sensitive diagnosis of COVID-19 with high throughput, portability, and low cost.

CdTe QDs-sensitized TiO2 nanocomposite for magnetic-assisted photoelectrochemical immunoassay of SARS-CoV-2 nucleocapsid protein.

A sensitive, reliable, and cost-effective detection for SARS-CoV-2 was urgently needed due to the rapid spread of COVID-19. Here, a "signal-on" magnetic-assisted PEC immunosensor was constructed for the quantitative detection of SARS-CoV-2 nucleocapsid (N) protein based on Z-scheme heterojunction. Fe3O4@SiO2@Au was used to connect the capture antibody to act as a capture probe (Fe3O4@SiO2@Au/Ab1). It can extract target analytes selectively in complex samples and multiple electrode rinsing and assembly steps were avoided effectively. CdTe QDs sensitized TiO2 coated on the surface of SiO2 spheres to form Z-scheme heterojunction (SiO2@TiO2@CdTe QDs), which broadened the optical absorption range and inhibited the quick recombination of photogenerated electron/hole of the composite. With fascinating photoelectric conversion performance, SiO2@TiO2@CdTe QDs were utilized as a signal label, thus further realizing signal amplification. The migration mechanism of photogenerated electrons was further deduced by active material quenching experiment and electron spin resonance (ESR) measurement. The elaborated immunosensor can detect SARS-CoV-2 N protein in the linear range of 0.005-50 ng mL-1 with a low detection limit of 1.8 pg mL-1 (S/N = 3). The immunosensor displays extraordinary sensitivity, strong anti-interference, and high reproducibility in detecting SARS-CoV-2 N protein, which envisages its potential application in the clinical diagnosis of COVID-19.

Hybridization Chain Reaction Lateral Flow Assays for Amplified Instrument-Free At-Home SARS-CoV-2 Testing.

The lateral flow assay format enables rapid, instrument-free, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience is maintained using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 min. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/µL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/µL, 1000 copies/µL, 2000 copies/µL, 2000 copies/µL, and 20,000 copies/µL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious diseases. As an alternative to viral protein detection, we further introduce an HCR lateral flow assay for viral RNA detection.

Inhaled ciclesonide does not affect production of antibodies or elimination of virus in patients with COVID-19: Subanalysis of a multicenter, open-label randomized trial.

During an earlier multicenter, open-label, randomized controlled trial designed to evaluate the effectiveness of high-dose inhaled ciclesonide in patients with asymptomatic or mild coronavirus disease 2019 (COVID-19), we observed that worsening of shadows on CT without worsening of clinical symptoms was more common with ciclesonide. The present study sought to determine if an association exists between worsening CT shadows and impaired antibody production in patients treated with inhaled ciclesonide. Eighty-nine of the 90 patients in the original study were prospectively enrolled. After exclusions, there were 36 patients each in the ciclesonide and control groups. We analyzed antibody titers against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein at various time points. Changes in viral load during treatment were compared. There was no significant difference in age, sex, body mass index, background clinical characteristics, or symptoms between the two groups. Although evaluation on day 8 suggested a greater tendency for worsening shadows on CT in the ciclesonide group (p = 0.072), there was no significant difference between them in the ability to produce antibodies (p = 0.379) or the maximum antibody titer during the clinical course. In both groups, worsening CT shadows and higher viral loads were observed on days 1-8, suggesting ciclesonide does not affect clearance of the virus (p = 0.134). High-dose inhaled ciclesonide did not impair production of antibodies against SARS-CoV-2 or affect elimination of the virus, suggesting that this treatment can be used safely in patients with COVID-19 patients who use inhaled steroids for asthma and other diseases.

Structural interactions of phytoconstituent(s) from cinnamon, bay leaf, oregano, and parsley with SARS-CoV-2 nucleocapsid protein: A comparative assessment for development of potential antiviral nutraceuticals.

SARS-CoV-2 has been responsible for causing 6,218,308 deaths globally till date and has garnered worldwide attention. The lack of effective preventive and therapeutic drugs against SARS-CoV-2 has further worsened the scenario and has bolstered research in the area. The N-terminal and C-terminal RNA binding domains (NTD and CTD) of SARS-CoV-2 nucleocapsid protein represent attractive therapeutic drug targets. Naturally occurring compounds are an excellent source of novel drug candidates due to their structural diversity and safety. Ten major bioactive compounds were identified in ethanolic extract (s) of Cinnamomum zeylanicum, Cinnamomum tamala, Origanum vulgare, and Petroselinum crispum using HPLC and their cytotoxic potential was determined against cancer and normal cell lines by MTT assay to ascertain their biological activity in vitro. To evaluate their antiviral potential, the binding efficacy to NTD and CTD of SARS-CoV-2 nucleocapsid protein was determined using in silico biology tools. In silico assessment of the phytocomponents revealed that most of the phytoconstituents displayed a druglike character with no predicted toxicity. Binding affinities were in the order apigenin > catechin > apiin toward SARS-CoV-2 nucleocapsid NTD. Toward nucleocapsid CTD, the affinity decreased as apigenin > cinnamic acid > catechin. Remdesivir displayed lesser affinity with NTD and CTD of SARS-CoV-2 nucleocapsid proteins than any of the studied phytoconstituents. Molecular dynamics (MD) simulation results revealed that throughout the 100 ns simulation, SARS-CoV-2 nucleocapsid protein NTD-apigenin complex displayed greater stability than SARS-CoV-2 nucleocapsid protein NTD-cinnamic acid complex. Hence, apigenin, catechin, apiin and cinnamic acid might prove as effective prophylactic and therapeutic candidates against SARS-CoV-2, if examined further in vitro and in vivo. PRACTICAL APPLICATIONS: Ten major bioactive compounds were identified in the extract(s) of four medicinally important plants viz. Cinnamomum zeylanicum, Cinnamomum tamala, Origanum vulgare and Petroselinum crispum using HPLC and their biological activity was also evaluated against cancer and normal cell lines. Interestingly, while all extract(s) wielded significant cytotoxicity against cancer cells, no significant toxicity was found against normal cells. The outcome of the results prompted evaluation of the antiviral potential of the ten bioactive compounds using in silico biology tools. The present study emphasizes on the application of computational approaches to understand the binding interaction and efficacy of the ten bioactive compounds from the above plants with SARS-CoV-2 nucleocapsid protein N-terminal and C-terminal RNA binding domains in preventing and/or treating COVID-19 using in silico tools. Druglikeness and toxicity profiles of the compounds were carried out to check the therapeutic application of the components. Additionally, molecular dynamics (MD) simulation was performed to check the stability of ligand-protein complexes. The results provided useful insights into the structural binding interaction(s) that can be exploited for the further development of potential antiviral agents targeting SARS-CoV-2 especially since no specific therapy is still available to combat the rapidly evolving virus and the existing treatment is more or less symptomatic which makes search for novel antiviral agents all the more necessary and crucial.

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids.

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.

Horseradish peroxidase-triggered direct in situ fluorescent immunoassay platform for sensing cardiac troponin I and SARS-CoV-2 nucleocapsid protein in serum.

Direct in situ fluorescent enzyme-linked immunosorbent assay (ELISA) is rarely investigated and reported. Herein, a direct in situ high-performance HRP-labeled fluorescent immunoassay platform was constructed. The platform was developed based on a rapid in situ fluorogenic reaction between Polyethyleneimine (PEI) and p-Phenylenediamine (PPD) analogues to generate fluorescent copolymer nanoparticles (FCNPs). The formation mechanism of FCNPs was found to be the oxidation of •OH radicals, which was further proved by nitrogen protection and scavenger of •OH radicals. Meantime, the fluorescence wavelength of FCNPs could be adjusted from 471 to 512 nm by introducing various substitution groups into the PPD structure. Using cardiac troponin I (cTnI) and SARS-CoV-2 nucleocapsid protein (N-protein) as the model antigens, the proposed fluorescent ELISA exhibited a wide dynamic range of 5-180 ng/mL and a low limit of detection (LOD) of 0.19 ng/mL for cTnI, and dynamic range of 0-120 ng/mL and a LOD of 0.33 ng/mL for SARS-CoV-2 N protein, respectively. Noteworthy, the proposed method was successful applied to evaluate the cTnI and SARS-CoV-2 N protein levels in serum with satisfied results. Therefore, the proposed platform paved ways for developing novel fluorescence-based HRP-labeled ELISA technologies and broadening biomarker related clinical diagnostics.

Electrochemical biosensors for the detection of SARS-CoV-2 pathogen and protein biomarkers.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV CoV-2) pathogen and protein biomarkers can improve the diagnosis accuracy for Coronavirus disease 2019 (COVID-19). Electrochemical biosensors have attracted extensive attention in the scientific community because of their simple design, fast response, good portability, high sensitivity and high selectivity. In this review, we summarized the progress in the electrochemical detection of COVID-19 pathogen and SARS-CoV-2 biomarkers, including SARS-CoV-2 spike protein and nucleocapsid protein and their antibodies.