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Antibodies are protein molecules whose primary function is to recognize antigens. However, recent studies have demonstrated their ability to hydrolyze specific substrates, such as proteins, oligopeptides, and nucleic acids. In 2023, two separate teams of researchers demonstrated the proteolytic activity of natural plasma antibodies from COVID-19 convalescents. These antibodies were found to hydrolyze the S-protein and corresponding oligopeptides. Our study shows that for antibodies with affinity to recombinant structural proteins of the SARS-CoV-2: S-protein, its fragment RBD and N-protein can only hydrolyze the corresponding protein substrates and are not cross-reactive. By using strict criteria, we have confirmed that this proteolytic activity is an intrinsic property of antibodies and is not caused by impurities co-eluting with them. This discovery suggests that natural proteolytic antibodies that hydrolyze proteins of the SARS-CoV-2 virus may have a positive impact on disease pathogenesis. It is also possible for these antibodies to work in combination with other antibodies that bind specific epitopes to enhance the process of virus neutralization. [ABSTRACT FROM AUTHOR]

Antibodies are protein molecules whose primary function is to recognize antigens. However, recent studies have demonstrated their ability to hydrolyze specific substrates, such as proteins, oligopeptides, and nucleic acids. In 2023, two separate teams of researchers demonstrated the proteolytic activity of natural plasma antibodies from COVID-19 convalescents. These antibodies were found to hydrolyze the S-protein and corresponding oligopeptides. Our study shows that for antibodies with affinity to recombinant structural proteins of the SARS-CoV-2: S-protein, its fragment RBD and N-protein can only hydrolyze the corresponding protein substrates and are not cross-reactive. By using strict criteria, we have confirmed that this proteolytic activity is an intrinsic property of antibodies and is not caused by impurities co-eluting with them. This discovery suggests that natural proteolytic antibodies that hydrolyze proteins of the SARS-CoV-2 virus may have a positive impact on disease pathogenesis. It is also possible for these antibodies to work in combination with other antibodies that bind specific epitopes to enhance the process of virus neutralization.

Early in the SARS-CoV-2 pandemic, polymerase chain reaction (PCR) testing was reserved for symptomatic patients. However, many infections were asymptomatic or mildly symptomatic. Positive serologic antibody (Ab) testing is theorized to be a marker of prior infection, allowing for identification of the true burden of the disease and the establishment of links between outbreaks. The objective was to assess serologic testing in subjects after PCR testing and compare seropositivity rates of household vs. non-household close contacts. We identified subjects who were PCR-positive between March 2020 and May 2021. Index cases and close contacts then underwent serologic testing for IgG against the SARS-CoV-2 nucleocapsid N-protein. One hundred and thirteen subjects underwent serologic testing with a sensitivity of 65.0%, specificity of 78.9%, positive predictive value of 92.9%, and negative predictive value of 34.9%. Index cases comprised 20.5% of subjects, and 73.9% were seropositive (p = 0.01). Close contacts included 72% household contacts and 28% non-household contacts. One seropositive case had no prior PCR and four were PCR negative. No statistical difference existed in seropositivity between household and non-household contact (53.1% vs. 43.8%, p = 0.4). Testing for IgG against SARS-CoV-2 N-protein may identify previously unrecognized infections in the community. Seropositivity rates of household vs. non-household contacts were not significantly different, and >50% of household contacts were seropositive.

The liquid−liquid phase separation phase separation phenomenon has been described for some biopolymers and has been studied in sufficient detail for a few proteins with intrinsically disordered regions. One example of such proteins is the nucleocapsid (N) protein of the severe acute respiratory syndrome coronavirus 2. In this review, we have analyzed available data on N-protein separation in the presence of viral RNA. Particular attention has been paid to transient contacts within the condensates and the N-protein/RNA fragments that form these contacts. The modern concepts of the role of the condensates in the SARS-CoV-2 life cycle and their influence on the host-protective machinery have been summarized. Finally, comments on the possibility of regulating the viral condensates using synthetic or natural small molecules (phase separation modulators), which can provide a new option in the design of antiviral agents, are given. [ABSTRACT FROM AUTHOR]

Relevance. Many countries around the world are developing effective vaccines against SARS-CoV-2. The measure of the effectiveness of the vaccination process has traditionally been antibody production. The frequency and intensity of adverse reactions is also an important factor in making a decision regarding a vaccine. This study presents the results of the evaluation of the formation of humoral immunity and the occurrence of reactions in response to the administration of Sputnik V (Gam-COVID-Vac), RF, and Sinopharm (BBIBP-CorV), PRC. Aim. Analyze immunogenicity and reactogenicity of COVID-19 vaccines used in the Republic of Belarus (Sputnik V and Sinopharm). Materials and methods. Evaluation of postvaccination immune response by enzyme immunoassay and differential enzyme immunoassay for class G immunoglobulins to S- and N-proteins SARS-CoV-2. Blood plasma of the study participants was used as biological material. Blood sampling was performed 3 times: immediately before the first vaccine dose, on day 42, and 6 months after the first vaccine dose. To evaluate the frequency and intensity of postvaccination reactions, study participants were questioned. Results. At 42 days after administration of both vaccines, antibody levels are rising, with a significantly higher quantitative IgG count for the Sputnik V vaccine. This trend is also observed 6 months after the first dose of both vaccines, both among those previously infected with SARS-CoV-2 and those without a history of COVID-19. The comparison of Sputnik V and Sinopharm vaccine groups in terms of IgG (BAU/ml) levels to S- and N-proteins revealed a statistically significant difference in IgG levels to S-protein: the Sputnik V vaccine group had significantly higher IgG levels to S-protein than the Sinopharm vaccine group (p = 0.0000196). The incidence of adverse reactions in this study was 45%. All reactions noted were mild to moderate in severity. The most common were soreness and redness at the injection site, elevated body temperature, and a combination of several reactions. The increased body temperature after vaccination was more common among those vaccinated with the Sputnik V vaccine. Conclusion. Compared to Sinopharm, Sputnik V vaccine produces higher antibody level. Adverse reactions were observed in both vaccinated groups. However, significant statistical differences were found with regard to fever in the Sputnik V vaccine group, which occurred more frequently.

Early in the SARS-CoV-2 pandemic, polymerase chain reaction (PCR) testing was reserved for symptomatic patients. However, many infections were asymptomatic or mildly symptomatic. Positive serologic antibody (Ab) testing is theorized to be a marker of prior infection, allowing for identification of the true burden of the disease and the establishment of links between outbreaks. The objective was to assess serologic testing in subjects after PCR testing and compare seropositivity rates of household vs. non-household close contacts. We identified subjects who were PCR-positive between March 2020 and May 2021. Index cases and close contacts then underwent serologic testing for IgG against the SARS-CoV-2 nucleocapsid N-protein. One hundred and thirteen subjects underwent serologic testing with a sensitivity of 65.0%, specificity of 78.9%, positive predictive value of 92.9%, and negative predictive value of 34.9%. Index cases comprised 20.5% of subjects, and 73.9% were seropositive (p = 0.01). Close contacts included 72% household contacts and 28% non-household contacts. One seropositive case had no prior PCR and four were PCR negative. No statistical difference existed in seropositivity between household and non-household contact (53.1% vs. 43.8%, p = 0.4). Testing for IgG against SARS-CoV-2 N-protein may identify previously unrecognized infections in the community. Seropositivity rates of household vs. non-household contacts were not significantly different, and >50% of household contacts were seropositive. [ABSTRACT FROM AUTHOR]

The article primarily discusses the ongoing evolution of SARS-CoV-2, focusing on its virulence, amino acid mutations, and the factors influencing its continued development. It explores how mutations in key proteins, namely the Spike and Nucleocapsid proteins, affect the virus's pathogenicity, fitness, and transmission. The discussion elaborates on how variations impact the virus's ability to infect, replicate, and its potential to cause diseases.

A highly sensitive and selective E-DNA biosensor was fabricated on a gold substrate using a reduced graphene oxide/polypyrrole/gold nanoparticle/oligonucleotide (RGO/PPy/Au*NPs/Apt) nanocomposite electrode to detect the nucleocapsid protein of SARS-CoV-2 in the patient blood plasma. The modified electrode was characterized by physicochemical techniques such as Fourier transform-infra red (FT-IR), Raman spectroscopy, X-ray diffraction (XRD), energy-dispersive X-ray (EDX) spectroscopy, X-ray photoelectron spectroscopy (XPS) analyses, Brunauer-Emmett-Teller (BET), and Barrett-Joyner-Halenda (BJH) analyses. Moreover, electrochemical analyses were employed to study the electrochemical performance of the electrodes including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV). Computational investigations of N protein bindings such as hydrogen bonding, Van der Waals binding, and Gibbs binding energies of the N-protein, and aptamer were studied by Molecular Dynamics Simulations (MDS). The unique synergistic effect of RGO, PPy, and the well-known effect of Au nanoparticles makes the DNA probe immobilized on the gold electrode surface. After optimizing the systems, the E-DNA biosensor exhibited a fast SWV response, higher sensitivity (33.77 μA.nM−1.cm−2) and selectivity, high efficiency, good storage stability, and acceptable repeatability for monitoring DNA. The results of real samples based on SWV indicated the correct functioning of the aptasensor in the presence of the SARS-CoV-2 virus. The limit of detection was 3.16×10−17 M and the limit of quantitation was 1.42×10−16 M. The MDS results indicated the stable dynamic folding of the aptamer for beneficial binding. The results indicated that the RGO/PPy/Au*NPs/Apt biosensor is promising for detecting of SARS-CoV-2 virus. [Display omitted] • E-DNA biosensor based on RGO/PPy/Au*NPs was fabricated. • The biosensor was used to detect the N-protein of SARS–CoV–2 in the real samples. • LOD, LOQ, and sensitivity were 3.16×10−17, 1.42×10−16 M, and 33.77 μA.nM−1.cm−2. • Computational investigations of N protein bindings were studied by MD simulations. • The binding interactions of the N-protein and aptamer were studied by MDs. [ABSTRACT FROM AUTHOR]

The new coronavirus SARS-CoV-2 has become a global challenge to medicine and, in particular, laboratory diagnostics. The study of the antibodies’ level to SARS-CoV-2 can be used as a confirmation test in the diagnosis of a disease, but it becomes of paramount importance in assessing population immunity resulting from a disease or vaccination, as well as in selection of convalescent plasma donors. The kits developed in our country and abroad for detecting antibodies to the SARS-CoV-2 virus differ both in the methods of testing and in the used coronavirus antigens to which the antibodies are directed. The aim of this study was to compare the diagnostic sensitivity and specificity of five kits for the detection of IgG antibodies to the SARS-CoV-2 virus, based on different diagnostic methods. Serum samples from 137 COVID-19 convalescents and 166 donors of blood and its components were examined. The control group consisted of 50 blood sera collected at the beginning of 2019 and 19 sera collected in 2018 (before the advent of the SARS-CoV-2 virus) and stored at -70 °C. Testing was carried out in analytical systems: rapid test “COVID-19 IgM/IgG Rapid Test (Colloidal Gold)” (China), on an automatic immunochemical analyzer Abbott Architect™ i2000 and kit “SARS-CoV2-IgG” (Abbot, Chicago , IL USA), by the chemiluminescence method using an automatic analyzer of the CL series and kits of the “Mindray” company (China) “SARS-CoV-2 IgM” and “SARS-CoV-2 IgG” and by the enzyme immunoassay method on the kits of the companies “Diagnostic Systems” Ltd (Russia, Nizhny Novgorod) “DS-IFA-ANTI-SARS-CoV-2-G”, “Xema” Ltd (Federal State Budgetary Institution “National Medical Research Center of Hematology” of the Ministry of Health of Russia) “SARS-CoV-2-IgG-IFA” and “Vector-Best” CJSC (Russia, Novosibirsk)” SARS-COV-2-IgM-IFA-BEST” and “SARS-COV-2-IgG-IFABEST”. When comparing the results of testing 137 plasma samples on the Vector-Best and Mindray kits for IgG antibodies, 127 samples were positive, 7 samples were negative on both kits, the discrepancy was 2.2%. In the study of IgM antibodies, 32.1% were positive, and 52.6% were negative in both kits. The discrepancy rate was 15.3%. Out of 166 samples, 1 serum (0.6%) was negative in 5 kits. On the Mindray kit, IgG antibodies to the antigens of the SARS-CoV-2 virus were detected in 165 samples (99.4%), on Vector-Best – in 164 sera (98.8%), on Diagnostic systems – in 151 (90.96%), on Xema – in 154 (92.8%), and on Abbott – in 155 samples (93.4%). At the same time, 135 (81.33%) samples were positive in all kits, while 30 samples had discordant results (18.07%), and in 9 sera, specific IgG was not detected in 2 or more kits. ROC analysis revealed a high diagnostic value of all tested kits (AUC from 0.908 to 0.998), which indicates a high quality of the separation model of positive and negative samples (p < 0.001). With the cut-off set by the manufacturers, the sensitivity and specificity ranged from 82.8% and 93.3% for the Diagnostic Systems kit to 99.4% and 95.8% for the VectorBest kit. The calculated correlation coefficients were higher between kits with a similar composition of the antigen used in the kits; therefore, it is better to monitor the dynamics of antibodies by diagnostic kits from the same manufacturer.

Throughout coronavirus disease (COVID‐19) outbreaks, the centers for disease control and prevention (CDCP) of a country require monitoring of particular territories to provide public health guidance. In this work, the Internet of Diseases (IoD) is suggested for continuous real‐time monitoring of infectious diseases for public health. Because converging information and communication technologies (ICTs) with point‐of‐care (POC) devices to enable the IoD for continuous real‐time health monitoring and processing of clinical records are crucial, an IoD platform associating a lab‐on‐a‐chip (LOC) device to diagnose severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) from oropharyngeal saliva samples have been developed and uploaded the resulted diagnostic data into a cloud‐based system to be connected with CDCP. Moreover, a choropleth IoD map to visualize provincial infection rate is proposed along with the IoD platform. The developed platform is applied for the quantification of SARS‐CoV‐2 N‐protein antigen with a LOD as low as 0.013 ng mL−1 and the infection rate of various provinces is projected with the IoD map successfully. Thus, the proposed IoD system has the potential to become an imperative tool for the disease control and prevention centers to restrain COVID‐19 outbreaks by identifying the severity of particular regions. [ABSTRACT FROM AUTHOR]

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.

SARS-CoV-2 nucleocapsid protein-based COVID-19 diagnosis is a promising alternative to the high-priced, time-consuming, and labor-intensive RT-PCR tests. Here, we developed a rapid, dip-type, wash-free plasmonic fiber optic absorbance biosensor (P-FAB) strategy for the point-of-care detection of SARS-CoV-2 N-protein, expressed abundantly during the infection. P-FAB involves a sandwich assay with plasmonic labels on the surface of a U-bent fiber optic sensor probe with a high evanescent wave absorbance (EWA) sensitivity. The SARS-CoV-2 N-protein is quantified in terms of the change in the intensity of the light propagating through the U-bent sensor probe coupled to a green LED and a photodetector. Firstly, the optical fiber material (silica vs. polymeric optical fiber), was evaluated to realize a sensitive sensor platform. The optimal size of AuNP labels (20, 40, and 60 nm) to achieve high sensitivity and a lower limit of detection (LoD) was investigated. Following the P-FAB strategy, fused silica/glass optical fiber (GOF) U-bent senor probe and citrate-capped AuNP labels (size ~40 nm) gave rise to an LoD down to ~2.5 ng/mL within 10 mins of read-out time. Further, studies on development and validation of a point of care (PoC) read-out device, and preclinical studies are in progress. [ABSTRACT FROM AUTHOR]

The ability of human coronaviruses to infect respiratory tracts and transmit through respiratory droplets makes them highly contagious and potential to become pandemic. The current pandemic situation developed due to COVID-19 infection warrants the rapid development of effective and safe antivirals for disease management. Natural products are considered as a reliable and valuable source for rapid drug discovery. Phenolics, a major class of plant secondary metabolites, have been screened on a large scale for their antiviral efficacy to combat emergent mutants of coronavirus. Phenolics have a lower risk for the development of toxicity comparing to synthetic compounds as we are naturally adapted to most of the plant Phenolics as part of our vegetarian diet. The available literature has shown that Phenolics could interfere with the various key enzymes associated with virus-host cell interactions and thus alleviate the severity of the disease. The review of structure-activity relationships indicated the roles of hydrophobic aliphatic side chains, catechol groups, pyran ring, glycosylation, flexible linkages between aromatic rings, etc in modulating the interactions of Phenolics with human coronavirus proteins. The summary of the current literature available in this review might be useful for the selection of Phenolics with diverse structural properties and to design semi-synthetic products with better drug properties against coronaviruses. [ABSTRACT FROM AUTHOR]

Abstract Background Rift Valley fever virus (RVFV), the causative agent of Rift Valley fever, is an enveloped single-stranded negative-sense RNA virus in the genus Phlebovirus, family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing abortion storms in pregnant ruminants, high neonatal mortality in animals, and morbidity and occasional fatalities in humans. The disease is endemic in parts of Africa and the Arabian Peninsula, but is described as emerging due to the wide range of mosquitoes that could spread the disease into non-endemic regions. There are different tests for determining whether animals are infected with or have been exposed to RVFV. The most common serological test is antibody ELISA, which detects host immunoglobulins M or G produced specifically in response to infection with RVFV. The presence of antibodies to RVFV nucleocapsid protein (N-protein) is among the best indicators of RVFV exposure in animals. This work describes an investigation of the feasibility of producing a recombinant N-protein in Nicotiana benthamiana and using it in an ELISA. Results The human-codon optimised RVFV N-protein was successfully expressed in N. benthamiana via Agrobacterium-mediated infiltration of leaves. The recombinant protein was detected as monomers and dimers with maximum protein yields calculated to be 500–558 mg/kg of fresh plant leaves. The identity of the protein was confirmed by liquid chromatography-mass spectrometry (LC-MS) resulting in 87.35% coverage, with 264 unique peptides. Transmission electron microscopy revealed that the protein forms ring structures of ~ 10 nm in diameter. Preliminary data revealed that the protein could successfully differentiate between sera of RVFV-infected sheep and from sera of those not infected with the virus. Conclusions To the best of our knowledge this is the first study demonstrating the successful production of RVFV N-protein as a diagnostic reagent by Agrobacterium-mediated transient heterologous expression in N. benthamiana. Preliminary testing of the antigen showed its ability to distinguish RVFV-positive animal sera from RVFV negative animal sera when used in an enzyme linked immunosorbent assay (ELISA). The cost-effective, scalable and simple production method has great potential for use in developing countries where rapid diagnosis of RVFV is necessary.

Background: The assessment of antibody responses to severe acute respiratory syndrome coronavirus-2 is potentially confounded by exposures to flaviviruses. The aims of the present research were to determine whether anti-dengue antibodies affect the viral load and the detection of anti-coronavirus nucleocapsid (N)-protein antibodies in coronavirus infectious disease 2019 (COVID-19) in Bangladesh. Methods: Viral RNA was evaluated in swab specimens from 115 COVID-19 patients by real-time reverse transcription polymerase chain reaction (rT-PCR). The anti-N-protein antibodies, anti-dengue virus E-protein antibodies and the dengue non-structural protein-1 were determined in serum from 115 COVID-19 patients, 30 acute dengue fever pre-COVID-19 pandemic and nine normal controls by ELISA. Results: The concentrations of viral RNA in the nasopharyngeal; Ct median (95% CI); 22 (21.9–23.3) was significantly higher than viral RNA concentrations in oropharyngeal swabs; and 29 (27–30.5) p < 0.0001. Viral RNA concentrations were not correlated with-dengue IgG levels. The anti-nucleocapsid antibodies were IgA 27% positive and IgG 35% positive at days 1 to 8 post-onset of COVID-19 symptoms versus IgA 0% and IgG 0% in dengue patients, p < 0.0001. The levels of anti- nucleocapsid IgA or IgG versus the levels of anti-dengue IgM or IgG revealed no significant correlations. Conclusions: Viral RNA and anti-nucleocapsid antibodies were detected in COVID-19 patients from dengue-endemic regions of Bangladesh, independently of the dengue IgG levels.

The appearance of new viruses and diseases has made the development of rapid and reliable diagnostic tests crucial. In light of it, we proposed a new method for assembling an electrochemical immunosensor, based on a one-step approach for selective layer formation. For this purpose, a mixture containing the immobilizing agent (polyxydroxybutyrate, PHB) and the recognition element (antibodies against SARS-CoV-2 nucleocapsid protein) was prepared and used to modify a screen-printed carbon electrode with electrodeposited graphene oxide, for the detection of SARS-CoV-2 nucleocapsid protein (N-protein). Under optimum conditions, N-protein was successfully detected in three different matrixes – saliva, serum, and nasal swab, with the lowest detectable values of 50 pg mL−1, 1.0 ng mL−1, and 50 pg mL−1, respectively. Selectivity was assessed against SARS-CoV-2 receptor-binding domain protein (RBD) and antibodies against yellow fever (YF), and no significant response was observed in presence of interferents, reinforcing the suitability of the proposed one-step approach for selective layer formation. The proposed biosensor was stable for up to 14 days, and the mixture was suitable for immunosensor preparation even after 60 days of preparation. The proposed assembly strategy reduces the cost, analysis time, and waste generation. This reduction is achieved through miniaturization, which results in the decreased use of reagents and sample volumes. Additionally, this approach enables healthcare diagnostics to be conducted in developing regions with limited resources. Therefore, the proposed one-step approach for selective layer formation is a suitable, simpler, and a reliable alternative for electrochemical immunosensing. [Display omitted] • New single-step method to assemble electrochemical immunosensors. • Highly selective and sensitive detection of the N protein from SARS-CoV-2. • Low-cost, feasible and disposable device with high sensitivity for point-of-care testing. • Innovative strategy highly reproducible and scalable for biosensors construction. • Excellent analytical performance towards detection of the target in saliva, serum, and swab samples. [ABSTRACT FROM AUTHOR]

Abstract: The severe acute respiratory syndrome (SARS) is a contagious disease that killed hundreds and sickened thousands of people worldwide between November 2002 and July 2003. The nucleocapsid (N) protein of the coronavirus responsible for this disease plays a critical role in viral assembly and maturation and is of particular interest because of its potential as an antiviral target or vaccine candidate. Refolding of SARS N-protein during production and purification showed the presence of two additional protein bands by SDS–PAGE. Mass spectroscopy (MALDI, SELDI, and LC/MS) confirmed that the bands are proteolytic products of N-protein and the cleavage sites are four SR motifs in the serine–arginine-rich region—sites not suggestive of any known protease. Furthermore, results of subsequent testing for contaminating protease(s) were negative: cleavage appears to be due to inherent instability and/or autolysis. The importance of N-protein proteolysis to viral life cycle and thus to possible treatment directions are discussed. [Copyright &y& Elsevier]

The carboxy-terminal domain of the transcription factor Escherichia coli NusA, NusACTD, interacts with the protein N of bacteriophage λ, λN, and the carboxyl terminus of the E. coli RNA polymerase α subunit, αCTD. We solved the solution structure of the unbound NusACTD with high-resolution nuclear magnetic resonance (NMR). Additionally, we investigated the binding sites of λN and αCTD on NusACTD using NMR titrations. The solution structure of NusACTD shows two structurally similar subdomains, NusA(353-416) and NusA(431-490), matching approximately two homologous acidic sequence repeats. Further characterization of NusACTD with 15N NMR relaxation data suggests that the interdomain region is only weakly structured and that the subdomains are not interacting. Both subdomains adopt an (HhH)2 fold. These folds are normally involved in DNA-protein and protein-protein interactions. NMR titration experiments show clear differences of the interactions of these two domains with αCTD and λN, in spite of their structural similarity. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

The breastfeeding of infants by mothers who are infected with SARS-CoV-2 has become a dramatic healthcare problem. The WHO recommends that infected women should not abandon breastfeeding; however, there is still the risk of contact transmission. Convalescent donor milk may provide a defense against the aforementioned issue and can eliminate the consequences of artificial feeding. Therefore, it is vital to characterize the epitope-specific immunological landscape of human milk from women who recovered from COVID-19. We carried out a comprehensive ELISA-based analysis of blood serum and human milk from maternity patients who had recovered from COVID-19 at different trimesters of pregnancy. It was found that patients predominantly contained SARS-CoV-2 N-protein-specific immunoglobulins and had manifested the antibodies for all the antigens tested in a protein-specific and time-dependent manner. Women who recovered from COVID-19 at trimester I–II showed a noticeable decrease in the number of milk samples with sIgA specific to the N-protein, linear NTD, and RBD-SD1 epitopes, and showed an increase in samples with RBD conformation-dependent sIgA. S-antigens were found to solely induce a sIgA1 response, whereas N-protein sIgA1 and sIgA2 subclasses were involved in 100% and 33% of cases. Overall, the antibody immunological landscape of convalescent donor milk suggests that it may be a potential defense agent against COVID-19 for infants, conferring them with a passive immunity. [ABSTRACT FROM AUTHOR]

Background: The assessment of antibody responses to severe acute respiratory syndrome coronavirus-2 is potentially confounded by exposures to flaviviruses. The aims of the present research were to determine whether anti-dengue antibodies affect the viral load and the detection of anti-coronavirus nucleocapsid (N)-protein antibodies in coronavirus infectious disease 2019 (COVID-19) in Bangladesh. Methods: Viral RNA was evaluated in swab specimens from 115 COVID-19 patients by real-time reverse transcription polymerase chain reaction (rT-PCR). The anti-N-protein antibodies, anti-dengue virus E-protein antibodies and the dengue non-structural protein-1 were determined in serum from 115 COVID-19 patients, 30 acute dengue fever pre-COVID-19 pandemic and nine normal controls by ELISA. Results: The concentrations of viral RNA in the nasopharyngeal; Ct median (95% CI); 22 (21.9–23.3) was significantly higher than viral RNA concentrations in oropharyngeal swabs; and 29 (27–30.5) p < 0.0001. Viral RNA concentrations were not correlated with-dengue IgG levels. The anti-nucleocapsid antibodies were IgA 27% positive and IgG 35% positive at days 1 to 8 post-onset of COVID-19 symptoms versus IgA 0% and IgG 0% in dengue patients, p < 0.0001. The levels of anti- nucleocapsid IgA or IgG versus the levels of anti-dengue IgM or IgG revealed no significant correlations. Conclusions: Viral RNA and anti-nucleocapsid antibodies were detected in COVID-19 patients from dengue-endemic regions of Bangladesh, independently of the dengue IgG levels. [ABSTRACT FROM AUTHOR]

Pertussis toxin, which specifically inactivates guanine nucleotide-binding proteins (N-proteins) involved in the signal transduction in various receptor systems, did not influence the positive inotropic effect of the alpha-adrenoceptor agonist phenylephrine in rat isolated left auricles. This indicates that the alpha-adrenoceptor-mediated positive inotropic effect does not involve a pertussis toxin-sensitive N-protein. [ABSTRACT FROM AUTHOR]

• Nucleocapsid protein of the Chandipura virus interacts with the (+) Leader RNA. • The interaction likely occurs through a helical motif in the C-terminus of the Nucleocapsid protein. • A peptide designed from the model, interacts specifically with the (+) Leader RNA. • The peptide specifically inhibits viral growth as shown by plaque and minigenome assays. • Nucleocapsid protein-(+)Leader RNA interaction may be a valid drug target for single-strand RNA viruses. The negative strand RNA virus family contains many human pathogens. Finding new antiviral drug targets against this class of human pathogens is one of the significant healthcare needs. Nucleocapsid proteins of negative strand RNA viruses wrap the viral genomic RNA and play essential roles in gene transcription and genome replication. Chandipura virus, a member of the Rhabdoviridae family, has a negative strand RNA genome. In addition to wrapping the genomic RNA, its nucleocapsid protein interacts with the positive strand leader RNA and plays a vital role in the virus life-cycle. We have designed a peptide, based on prior knowledge and demonstrated that the peptide is capable of binding specifically to the positive strand leader RNA. When the peptide was transported inside the cell, it inhibited viral growth with IC 50 values in the low micromolar range. Given the widespread occurrence of leader RNAs in negative strand RNA viruses and its interaction with the nucleocapsid protein, it is likely that this interaction could be a valid drug target for other negative strand RNA viruses. [ABSTRACT FROM AUTHOR]