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Host–virus protein interactions are critical for intracellular viral propagation. Understanding the interactions between cellular and viral proteins may help us develop new antiviral strategies. Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe damage to the global swine industry. Here, we employed co-immunoprecipitation and liquid chromatography-mass spectrometry to characterize 426 unique PEDV nucleocapsid (N) protein-binding proteins in infected Vero cells. A protein–protein interaction network (PPI) was created, and gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses revealed that the PEDV N-bound proteins belong to different cellular pathways, such as nucleic acid binding, ribonucleoprotein complex binding, RNA methyltransferase, and polymerase activities. Interactions of the PEDV N protein with 11 putative proteins: tripartite motif containing 21, DEAD-box RNA helicase 24, G3BP stress granule assembly factor 1, heat shock protein family A member 8, heat shock protein 90 alpha family class B member 1, YTH domain containing 1, nucleolin, Y-box binding protein 1, vimentin, heterogeneous nuclear ribonucleoprotein A2/B1, and karyopherin subunit alpha 1, were further confirmed by in vitro co-immunoprecipitation assay. In summary, studying an interaction network can facilitate the identification of antiviral therapeutic strategies and novel targets for PEDV infection. [ABSTRACT FROM AUTHOR]

With the outbreak and spread of COVID-19, a deep investigation of SARS-CoV-2 is urgent. Direct usage of this virus for scientific research could provide reliable results and authenticity. However, it is strictly constrained and unrealistic due to its high pathogenicity and infectiousness. Considering its biosafety, different systems and technologies have been employed in immunology and biomedical studies. In this study, phage display technology was used to construct a nonpathogenic model for COVID-19 research. The nucleocapsid protein of SARS-CoV-2 was fused with the M13 phage capsid p3 protein and expressed on the M13 phages. After validation of its successful expression, its potential as the standard for qPCR quantification and affinity with antibodies were confirmed, which may show the possibility of using this nonpathogenic bacteriophage to replace the pathogenic virus in scientific research concerning SARS-CoV-2. In addition, the model was used to develop a system for the classification and identification of different samples using ATR–FTIR, which may provide an idea for the development and evaluation of virus monitoring equipment in the future. [ABSTRACT FROM AUTHOR]

The global pandemic of COVID-19 has created an unrivalled need for sensitive and rapid point-of-care testing (POCT) methods for the detection of infectious viruses. For the novel coronavirus SARS-CoV-2, the nucleocapsid protein (N-protein) is one of the most abundant structural proteins of the virus and it serves as a useful diagnostic marker for detection. Herein, we report a fiber optic particle plasmon resonance (FOPPR) biosensor which employed a single-stranded DNA (ssDNA) aptamer as the recognition element to detect the SARS-CoV-2 N-protein in 15 min with a limit of detection (LOD) of 2.8 nM, meeting the acceptable LOD of 106 copies/mL set by the WHO target product profile. The sensor chip is a microfluidic chip based on the balance between the gravitational potential and the capillary force to control fluid loading, thus enabling the power-free auto-flowing function. It also has a risk-free self-contained design to avoid the risk of the virus leaking into the environment. These findings demonstrate the potential for designing a low-cost and robust POCT device towards rapid antigen detection for early screening of SARS-CoV-2 and its related mutants. [ABSTRACT FROM AUTHOR]

Coronaviruses have long posed a major threat not only to human health but also to agriculture. Outbreaks of an animal coronavirus such as porcine epidemic diarrhea virus (PEDV) can cause up-to-100% mortality in suckling piglets, resulting in devastating effects on the livestock industry. Understanding how the virus evades its host's defense can help us better manage the infection. Zinc-finger antiviral protein (ZAP) is an important class of host antiviral factors against a variety of viruses, including the human coronavirus. In this study, we have shown that a representative porcine coronavirus, PEDV, can be suppressed by endogenous or porcine-cell-derived ZAP in VeroE6 cells. An uneven distribution pattern of CpG dinucleotides in the viral genome is one of the factors contributing to suppression, as an increase in CpG content in the nucleocapsid (N) gene renders the virus more susceptible to ZAP. Our study revealed that the virus uses its own nucleocapsid protein (pCoV-N) to interact with ZAP and counteract the activity of ZAP. The insights into coronavirus-host interactions shown in this work could be used in the design and development of modern vaccines and antiviral agents for the next pandemic. [ABSTRACT FROM AUTHOR]

Understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to contain the COVID-19 pandemic. Using a multiplex approach, serum IgG responses against the whole SARSCoV-2 proteome and the nucleocapsid proteins of endemic human coronaviruses (HCoVs) were measured in SARS-CoV-2-infected donors and healthy controls. COVID-19 severity strongly correlated with IgG responses against the nucleocapsid (N) of SARS-CoV-2 and possibly with the number of viral antigens targeted. Furthermore, a strong correlation between COVID-19 severity and serum responses against N of endemic alpha-but not betacoronaviruses was detected. This correlation was neither caused by cross-reactivity of antibodies, nor by a general boosting effect of SARS-CoV-2 infection on pre-existing humoral immunity. These findings raise the prospect of a potential disease progression marker for COVID-19 severity that allows for early stratification of infected individuals. [ABSTRACT FROM AUTHOR]

The nucleocapsid protein (NP) is one of the main proteins out of four structural proteins of coronaviruses including the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, discovered in 2019. NP packages the viral RNA during virus assembly and is, therefore, indispensable for virus reproduction. NP consists of two domains, i.e., the N- and C-terminal domains. RNA-binding is mainly performed by a binding pocket within the N-terminal domain (NTD). NP represents an important target for drug discovery to treat COVID-19. In this project, we used the Vina LC virtual drug screening software and a ZINC-based database with 210,541 natural and naturally derived compounds that specifically target the binding pocket of NTD of NP. Our aim was to identify coronaviral inhibitors that target NP not only of SARS-CoV-2 but also of other diverse human pathogenic coronaviruses. Virtual drug screening and molecular docking procedures resulted in 73 candidate compounds with a binding affinity below −9 kcal/mol with NP NTD of SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-NL63, HoC-229E, and HCoV-HKU1. The top five compounds that met the applied drug-likeness criteria were then tested for their binding in vitro to the NTD of the full-length recombinant NP proteins using microscale thermophoresis. Compounds (1), (2), and (4), which belong to the same scaffold family of 4-oxo-substituted-6-[2-(4a-hydroxy-decahydroisoquinolin-2-yl)2H-chromen-2-ones and which are derivates of coumarin, were bound with good affinity to NP. Compounds (1) and (4) were bound to the full-length NP of SARS-CoV-2 (aa 1–419) with Kd values of 0.798 (±0.02) µM and 8.07 (±0.36) µM, respectively. Then, these coumarin derivatives were tested with the SARS-CoV-2 NP NTD (aa 48–174). Compounds (1) and (4) revealed Kd-values of 0.95 (±0.32) µM and 7.77 (±6.39) µM, respectively. Compounds (1) and (4) caused low toxicity in human A549 and MRC-5 cell lines. These compounds may represent possible drug candidates, which need further optimization to be used against COVID-19 and other coronaviral infections. [ABSTRACT FROM AUTHOR]

Background: We conducted a longitudinal study to estimate immunity produced in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among university students over seven months. Methods: All participants were attending a public university and resided in Pitt County, North Carolina. University students enrolled weekly for 10 weeks between 26 August 2020 and 28 October 2020, resulting in 136 young adults completing at least one study visit by 17 November 2020. Enrolled students completed an online survey and nasal swab collection at two-week intervals and monthly blood collection between 26 August 2020 and 31 March 2021. Results: Amongst 695 serum samples tested during follow-up, the prevalence of a positive result for anti-nucleocapsid antibodies (N-IgG) was 9.78%. In 22 students with more than one positive N-IgG serum sample, 68.1% of the group lost persistence of N-IgG below the positive threshold over 140 days. Anti-spike IgG antibodies were significantly higher among 11 vaccinated compared to 10 unvaccinated. Conclusions: In healthy young adults, N-IgG wanes below the detectable threshold within five months. S-IgG titer remained consistently elevated months after infection, and significantly increased after vaccination. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) nucleocapsid protein is an essential structural component of mature virions, encapsulating the genomic RNA and modulating RNA transcription and replication. Several of its activities might be associated with the protein's ability to undergo liquid–liquid phase separation. NSARS‐CoV‐2 contains an intrinsically disordered region at its N‐terminus (NTE) that can be phosphorylated and is affected by mutations found in human COVID‐19 infections, including in the Omicron variant of concern. Here, we show that NTE deletion decreases the range of RNA concentrations that can induce phase separation of NSARS‐CoV‐2. In addition, deletion of the prion‐like NTE allows NSARS‐CoV‐2 droplets to retain their liquid‐like nature during incubation. We further demonstrate that RNA‐binding engages multiple parts of the NTE and changes NTE's structural properties. The results form the foundation to characterize the impact of N‐terminal mutations and post‐translational modifications on the molecular properties of the SARS‐CoV‐2 nucleocapsid protein. Statement: The nucleocapsid protein of SARS‐CoV‐2 plays an important role in both genome packaging and viral replication upon host infection. Replication has been associated with RNA‐induced liquid–liquid phase separation of the nucleocapsid protein. We present insights into the role of the N‐terminal part of the nucleocapsid protein in the protein's RNA‐mediated liquid–liquid phase separation. [ABSTRACT FROM AUTHOR]

The capability to detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and identify immune responses among the population is crucial for managing the outbreak of the COVID-19 pandemic. Although PCR-based nucleic acid detection techniques are utilized to detect viral infection in people, alternative tests capable of distinguishing between exposure and infection are urgently needed beyond this restricted window of detectable viral replication. Antibodies are produced in human sera within a few days after viral infection, providing longer period for performing tests to acquire reliable database. Herewith, we provide the results of our in-house developed ELISA (Enzyme-Linked Immunosorbent Assay) that displays all of the properties necessary for high-throughput of human sera sample analysis. This test does not involve the handling of live viruses, although it detects a variety of antibody types in serum and plasma of human after exposure to the virus. For in-house development of the kit, the nucleocapsid (N) gene of SARS-CoV-2 virus was cloned in the prokaryotic expression vector pGEX-6P-1, and purified N protein was used to detect IgG antibodies in human sera samples. In total 76 human serum samples that were collected before novel coronavirus registry in Mongolia in March 2020, as well as 200 serum samples from patients who had been infected by SARS-CoV-2 virus, were used. Among 200 serum samples, 188 were positive and 12 were false negative, while in non-infected cases 69 were negative and 7 were false positive, suggesting 94 per cent sensitivity and 90.7 per cent specificity of the kit, with p-values of 0.02.

ABSTRACT Accurate, highly specific immunoassays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to evaluate seroprevalence. This study investigated the concordance of results across four immunoassays targeting different antigens for sera collected at the beginning of the SARS-CoV-2 pandemic in the United States. Specimens from All of Us participants contributed between January and March 2020 were tested using the Abbott Architect SARS-CoV-2 IgG (immunoglobulin G) assay (Abbott) and the EuroImmun SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA) (EI). Participants with discordant results, participants with concordant positive results, and a subset of concordant negative results by Abbott and EI were also tested using the Roche Elecsys anti-SARS-CoV-2 (IgG) test (Roche) and the Ortho-Clinical Diagnostics Vitros anti-SARS-CoV-2 IgG test (Ortho). The agreement and 95% confidence intervals were estimated for paired assay combinations. SARS-CoV-2 antibody concentrations were quantified for specimens with at least two positive results across four immunoassays. Among the 24,079 participants, the percent agreement for the Abbott and EI assays was 98.8% (95% confidence interval, 98.7%, 99%). Of the 490 participants who were also tested by Ortho and Roche, the probability-weighted percentage of agreement (95% confidence interval) between Ortho and Roche was 98.4% (97.9%, 98.9%), that between EI and Ortho was 98.5% (92.9%, 99.9%), that between Abbott and Roche was 98.9% (90.3%, 100.0%), that between EI and Roche was 98.9% (98.6%, 100.0%), and that between Abbott and Ortho was 98.4% (91.2%, 100.0%). Among the 32 participants who were positive by at least 2 immunoassays, 21 had quantifiable anti-SARS-CoV-2 antibody concentrations by research assays. The results across immunoassays revealed concordance during a period of low prevalence. However, the frequency of false positivity during a period of low prevalence supports the use of two sequentially performed tests for unvaccinated individuals who are seropositive by the first test. IMPORTANCE What is the agreement of commercial SARS-CoV-2 immunoglobulin G (IgG) assays during a time of low coronavirus disease 2019 (COVID-19) prevalence and no vaccine availability? Serological tests produced concordant results in a time of low SARS-CoV-2 prevalence and no vaccine availability, driven largely by the proportion of samples that were negative by two immunoassays. The CDC recommends two sequential tests for positivity for future pandemic preparedness. In a subset analysis, quantified antinucleocapsid and antispike SARS-CoV-2 IgG antibodies do not suggest the need to specify the antigen targets of the sequential assays in the CDC’s recommendation because false positivity varied as much between assays targeting the same antigen as it did between assays targeting different antigens.

Coronaviruses have long posed a major threat not only to human health but also to agriculture. Outbreaks of an animal coronavirus such as porcine epidemic diarrhea virus (PEDV) can cause up-to-100% mortality in suckling piglets, resulting in devastating effects on the livestock industry. Understanding how the virus evades its host’s defense can help us better manage the infection. Zinc-finger antiviral protein (ZAP) is an important class of host antiviral factors against a variety of viruses, including the human coronavirus. In this study, we have shown that a representative porcine coronavirus, PEDV, can be suppressed by endogenous or porcine-cell-derived ZAP in VeroE6 cells. An uneven distribution pattern of CpG dinucleotides in the viral genome is one of the factors contributing to suppression, as an increase in CpG content in the nucleocapsid (N) gene renders the virus more susceptible to ZAP. Our study revealed that the virus uses its own nucleocapsid protein (pCoV-N) to interact with ZAP and counteract the activity of ZAP. The insights into coronavirus-host interactions shown in this work could be used in the design and development of modern vaccines and antiviral agents for the next pandemic.

Understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to contain the COVID-19 pandemic. Using a multiplex approach, serum IgG responses against the whole SARS-CoV-2 proteome and the nucleocapsid proteins of endemic human coronaviruses (HCoVs) were measured in SARS-CoV-2-infected donors and healthy controls. COVID-19 severity strongly correlated with IgG responses against the nucleocapsid (N) of SARS-CoV-2 and possibly with the number of viral antigens targeted. Furthermore, a strong correlation between COVID-19 severity and serum responses against N of endemic alpha- but not betacoronaviruses was detected. This correlation was neither caused by cross-reactivity of antibodies, nor by a general boosting effect of SARS-CoV-2 infection on pre-existing humoral immunity. These findings raise the prospect of a potential disease progression marker for COVID-19 severity that allows for early stratification of infected individuals.

Recently, the world is facing the outbreaks of severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 and the number of infected patients is increasing every day. Researchers are doing their best to find the most effective treatment to tackle this deathly virus. Several approaches had been proposed to be tested in the lab for the efficacy but none of them are qualified to be use as the treatment of the COVID-19. Therefore, the aim of this study is to design a vaccine based on epitope, which were obtained from the nucleocapsid phosphoprotein (N protein). In addition, 38 samples of SARS-CoV-2 Isolates were being retrieved from the GISAID Database and NCBI GenBank. Later on, these samples will be used for checking the evolutionary relationship of the SARS-CoV-2 and also, to determine whether this nucleocapsid proteins are well-conserved (less or even no mutations occur at all) and whether there was any evolutionary relationship between the recent coronavirus with the previous coronavirus by conducting the phylogenetic analysis. Then, we wanted to see the molecular interaction between the human BCR/FAB receptor with the predicted peptides through the molecular docking process. All of the peptides were generated by the IEDB analysis tools and have already been tested for the antigenicity, so the one that was being docked are the peptide that has antigen properties. Based on the analysis that had been done, we would like to recommend the PEP1 as an epitope-based peptide vaccine candidate to deal with the SARS-CoV-2 outbreaks.

Coronaviruses pose serious threats to human and animal health worldwide, of which their structural nucleocapsid (N) proteins play multiple key roles in viral replication. However, the structures of animal coronavirus N proteins are poorly understood, posing challenges for research on their functions and pathogenic mechanisms as well as the development of N protein-based antiviral drugs. Therefore, N proteins must be further explored as potential antiviral targets. We determined the structure of the NNTD of feline infectious peritonitis virus (FIPV) and identified 3,6-dihydroxyflavone (3,6- DHF) as an effective N protein inhibitor. 3,6-DHF successfully inhibited FIPV replication in CRFK cells, showing broad-spectrum activity and effectiveness against drugresistant strains. Our study provides important insights for developing novel broadspectrum anti-coronavirus drugs and treating infections caused by drug-resistant mutant strains. • This study determined the N-NTD structure of FIPV for the first time. • Virtual screening and BLI revealed 3,6-DHF as an inhibitor of the N protein. • 3,6-DHF effectively inhibited FIPV propagation in CRFK cells. • The inhibitor 3,6-DHF showed broad-spectrum activity against various coronaviruses. • 3,6-DHF demonstrated inhibitory effects on drug-resistant strains. [ABSTRACT FROM AUTHOR]

Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus that can cause diarrhea in pigs of all ages with varying severity. Host–virus protein interactions are critical for intracellular viral replication. Elucidating the interactions between cellular and viral proteins can help us to design antiviral strategies. PDCoV N protein is the most abundant and vital regulator in virus replication. In this study, 604 host proteins were identified to interact with PDCoV N protein by Co-IP combined with LC-MS, of which 243 proteins were specifically bound to N protein. PPI analysis revealed that the N-interacting host proteins are categorized into three groups: ribonucleoprotein complex biogenesis modulation, cellular nitrogen compound metabolism, and nucleic acid binding. GO and KEGG analyses showed that the host proteins are primarily involved in mRNA splicing, stress granule assembly, spliceosomal snRNP assembly. Additionally, four host proteins-TRIM25, HNRNPUL1, RPS27A, and SLC3A2-were selected to validate the interactome data through Co-IP and Confocal assays. This study can help in designing anti-PDCoV strategies and understanding the replication mechanism of PDCoV. • 243 host proteins were identified to specifically interact with PDCoV N protein. • The host proteins are divided into three clusters and mainly regulate RNA translation and mRNA splice activity. • The function of the host proteins are involved in mRNA splicing, stress granule assembly, and spliceosomal snRNP assembly. [ABSTRACT FROM AUTHOR]

Despite the success of first-generation COVID-19 vaccines targeting the spike (S) protein, emerging SARS-CoV-2 variants have led to immune escape, reducing the efficacy of these vaccines. Additionally, some individuals are unable to mount an effective immune response to S protein-based vaccines. This has created a need for alternative vaccine strategies that are less susceptible to mutations and capable of providing broad and durable protection. This study aimed to evaluate the efficacy and safety of a novel COVID-19 vaccine based on the full-length recombinant nucleocapsid (N) protein of SARS-CoV-2. We conducted a prospective, multicenter, randomized, double-blind, placebo-controlled phase 3 clinical trial (NCT05726084) in Russia. Participants (n = 5229) were adults aged 18 years and older, with a BMI of 18.5–30 kg/m², and without significant clinical abnormalities. They were randomized in a 2:1 ratio to receive a single intramuscular dose of either the N protein-based vaccine (50 µg) or placebo. Randomization was done through block randomization, and masking was ensured by providing visually identical formulations of vaccine and placebo. The primary outcome was the incidence of symptomatic COVID-19 confirmed by PCR more than 15 days after vaccination within a 180-day observation period, analyzed on an intention-to-treat basis. Between May 18, 2023, and August 9, 2023, 5229 participants were randomized, with 3486 receiving the vaccine and 1743 receiving the placebo. Eight cases of PCR-confirmed symptomatic COVID-19 occurred in the vaccine group (0.23%) compared to 27 cases in the placebo group (1.55%), yielding a vaccine efficacy of 85.2% (95% CI: 67.4–93.3; p < 0.0001). Adverse events were mostly mild and included local injection site reactions. There were no vaccine-related serious adverse events. The N protein-based COVID-19 vaccine demonstrated significant efficacy and a favorable safety profile, suggesting it could be a valuable addition to the global vaccination effort, particularly in addressing immune escape variants and offering an alternative for those unable to respond to S protein-based vaccines. These results support the continued development and potential deployment of N protein-based vaccines in the ongoing fight against COVID-19. [Display omitted] • Novel N-protein Convacell vaccine targets conserved nucleocapsid protein, offering variant resilience. • First human trial shows N-protein COVID-19 Convacell vaccine efficacy with 85.2% protection rate. • N-protein Convacell vaccine induces robust immune response against symptomatic SARS-CoV-2 infection. • N-protein Convacell vaccine offers new approach in COVID-19 prevention. [ABSTRACT FROM AUTHOR]

Diagnostics employing multiple modalities have been essential for controlling and managing COVID-19, caused by SARS-CoV-2. However, scaling up Reverse Transcription–Quantitative Polymerase Chain Reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection, remains challenging in low and middle-income countries. Cost-effective and high-throughput alternatives like enzyme-linked immunosorbent assay (ELISA) could address this issue. We developed an in-house SARS-CoV-2 nucleocapsid capture ELISA, and validated on 271 nasopharyngeal swab samples from humans (n = 252), bovines (n = 10), and dogs (n = 9). This ELISA has a detection limit of 195 pg/100 µL of nucleocapsid protein and does not cross-react with related coronaviruses, ensuring high specificity to SARS-CoV-2. Diagnostic performance was evaluated using receiver operating characteristic curve analysis, showing a diagnostic sensitivity of 67.78 % and specificity of 100 %. Sensitivity improved to 74.32 % when excluding positive clinical samples with RT-qPCR Ct values > 25. Furthermore, inter-rater reliability analysis demonstrated substantial agreement (κ values = 0.73–0.80) with the VIRALDTECT II Multiplex RT-qPCR kit and perfect agreement with the CoVeasy™ COVID-19 rapid antigen self-test (κ values = 0.89–0.93). Our findings demonstrated that the in-house nucleocapsid capture ELISA is suitable for SARS-CoV-2 testing in humans and animals, meeting the necessary sensitivity and specificity thresholds for cost-effective, large-scale screening. • Developed and validated a cost-effective SARS-CoV-2 nucleocapsid capture ELISA. • The assay can be performed in a conventional BSL-2 laboratory. • Demonstrated a substantial agreement with RT-qPCR and rapid antigen tests. • Suitable for large-scale SARS-CoV-2 screening in low and middle-income countries. [ABSTRACT FROM AUTHOR]

To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus. • We generated two FIV Gag chimeric proteins containing different SIV nucleocapsid (NC) sequences. • The FIV Gag NC1 chimera, which carries the two SIV zinc fingers motifs, does not assemble into virus-like particles. • The FIV Gag NC2 chimera, which contains the SIV NC-SP2 module, is assembly competent. • NC2 virions are partially competent for genomic RNA packaging. [ABSTRACT FROM AUTHOR]

Newcastle disease (ND) is an acute and highly contagious infectious disease found in poultry. Although commercial ND virus (NDV) vaccines are universally used, some case reports persistently documented vaccination failure. Therefore, novel strategies are still required to control the occurrence of the disease in chickens. Recently, nanobodies (Nbs), which have the advantages of small molecular weight and low production costs, have been shown to be promising therapeutics against viral infection. In the present study, a total of 16 Nbs against NDV nucleocapsid protein (NP) were screened from two libraries against NDV using phage display technology. Of the 16 screened Nbs, eight were prevented from binding to NDV NP protein through administering positive chicken sera for anti-NDV antibodies, indicating that the epitopes recognized by these eight Nbs were able to induce the immune response after the chickens were infected with NDV stock. Subsequently, transfection assay, construction of recombinant DF-1 cells capable of expressing different nanobodies and viral inhibition assay were used to screen the nanobodies inhibiting NDV replication. The results demonstrated that Nb18, Nb30, and Nb88 significantly inhibited the replication of Class I and different genotypes of Class II NDV strains in DF-1 cells when they were expressed in the cytoplasm. Collectively, these nanobodies provided new tools for researching the functions of NDV NP protein and may be used as a novel strategy for designing drugs against NDV infection in chickens. [ABSTRACT FROM AUTHOR]

The present study aimed to explore if bovine coronavirus nucleocapsid (BCoV N) impacts IFN-β production in the host cells and to reveal further molecular mechanism of BCoV pathogenesis. Human embryonic kidney (HEK) 293 T cells were transiently transfected with pMyc-BCoV-N recombinant plasmids, then infected with the vesicular stomatitis virus (VSV). Expression levels of beta interferon (IFN-β) mRNA were detected using RT-qPCR. The results showed that BCoV N gene was 1347 bp that was consistent with the expected size. pMyc-BCoV-N recombinant protein was 1347 bp which was successfully transcribed and overexpressed in HEK 293 T cells. BCoV-N recombinant protein inhibited dose-dependently VSV-induced IFN-β production (p < 0.01). MDA5, MAVS, TBK1 and IRF3 could promote transcription levels of IFN-β mRNA. But, BCoV-N protein demoted IFN-β transcription levels induced by MDA5, MAVS, TBK1 and IRF3. Furthermore, expression levels of MDA5, MAVS, TBK1 and IRF3 mRNAs were reduced in RIG-I-like receptor (RLR) pathway. In conclusion, BCoV-N reduced IFN-β levels in RIG-I-like receptor (RLR) pathway in HEK 293 T cells which were induced by MDA5, MAVS, TBK1 and IRF3(5D). BCoV-N protein inhibited IFN-β production and activation of RIG-I-like receptors (RLRs) signal pathway. Our findings demonstrated BCoV N protein is an IFN-β antagonist through inhibition of MDA5, MAVS, TBK1 and IRF3(5D) in RLRs pathway, also revealed a new mechanism of BCoV N protein to evade host innate immune response by inhibiting type I IFN production, which is beneficial to developing novel prevention strategy for BCoV disease in the animals and humans. [ABSTRACT FROM AUTHOR]

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen which mainly causes diarrhea, dehydration and death in nursing piglets, threatening the global swine industry. Moreover, it can infect multiple animal species and humans. Hence, reliable diagnostic assays are needed to better control this zoonotic pathogen. Here, a blocking ELISA was developed using a recombinant nucleocapsid (N) protein as the coating antigen paired with an N-specific monoclonal antibody (mAb) as the detection antibody. The percent inhibition (PI) of the ELISA was determined using 384 swine serum samples, with an indirect immunofluorescence assay (IFA) as the reference method. Through receiver operating characteristic analysis in conjunction with Youden's index, the optimal PI cut-off value was determined to be 51.65%, which corresponded to a diagnostic sensitivity of 98.79% and a diagnostic specificity of 100%. Of the 330 serum samples tested positive via IFA, 326 and 4 were tested positive and negative via the ELISA, respectively, while the 54 serum samples tested negative via IFA were all negative via the ELISA. The overall coincidence rate between the two assays was 98.96% (380/384). The ELISA exhibited good repeatability and did not cross-react with antisera against other swine pathogens. Overall, this is the first report on developing a blocking ELISA for PDCoV serodiagnosis. [ABSTRACT FROM AUTHOR]

It is an acknowledged fact that SARS-CoV-2 exhibits tropism for the human placenta. A possible mechanism of SARS-CoV-2 entry into host cells is via angiotensin-converting enzyme 2 (ACE2) receptors, which are expressed in trophoblasts, endothelial cells, and macrophages. The present study describes a case of spontaneous miscarriage in the 20th gestational week after maternal SARS-CoV-2 infection. The placenta and various fetal organs were examined for structural alterations and expression of the viral nucleocapsid protein and several immune cell markers via immunohistochemistry (IHC). Histopathological examination of the placenta revealed acute chorioamnionitis, acute subamnionic placentitis, multiple intervillous thrombi, increased fibrinoid deposition, and necrotic changes of the chorionic villi. Immunohistochemistry confirmed the presence of SARS-CoV-2 nucleocapsid protein regions predominantly in the syncytiotrophoblast. Staining of the placental tissue for different markers helped elucidate the distribution of immune cells. Pathomorphological examination of the fetal organs demonstrated changes in microcirculation with the presence of sludge phenomenon and diapedesis haemorrhages, mostly in the lungs, brain, and myocardium. IHC staining of fetal organs revealed expression of SARS-CoV-2 nucleocapsid protein, which was detected to the highest extent in the brain, lungs, and liver. The findings of the present report support the hypothesis of possible vertical transmission of SARS-CoV-2 from mother to fetus. [ABSTRACT FROM AUTHOR]

Vaccination certainly is the best way to fight against the COVID‐19 pandemic. In this study, the seroconversion effectiveness of two vaccines against severe acute respiratory syndrome coronavirus 2 was assessed in healthcare workers: virus‐inactivated CoronaVac (CV, n = 303), and adenovirus‐vectored Oxford–AstraZeneca (AZ, n = 447). The immunoglobulin G (IgG) antibodies anti‐spike glycoprotein and anti‐nucleocapsid protein were assessed by enzyme‐linked immunosorbent assay at the time before vaccination (T1), before the second dose (T2), and 30 days after the second dose (T3). Of all individuals vaccinated with AZ, 100% (n = 447) exhibited seroconversion, compared to 91% (n = 276) that were given CV vaccine. Among individuals who did not respond to the CV, only three individuals showed a significant increase in the antibody level 4 months later the booster dose. A lower seroconversion rate was observed in elders immunized with the CV vaccine probably due to the natural immune senescence, or peculiarity of this vaccine. The AZ vaccine induced a higher humoral response; however, more common side effects were also observed. Nonvaccinated convalescent individuals revealed a similar rate of anti‐spike IgG to individuals that were given two doses of CV vaccine, which suggests that only a one‐shot COVID‐19 vaccine could produce an effective immune response in convalescents. [ABSTRACT FROM AUTHOR]

The nucleocapsid (N) protein of SARS-CoV-2 has been reported to have a high ability of liquid-liquid phase separation, which enables its incorporation into stress granules (SGs) of host cells. However, whether SG invasion by N protein occurs in the scenario of SARS-CoV-2 infection is unknow, neither do we know its consequence. Here, we used SARS-CoV-2 to infect mammalian cells and observed the incorporation of N protein into SGs, which resulted in markedly impaired self-disassembly but stimulated cell cellular clearance of SGs. NMR experiments further showed that N protein binds to the SG-related amyloid proteins via non-specific transient interactions, which not only expedites the phase transition of these proteins to aberrant amyloid aggregation in vitro, but also promotes the aggregation of FUS with ALS-associated P525L mutation in cells. In addition, we found that ACE2 is not necessary for the infection of SARS-CoV-2 to mammalian cells. Our work indicates that SARS-CoV-2 infection can impair the disassembly of host SGs and promote the aggregation of SG-related amyloid proteins, which may lead to an increased risk of neurodegeneration. [ABSTRACT FROM AUTHOR]

Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the world and has had a devastating impact on health and economy. The biochemical characterization of SARS-CoV-2 proteins is important for drug design and development. In this study, we discovered that the SARS-CoV-2 nucleocapsid protein can melt double-stranded DNA (dsDNA) in the 5'-3' direction, similar to SARSCoV-2 nonstructural protein 13. However, the unwinding activity of SARS-CoV-2 nucleocapsid protein was found to be more than 22 times weaker than that of SARSCoV-2 nonstructural protein 13, and the melting process was independent of nucleoside triphosphates and Mg2+. Interestingly, at low concentrations, the SARS-CoV-2 nucleocapsid protein exhibited a stronger annealing activity than SARS-CoV-2 nonstructural protein 13; however, at high concentrations, it promoted the melting of dsDNA. These findings have deepened our understanding of the SARS-CoV-2 nucleocapsid protein and will help provide novel insights into antiviral drug development. [ABSTRACT FROM AUTHOR]

The pandemic caused by the SARS-CoV-2 virus, the agent responsible for the COVID-19 disease, has affected millions of people worldwide. There is constant search for new therapies to either prevent or mitigate the disease. Fortunately, we have observed the successful development of multiple vaccines. Most of them are focused on one viral envelope protein, the spike protein. However, such focused approaches may contribute for the rise of new variants, fueled by the constant selection pressure on envelope proteins, and the widespread dispersion of coronaviruses in nature. Therefore, it is important to examine other proteins, preferentially those that are less susceptible to selection pressure, such as the nucleocapsid (N) protein. Even though the N protein is less accessible to humoral response, peptides from its conserved regions can be presented by class I Human Leukocyte Antigen (HLA) molecules, eliciting an immune response mediated by T-cells. Given the increased number of protein sequences deposited in biological databases daily and the N protein conservation among viral strains, computational methods can be leveraged to discover potential new targets for SARSCoV-2 and SARS-CoV-related viruses. Here we developed SARS-Arena, a user-friendly computational pipeline that can be used by practitioners of different levels of expertise for novel vaccine development. SARS-Arena combines sequence-based methods and structure-based analyses to (i) perform multiple sequence alignment (MSA) of SARSCoV-related N protein sequences, (ii) recover candidate peptides of different lengths from conserved protein regions, and (iii) model the 3D structure of the conserved peptides in the context of different HLAs. We present two main Jupyter Notebook workflows that can help in the identification of new T-cell targets against SARS-CoV viruses. In fact, in a cross-reactive case study, our workflows identified a conserved N protein peptide (SPRWYFYYL) recognized by CD8+ T-cells in the context of HLA-B7+. SARS-Arena is available at https://github.com/KavrakiLab/SARS-Arena. [ABSTRACT FROM AUTHOR]

The ongoing outbreak of COVID‐19 caused by SARS‐CoV‐2 has resulted in a serious public health threat globally. Nucleocapsid protein is a major structural protein of SARS‐CoV‐2 that plays important roles in the viral RNA packing, replication, assembly, and infection. Here, we report two crystal structures of nucleocapsid protein C‐terminal domain (CTD) at resolutions of 2.0 Å and 3.1 Å, respectively. These two structures, crystallized under different conditions, contain 2 and 12 CTDs in asymmetric unit, respectively. Interestingly, despite different crystal packing, both structures show a similar dimeric form as the smallest unit, consistent with its solution form measured by the size‐exclusion chromatography, suggesting an important role of CTD in the dimerization of nucleocapsid proteins. By analyzing the surface charge distribution, we identified a stretch of positively charged residues between Lys257 and Arg262 that are involved in RNA‐binding. Through screening a single‐domain antibodies (sdAbs) library, we identified four sdAbs targeting different regions of nucleocapsid protein with high affinities that have future potential to be used in viral detection and therapeutic purposes. [ABSTRACT FROM AUTHOR]

Introduction: Assay kits for detection of Immunoglobulin G (IgG) against the nucleocapsid protein (anti-nucleocapsid IgG) and spike proteins (anti-spike IgG) of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) were commercially provided by several manufacturers. These assay kits should be verified by measuring the same sample. Aim: To compare the diagnostic value of three Coronavirus Disease-2019 (COVID-19) kits in evaluating six immunoassays developed by three manufacturers (Abbott, Euglena, and Roche) to detect anti-nucleocapsid IgG and anti-spike IgG. Materials and Methods: Present study was an observational cross-sectional study conducted from June 2020 to December 2020. Antibody titers for anti-nucleocapsid IgG and anti-spike IgG among 429 Healthcare Workers (HCWs) in a Tone Central Hospital, Japan where a nosocomial infection of the COVID-19 occurred were measured by six immunoassays with kits developed by three different manufacturers. The sensitivity and specificity of each kit was compared to real-time Reverse Transcription-Polymerase Chain Reaction (RT-qPCR). Results: Six of the HCWs tested positive for SARS-CoV-2 via RT-qPCR, and the rest tested negative. The severity of COVID-19 among these six HCWs ranged from mild to moderate. The sensitivity and specificity values against RT-qPCR were, 100% and 99.5% for Abbott, 83.3% and 100% for Euglena, and 100% and 100% for Roche when using the nucleocapsid protein assay and 100% and 99.8% for Abbott, 100% and 100% for Euglena, and 100% and 100% for Roche when using the spike protein assay kit. Conclusion: The commercial kits provided by three manufacturers reflected the immune status of individuals. There were no major differences in the performance of these test kits. Discordant results with the antibody titer for anti-nucleocapsid IgG and anti-spike IgG were detected by using assay kits provided by Abbott and Euglena. To evaluate the past history of COVID-19, it should be noted that the single measurement of anit-nucleocapsid IgG or anti-spike IgG could not exclude false negative or positives. [ABSTRACT FROM AUTHOR]

Hantaan virus (HTNV) is the main cause of hemorrhagic fever with renal syndrome (HFRS) around the world, which results in profound morbidity and mortality. However, there are currently no FDA-approved therapeutics or vaccines against HFRS. To find new anti-HTNV drugs, the inhibitory activity of 901 small molecule kinase inhibitors against HTNV is analyzed. Among these compounds, compound 8G1 inhibits HTNV with a relatively high inhibition rate and lower toxicity. The viral titer and nucleocapsid protein of HTNV are reduced after compound 8G1 treatment in a dose-dependent manner at concentrations ranging from 1 to 20 μM. In addition, the administration of compound 8G1 at the early stage of HTNV infection can inhibit the replication of HTNV. The molecular docking result reveals that compound 8G1 forms interactions with the key amino acid residues of serine/threonine-protein kinase B (Akt), which is responsible for the observed affinity. Then, the mammalian target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E (eIF4E) signaling pathways are inhibited. Our results may help to design novel targets for therapeutic intervention against HTNV infection and to understand the anti-HTNV mechanism of protein kinase inhibitors. [ABSTRACT FROM AUTHOR]

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

Since autumn 2020, rapid antigen tests (RATs) have been implemented in several countries as an important pillar of the national testing strategy to rapidly screen for infections on site during the SARS-CoV-2 pandemic. The current surge in infection rates around the globe is driven by the variant of concern (VoC) omicron (B.1.1.529). Here, we evaluated the performance of nine SARS-CoV-2 RATs in a single-centre laboratory study. We examined a total of 115 SARS-CoV-2 PCR-negative and 166 SARS-CoV-2 PCR-positive respiratory swab samples (101 omicron, 65 delta (B.1.617.2)) collected from October 2021 until January 2022 as well as cell culture-expanded clinical isolates of both VoCs. In an assessment of the analytical sensitivity in clinical specimen, the 50% limit of detection (LoD50) ranged from 1.77 × 106 to 7.03 × 107 RNA copies subjected to the RAT for omicron compared to 1.32 × 105 to 2.05 × 106 for delta. To score positive in these point-of-care tests, up to 10-fold (LoD50) or 101-fold (LoD95) higher virus loads were required for omicron- compared to delta-containing samples. The rates of true positive test results for omicron samples in the highest virus load category (Ct values < 25) ranged between 31.4 and 77.8%, while they dropped to 0–8.3% for samples with intermediate Ct values (25–30). Of note, testing of expanded virus stocks suggested a comparable RAT sensitivity of both VoCs, questioning the predictive value of this type of in vitro-studies for clinical performance. Given their importance for national test strategies in the current omicron wave, awareness must be increased for the reduced detection rate of omicron infections by RATs and a short list of suitable RATs that fulfill the minimal requirements of performance should be rapidly disclosed. [ABSTRACT FROM AUTHOR]

Compared to the parental SARS-CoV-2 virus, infections by the now dominant Delta variant of SARS-CoV-2 appear to be more common and more severe in pregnant women. The need for a robust, cheap, and quick method for diagnosing placental infection by SARS-CoV-2 has thus become more acute. Here, we describe a highly sensitive and specific immunohistochemical assay for SARS-CoV-2 nucleocapsid protein for routine use in placental pathology practice. [ABSTRACT FROM AUTHOR]

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the pathogenic agent leading to COVID-19. Due to high speed of transmission and mutation rates, universal diagnosis and appropriate prevention are still urgently needed. The nucleocapsid protein of SARS-CoV-2 is considered more conserved than spike proteins and is abundant during the virus' life cycle, making it suitable for diagnostic applications. Here, we designed and developed a fluorescent immunochromatography assay (FICA) for the rapid detection of SARS-CoV-2-specific antibodies using ZnCdSe/ZnS QDs-conjugated nucleocapsid (N) proteins as probes. The nucleocapsid protein was expressed in E.coli and purified via Ni-NTA affinity chromatography with considerable concentration (0.762 mg/mL) and a purity of more than 90%, which could bind to specific antibodies and the complex could be captured by Staphylococcal protein A (SPA) with fluorescence displayed. After the optimization of coupling and detecting conditions, the limit of detection was determined to be 1:1.024 × 105 with an IgG concentration of 48.84 ng/mL with good specificity shown to antibodies against other zoonotic coronaviruses and respiratory infection-related viruses (n = 5). The universal fluorescent immunochromatography assay simplified operation processes in one step, which could be used for the point of care detection of SARS-CoV-2-specific antibodies. Moreover, it was also considered as an efficient tool for the serological screening of potential susceptible animals and for monitoring the expansion of virus host ranges. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) is the etiological agent of porcine epidemic diarrhea (PED) characterized by vomit, watery diarrhea, dehydration and high mortality. Outbreaks of highly pathogenic variant strains of PEDV have resulted in extreme economic losses to the swine industry all over the world. The study of host–virus interaction can help to better understand the viral pathogenicity. Many studies have shown that poly(A)-binding proteins are involved in the replication process of various viruses. Here, we found that the infection of PEDV downregulated the expression of poly(A)-binding protein cytoplasmic 1 (PABPC1) at the later infection stage in Vero cells. The overexpression of PABPC1 inhibited the proliferation of PEDV at transcription and translation level, and siRNA-mediated depletion of PABPC1 promoted the replication of PEDV. Furthermore, mass spectrometry analysis and immunoprecipitation assay confirmed that PABPC1 interacted with the nucleocapsid (N) protein of PEDV. Confocal microscopy revealed the co-localizations of PABPC1 with N protein in the cytoplasm. Taken together, these results demonstrate the antiviral effect of PABPC1 against PEDV replication by interacting with N protein, which increases understanding of the interaction between PEDV and host. [ABSTRACT FROM AUTHOR]

The pandemic caused by the SARS-CoV-2 virus, the agent responsible for the COVID-19 disease, has affected millions of people worldwide. There is constant search for new therapies to either prevent or mitigate the disease. Fortunately, we have observed the successful development of multiple vaccines. Most of them are focused on one viral envelope protein, the spike protein. However, such focused approaches may contribute for the rise of new variants, fueled by the constant selection pressure on envelope proteins, and the widespread dispersion of coronaviruses in nature. Therefore, it is important to examine other proteins, preferentially those that are less susceptible to selection pressure, such as the nucleocapsid (N) protein. Even though the N protein is less accessible to humoral response, peptides from its conserved regions can be presented by class I Human Leukocyte Antigen (HLA) molecules, eliciting an immune response mediated by T-cells. Given the increased number of protein sequences deposited in biological databases daily and the N protein conservation among viral strains, computational methods can be leveraged to discover potential new targets for SARS-CoV-2 and SARS-CoV-related viruses. Here we developed SARS-Arena, a user-friendly computational pipeline that can be used by practitioners of different levels of expertise for novel vaccine development. SARS-Arena combines sequence-based methods and structure-based analyses to (i) perform multiple sequence alignment (MSA) of SARS-CoV-related N protein sequences, (ii) recover candidate peptides of different lengths from conserved protein regions, and (iii) model the 3D structure of the conserved peptides in the context of different HLAs. We present two main Jupyter Notebook workflows that can help in the identification of new T-cell targets against SARS-CoV viruses. In fact, in a cross-reactive case study, our workflows identified a conserved N protein peptide (SPRWYFYYL) recognized by CD8+ T-cells in the context of HLA-B7+. SARS-Arena is available at https://github.com/KavrakiLab/SARS-Arena.

ABSTRACT The study aimed to establish the performance of the SARS-CoV-2 Rapid Antibody Test (IgG and IgM) and the Elecsys Anti-SARS-CoV-2 S assay in vaccinated individuals. A panel of serum samples from Boca Biolistics was utilized to assess antibodies following vaccination, consisting of samples drawn prior to vaccination, after the first dose, or at least 14 days after the second dose of Moderna mRNA-1273 or Pfizer-BioNTech BNT162b2 COVID-19 vaccines. Agreement between the two methods was measured and stratified by test evaluator and assay lot. Agreement between the SARS-CoV-2 Rapid Antibody Test (IgG) and Elecsys Anti-SARS-CoV-2 S assay qualitative measurements at the different assessment points for both mRNA-1273 and BNT162b2 ranged between 97.06% (95% confidence interval [CI] 84.67, 99.93) to 100% (95% CI 82.35, 100). Agreement of the SARS-CoV-2 Rapid Antibody Test (IgG) with the Elecsys Anti-SARS-CoV-2 S assay was not highly influenced by either lot or evaluator. There was a medium-to-strong correlation between the semiquantitative SARS-CoV-2 Rapid Antibody Test (IgG) result and quantitative Elecsys Anti-SARS-CoV-2 S assay in samples taken after both doses of the vaccines, with higher intensity bands being associated with higher total anti-S antibody titer (mRNA-1273, P = 0.0019; BNT162b2, P < 0.0001). Conclusion Semiquantitative SARS-CoV-2 Rapid Antibody Test (IgG) and quantitative Elecsys Anti-SARS-CoV-2 S assay correlated well, suggesting that the SARS-CoV-2 Rapid Antibody Test (IgG) is helpful in understanding the immune response postvaccination. The current data support the use of the SARS-CoV-2 Rapid Antibody Test (IgG) in the vaccinated population. IMPORTANCE Serologic assays are an essential tool for seroprevalence surveys, for quality control of vaccines, and to determine the response to vaccination. Although a correlate of immunity has not yet been established for COVID-19 vaccines, antibody titers after natural infection and vaccination have been associated with protection from symptomatic SARS-CoV-2 infection. Rapid point-of-care assays can be of use in this context with advantages over centralized testing, such as speed and ease of use. The point-of-care SARS-CoV-2 Rapid Antibody Test (IgG) compared favorably to the Elecsys Anti-SARS-CoV-2 S assay with agreement rates above 97.06%, after one or two doses of Moderna mRNA-1273 or Pfizer-BioNTech BNT162b2. Semiquantitative SARS-CoV-2 Rapid Antibody Test (IgG) and quantitative Elecsys Anti-SARS-CoV-2 S assay results correlated well, suggesting that SARS-CoV-2 Rapid Antibody Test (IgG) is helpful in understanding the immune response postvaccination. The current data support the use of the SARS-CoV-2 Rapid Antibody Test (IgG) in the vaccinated population.

Hantaan virus (HTNV) is the main cause of hemorrhagic fever with renal syndrome (HFRS) around the world, which results in profound morbidity and mortality. However, there are currently no FDA-approved therapeutics or vaccines against HFRS. To find new anti-HTNV drugs, the inhibitory activity of 901 small molecule kinase inhibitors against HTNV is analyzed. Among these compounds, compound 8G1 inhibits HTNV with a relatively high inhibition rate and lower toxicity. The viral titer and nucleocapsid protein of HTNV are reduced after compound 8G1 treatment in a dose-dependent manner at concentrations ranging from 1 to 20 μM. In addition, the administration of compound 8G1 at the early stage of HTNV infection can inhibit the replication of HTNV. The molecular docking result reveals that compound 8G1 forms interactions with the key amino acid residues of serine/threonine-protein kinase B (Akt), which is responsible for the observed affinity. Then, the mammalian target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E (eIF4E) signaling pathways are inhibited. Our results may help to design novel targets for therapeutic intervention against HTNV infection and to understand the anti-HTNV mechanism of protein kinase inhibitors.

The SARS-CoV-2 nucleocapsid protein (NP) is an important indicator for the virus infection, highlighting the crucial role of NP-specific monoclonal antibodies (mAbs) used in multiple biochemical assays and clinical diagnosis for detecting the NP antigen. Here, we reported a pair of noncompeting human NP-specific mAbs, named P301-F7 and P301-H5, targeting two distinct linear epitopes on SARS-CoV-2 or SARS-CoV. We evaluated the application of P301-F7 in the analysis of enzyme linked immunosorbent assay, western blot, flow cytometry, immunofluorescence, and focus reduction neutralization test. We for the first time report a broad mAb effectively recognizing various live viruses of SARS-CoV-2 variants including Alpha, Beta, Delta, and Omicron, indicating a wide range of application prospects. [ABSTRACT FROM AUTHOR]

In global infection and serious morbidity and mortality, porcine epidemic diarrhea virus (PEDV) has been regarded as a dreadful porcine pathogen, but the existing commercial vaccines are not enough to fully protect against the epidemic strains. Therefore, it is of great necessity to feature the PEDV-host interaction and develop efficient countermeasures against viral infection. As an RNA/DNA protein, the trans-active response DNA binding protein (TARDBP) plays a variety of functions in generating and processing RNA, including transcription, splicing, transport, and mRNA stability, which have been reported to regulate viral replication. The current work aimed to detect whether and how TARDBP influences PEDV replication. Our data demonstrated that PEDV replication was significantly suppressed by TARDBP, regulated by KLF16, which targeted its promoter. We observed that through the proteasomal and autophagic degradation pathway, TARDBP inhibited PEDV replication via the binding as well as degradation of PEDV-encoded nucleocapsid (N) protein. Moreover, we found that TARDBP promoted autophagic degradation of N protein via interacting with MARCHF8, an E3 ubiquitin ligase, as well as NDP52, a cargo receptor. We also showed that TARDBP promoted host antiviral innate immune response by inducing interferon (IFN) expression through the MyD88-TRAF3-IRF3 pathway during PEDV infection. In conclusion, these data revealed a new antiviral role of TARDBP, effectively suppressing PEDV replication through degrading virus N protein via the proteasomal and autophagic degradation pathway and activating type I IFN signaling via upregulating the expression of MyD88. [ABSTRACT FROM AUTHOR]

Background: Finding the key amino acid sites that could affect viral biological properties or protein functions has always been a topic of substantial interest in virology. The nucleocapsid (N) protein is one of the principal proteins of the porcine reproductive and respiratory syndrome virus (PRRSV) and plays a vital role in the virus life cycle. The N protein has only 123 or 128 amino acids, some of key amino acid sites which could affect the protein functions or impair the viral biological characteristics have been identified. In this research, our objective was to find out whether there are other novel amino acid sites of the N protein can affect N protein functions or PRRSV-2 replication. Results: In this study, we found mutated the serine78 and serine 99of the nucleocapsid (N) protein can reduce the N-induced expression of IL-10 mRNA; Then, by using reverse genetics system, we constructed and rescued the mutant viruses, namely, A78 and A99.The IFA result proved that the mutations did not affect the rescue of the PRRSV-2. However, the results of the multistep growth kinetics and qPCR assays indicated that, compared with the viral replication ability, the titres and gRNA levels of A78 were significantly decreased compared with the wild-type. Further study showed that a single amino acid change from serine to alanine at position 78 of the N protein could abrogates the level of viral genomic and subgenomic RNAs. It means the mutation could significant decrease the viral replication efficiency in vitro. Conclusions: Our results suggest that the serine78 of N protein is a key site which could affect the N protein function and PRRSV replication ability. [ABSTRACT FROM AUTHOR]

Objective Real-time reverse transcription-polymerase chain reaction is the gold standard for the diagnosis of COVID-19, but it is necessary to utilize other tests to determine the burden of the disease and the spread of the outbreak such as IgG-, IgM-, and IgA-based antibody detection using enzyme-linked immunosorbent assay (ELISA). Materials and Methods We developed an indirect ELISA assay to quantitatively measure the amount of COVID-19 IgG, IgM, and IgA antibodies present in patient serum, dried blood, and plasma. Results The population cutoff values for positivity were determined by receiver operating characteristic curves to be 1.23 U/mL, 23.09 U/mL, and 6.36 U/mL for IgG, IgM, and IgA, respectively. After albumin subtraction, the specificity remained >98% and the sensitivity was 95.72%, 83.47%, and 82.60%, respectively, for IgG, IgM, and IgA antibodies to the combined spike subunit 1 receptor binding domain and N proteins in serum. Plasma and dried blood spot specimens were also validated on this assay. Conclusion This assay may be used for determining the seroprevalence of SARS-CoV-2 in a population exposed to the virus or in vaccinated individuals. [ABSTRACT FROM AUTHOR]

Rice stripe virus (RSV) transmitted by the small brown planthopper causes severe rice yield losses in Asian countries. Although viral nuclear entry promotes viral replication in host cells, whether this phenomenon occurs in vector cells remains unknown. Therefore, in this study, we systematically evaluated the presence and roles of RSV in the nuclei of vector insect cells. We observed that the nucleocapsid protein (NP) and viral genomic RNAs were partially transported into vector cell nuclei by utilizing the importin α nuclear transport system. When blocking NP nuclear localization, cytoplasmic RSV accumulation significantly increased. In the vector cell nuclei, NP bound the transcription factor YY1 and affected its positive regulation to FAIM. Subsequently, decreased FAIM expression triggered an antiviral caspase-dependent apoptotic reaction. Our results reveal that viral nuclear entry induces completely different immune effects in vector and host cells, providing new insights into the balance between viral load and the immunity pressure in vector insects. [ABSTRACT FROM AUTHOR]

The timely detecting of SARS-CoV-2 coronavirus antigens for infection validation is an urgent request for COVID-19 pandemic control. This study constructed label-free electrochemical impedance spectroscopy (EIS)-based immunosensors based on gold nanostructured screen-printed carbon electrodes (AuNS/SPCEs) to detect the SARS-CoV-2 nucleocapsid protein (N-protein) in saliva. Using short-chain 3-mercaptopropionic acid (MPA) as a linker to covalently bond streptavidin (SA) and bovine serum albumin (BSA) for controlling the oriented immobilization of the biotinylated anti-N-protein antibody (BioAb) can offer a greater sensitivity, a lower limit of detection (LOD), and better reproducibility of immunosensors (defined as BioAb/SA-BSA/MPA/AuNS/SPCEs) than the antibody randomly immobilized immunosensors and the long-chain 11-mercaptoundecanoic acid (MUA)-modified immunosensors (BioAb/SA-BSA/MUA/AuNS/SPCEs). The BioAb/SA-BSA/MPA/AuNS/SPCE-based immunosensors presented good linearity from 0.01 ng/mL to 100 ng/mL and a low LOD of 6 pg/mL in a phosphate buffer solution (PBS) and PBS-diluted saliva. Moreover, the immunosensor exhibited little cross-activity with other viral antigens such as MERS-CoV N-protein, influenza A N-protein, influenza B N-protein, and SARS-CoV-2 spike protein, indicating the high specificity of the immunosensors. The disposable label-free EIS-based immunosensors have promising potential in facilitating the rapid and sensitive tests of saliva-based COVID-19 diagnostics. [ABSTRACT FROM AUTHOR]

Human coronavirus HKU1 (HCoV-HKU1) is one of the four endemic coronaviruses. It has been suggested that there is a difference in incidence, with PCR-confirmed HCoV-NL63 and HCoV-OC43 infections occurring more commonly, whereas HCoV-HKU1 is the least seen. Lower incidence of HCoV-HKU1 infection has also been observed in serological studies. The current study aimed to investigate antibody dynamics during PCR-confirmed HCoV-HKU1 infections using serum collected during infection and 1 month later. We expressed a new HCoV-HKU1 antigen consisting of both the linker and carboxy-terminal domain of the viral nucleocapsid protein and implemented it in ELISA. We also applied a spike-based Luminex assay on serum samples from PCR-confirmed infections by the four endemic HCoVs. At least half of HCoV-HKU1-infected subjects consistently showed no antibody rise via either assay, and some subjects even exhibited substantial antibody decline. Investigation of self-reported symptoms revealed that HCoV-HKU1-infected subjects rated their illness milder than subjects infected by other HCoVs. In conclusion, HCoV-HKU1 infections reported in this study displayed atypical antibody dynamics and milder symptoms when compared to the other endemic HCoVs. [ABSTRACT FROM AUTHOR]