Autophagy plays an antiviral defence role against tomato spotted wilt orthotospovirus and is counteracted by viral effector NSs.

Autophagy, an intracellular degradation process, has emerged as a crucial innate immune response against various plant pathogens, including viruses. Tomato spotted wilt orthotospovirus (TSWV) is a highly destructive plant pathogen that infects over 1000 plant species and poses a significant threat to global food security. However, the role of autophagy in defence against the TSWV pathogen, and whether the virus counteracts this defence, remains unknown. In this study, we report that autophagy plays an important role in antiviral defence against TSWV infection; however, this autophagy-mediated defence is counteracted by the viral effector NSs. Transcriptome profiling revealed the up-regulation of autophagy-related genes (ATGs) upon TSWV infection. Blocking autophagy induction by chemical treatment or knockout/down of ATG5/ATG7 significantly enhanced TSWV accumulation. Notably, the TSWV nucleocapsid (N) protein, a major component of the viral replication unit, strongly induced autophagy. However, the TSWV nonstructural protein NSs was able to effectively suppress N-induced autophagy in a dose-dependent manner. Further investigation revealed that NSs inhibited ATG6-mediated autophagy induction. These findings provide new insights into the defence role of autophagy against TSWV, a representative segmented negative-strand RNA virus, as well as the tospoviral pathogen counterdefence mechanism.

A novel double antibody sandwich quantitative ELISA for detecting porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0.05 ng/mL and 103.02 50% tissue culture infective dose per mL (TCID50/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92.55% with a kappa value of 0.85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94.29% with a kappa value of 0.88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. KEY POINTS: • A new anti-PEDV N protein monoclonal antibody strain was prepared • Establishment of a more sensitive double antibody sandwich quantitative ELISA • DAS-qELISA was found to be useful for controlling the PEDV spread.

Control of nuclear localization of the nucleocapsid protein of SARS-CoV-2.

The nucleocapsid (N) protein of coronaviruses is a structural protein that binds viral RNA for assembly into the mature virion, a process that occurs in the cytoplasm. Several coronavirus N proteins also localize to the nucleus. Herein, we identify that two sequences (NLSs) are required for nuclear localization of the SARS-CoV-2 N protein. Deletion or mutation of these two sequences creates an N protein that does not localize to the nucleus in HEK293T cells. Overexpression of both wild-type and NLS-mutated N proteins dysregulate a largely overlapping set of mRNAs in HEK293T cells, suggesting that these N proteins do not have direct nuclear effects on transcription. Consistent with that hypothesis, both N proteins induce nuclear localization of NF-κB p65 and dysregulate a set of previously identified NF-κB-dependent genes. The effects of N on nuclear properties are proposed to alter host cell functions that contribute to viral pathogenesis or replication.

Identification of novel broad-spectrum antiviral drugs targeting the N-terminal domain of the FIPV nucleocapsid protein.

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.

AlphaFold2 Reveals Structural Patterns of Seasonal Haplotype Diversification in SARS-CoV-2 Nucleocapsid Protein Variants.

The COVID-19 pandemic saw the emergence of various Variants of Concern (VOCs) that took the world by storm, often replacing the ones that preceded them. The characteristic mutant constellations of these VOCs increased viral transmissibility and infectivity. Their origin and evolution remain puzzling. With the help of data mining efforts and the GISAID database, a chronology of 22 haplotypes described viral evolution up until 23 July 2023. Since the three-dimensional atomic structures of proteins corresponding to the identified haplotypes are not available, ab initio methods were here utilized. Regions of intrinsic disorder proved to be important for viral evolution, as evidenced by the targeted change to the nucleocapsid (N) protein at the sequence, structure, and biochemical levels. The linker region of the N-protein, which binds to the RNA genome and self-oligomerizes for efficient genome packaging, was greatly impacted by mutations throughout the pandemic, followed by changes in structure and intrinsic disorder. Remarkably, VOC constellations acted co-operatively to balance the more extreme effects of individual haplotypes. Our strategy of mapping the dynamic evolutionary landscape of genetically linked mutations to the N-protein structure demonstrates the utility of ab initio modeling and deep learning tools for therapeutic intervention.

N-protein vaccine is effective against COVID-19: Phase 3, randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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.

Cellular immunity to nucleoproteins (NP) of Crimean-Congo hemorrhagic fever virus (CCHFV) and Hazara Virus (HAZV).

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a globally significant vector-borne pathogen with no internationally-licensed preventative and therapeutic interventions. Hazara virus (HAZV), on the other hand, a related Orthonairovirus, has not been reported as a human pathogen. HAZV has been proposed as a surrogate model for studying CCHFV, bisosafety level 4 (BSL-4) agent. Previously, we investigated the humoral immune responses between NPs of these viruses and in this study, we extended the scrutiny to cellular immune responses elicited by NPs of CCHFV and HAZV. Here, mice were immunized with recombinant CCHFV NP and HAZV NP to evaluate the correlates of cell-mediated immunity (CMI). Delayed-type hypersensitivity (DTH) responses were assessed by challenging immunized mice with CCHFV-rNP or HAZV-rNP on the footpad and lymphocyte proliferation assays (LPAs) were performed by stimulating splenocytes in vitro with CCHFV-rNP or HAZV-rNP to compare cellular immune responses. In all test groups, strong DTH and LPA responses were detected against homologous and heterologous challenging antigens. To assess the cytokine response, an RT-qPCR -specific for cytokine mRNAs was utilized. Interestingly, CCHFV NP stimulated groups exhibited a significantly elevated mRNA level of interleukin 17 A (IL-17) compared to HAZV NP, indicating a notable difference in immune responses. This study presents comparison between CMI elicited by NPs of CCHFV and HAZV and contributes to the understanding of a highly pathogenic virus, particularly in the context of the declaration of CCHFV by World Health Organization's (WHO) as a major viral threat to the world.

Viral, Immunologic, and Laboratory Parameters in Patients With and Without Post-Acute Sequelae of SARS-CoV-2 Infection (PASC).

BACKGROUND: The pathophysiological mechanisms underlying the post-acute sequelae of severe acute respiratory syndrome coronavirus 2 infection (PASC) are not well understood. Our study aimed to investigate various aspects of theses mechanisms, including viral persistence, immunological responses, and laboratory parameters in patients with and without PASC. METHODS: We prospectively enrolled adults aged ≥ 18 years diagnosed with coronavirus disease 2019 (COVID-19) between August 2022 and July 2023. Blood samples were collected at three time-points: within one month of diagnosis (acute phase) and at 1 month, and 3 months post-diagnosis. Following a recent well-designed definition of PASC, PASC patients were defined as those with a questionnaire-based PASC score ≥ 12 persisting for at least 4 weeks after the initial COVID-19 diagnosis. RESULTS: Of 57 eligible COVID-19 patients, 29 (51%) had PASC, and 28 (49%) did not. The PASC group had significantly higher nucleocapsid protein (NP) antigenemia 3 months after COVID-19 diagnosis (P = 0.022). Furthermore, several cytokines, including IL-2, IL-17A, VEGF, RANTES, sCD40L, IP-10, I-TAC, and granzyme A, were markedly elevated in the PASC group 1 and/or 3 month(s) after COVID-19 diagnosis. In contrast, the median values of several serological markers, including thyroid markers, autoimmune indicators, and stress-related hormones, were within the normal range. CONCLUSION: Levels of NP antigen and of various cytokines involved in immune responses become significantly elevated over time after COVID-19 diagnosis in PASC patients compared to non-PASC patients. This suggests that PASC is associated with prolonged immune dysregulation resulting from heightened antigenic stimulation.

Structural basis for the participation of the SARS-CoV-2 nucleocapsid protein in the template switch mechanism and genomic RNA reorganization.

The COVID-19 pandemic has resulted in a significant toll of deaths worldwide, exceeding seven million individuals, prompting intensive research efforts aimed at elucidating the molecular mechanisms underlying the pathogenesis of SARS-CoV-2 infection. Despite the rapid development of effective vaccines and therapeutic interventions, COVID-19 remains a threat to humans due to the emergence of novel variants and largely unknown long-term consequences. Among the viral proteins, the nucleocapsid protein (N) stands out as the most conserved and abundant, playing the primary role in nucleocapsid assembly and genome packaging. The N protein is promiscuous for the recognition of RNA, yet it can perform specific functions. Here, we discuss the structural basis of specificity, which is directly linked to its regulatory role. Notably, the RNA chaperone activity of N is central to its multiple roles throughout the viral life cycle. This activity encompasses double-stranded RNA (dsRNA) annealing and melting and facilitates template switching, enabling discontinuous transcription. N also promotes the formation of membraneless compartments through liquid‒liquid phase separation (LLPS), thereby facilitating the congregation of the replication and transcription complex (RTC). Considering the information available regarding the catalytic activities and binding signatures of the N protein‒RNA interaction, this review focuses on the regulatory role of the SARS-CoV‒2 N protein. We emphasize the participation of the N protein in discontinuous transcription, template switching, and RNA chaperone activity, including double-stranded RNA melting and annealing activities.

Development of a double-antibody sandwich ELISA for detection of SARS-CoV-2 variants based on nucleocapsid protein-specific antibodies.

The COVID-19 pandemic, driven by the SARS-CoV-2 virus, has posed a severe threat to global public health. Rapid, reliable, and easy-to-use detection methods for SARS-CoV-2 variants are critical for effective epidemic prevention and control. The N protein of SARS-CoV-2 serves as an ideal target for antigen detection. In this study, we achieved soluble expression of the recombinant SARS-CoV-2 N protein using an Escherichia coli expression system and generated specific monoclonal antibodies by immunizing BALB/c mice. We successfully developed 10 monoclonal antibodies against the N protein, designated 5B7, 5F2-C11, 5E2-E8, 6C3-D8, 7C8, 9F2-E9, 12H5-D11, 13G2-C10, 14E9-F6, and 15H3-E10. Using these antibodies, we established a sandwich ELISA with 6C3-D8 as the capture antibody and 5F2-C11 as the detection antibody. The assay demonstrated a sensitivity of 0.78 ng/mL and showed no cross-reactivity with MERS-CoV, HCoV-OC43, HCoV-NL63, and HCoV-229E. Furthermore, this method successfully detected both wild-type SARS-CoV-2 and its variants, including Alpha, Beta, Delta, and Omicron. These findings indicate that our sandwich ELISA exhibits excellent sensitivity, specificity, and broad-spectrum applicability, providing a robust tool for detecting SARS-CoV-2 variants.

Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements.

This study presents a graphene field-effect transistor (gFET) biosensor with dual detection capabilities for SARS-CoV-2: one RNA detection assay to confirm viral positivity and the other for nucleocapsid (N-)protein detection as a proxy for infectiousness of the patient. This technology can be rapidly adapted to emerging infectious diseases, making an essential tool to contain future pandemics. To detect viral RNA, the highly conserved E-gene of the virus was targeted, allowing for the determination of SARS-CoV-2 presence or absence using nasopharyngeal swab samples. For N-protein detection, specific antibodies were used. Tested on 213 clinical nasopharyngeal samples, the gFET biosensor showed good correlation with RT-PCR cycle threshold values, proving its high sensitivity in detecting SARS-CoV-2 RNA. Specificity was confirmed using 21 pre-pandemic samples positive for other respiratory viruses. The gFET biosensor had a limit of detection (LOD) for N-protein of 0.9 pM, establishing a foundation for the development of a sensitive tool for monitoring active viral infection. Results of gFET based N-protein detection corresponded to the results of virus culture in all 16 available clinical samples and thus it also proved its capability to serve as a proxy for infectivity. Overall, these findings support the potential of the gFET biosensor as a point-of-care device for rapid diagnosis of SARS-CoV-2 infection and indirect assessment of infectiousness in patients, providing additional information for clinical and public health decision-making.

N-terminus of SARS-CoV-2 nonstructural protein 3 interrupts RNA-driven phase separation of N protein by displacing RNA.

The connection between SARS-CoV-2 replication-transcription complexes (RTCs) and nucleocapsid (N) protein is critical for regulating genomic RNA replication and virion packaging over the viral life cycle. However, the mechanism that dynamically regulates genomic RNA packaging and replication remains elusive. Here, we demonstrate that the N-terminal domain (NTD) of SARS-CoV-2 nonstructural protein 3 (Nsp3), a core component of viral RTCs, binds N protein and displaces RNA in a concentration-dependent manner. This interaction disrupts liquid-liquid phase separation of N protein driven by N protein-RNA interactions which is crucial for virion packaging and viral replication. We also report a high-resolution crystal structure of the Nsp3 ubiquitin-like domain 1 (Ubl1) at 1.49 Å, which reveals abundant negative charges on the protein surface. Sequence and structural analyses identify several conserved motifs at the Ubl1-N protein interface and a previously unexplored highly negative groove, providing insights into the molecular mechanism of Ubl1-mediated modulation of N protein-RNA binding. Our findings elucidate the mechanism of dynamic regulation of SARS-CoV-2 genomic RNA replication and packaging over the viral life cycle. Targeting the conserved Ubl1-N protein interaction hotspots also promises to aid in the development of broad-spectrum antivirals against pathogenic coronaviruses.

Utilizing artificial intelligence for precision exploration of N protein targeting phenanthridine sars-cov-2 inhibitors: A novel approach.

The persistent mutation of the novel coronavirus presents a continual threat of infections and associated illnesses. While considerable research efforts have concentrated on the functional proteins of SARS-CoV-2 in the development of anti-COVID-19 therapeutics, the structural proteins, particularly the N protein, have received comparatively less attention. This study focuses on the N protein, a critical structural component of the virus, and employs advanced deep learning models, including EMPIRE and DeepFrag, to optimize the structures of phenanthridine-based compounds. More than 10,000 small molecules, derived through deep learning, underwent high-throughput virtual screening, resulting in the synthesis of 44 compounds. Compound 38 showed a binding potential energy of -8.2 kcal/mol in molecular docking. Surface Plasmon Resonance (SPR) and Microscale Thermophoresis (MST) validation yielded dissociation constants of 353 nM and 726 nM, confirming strong binding to the N protein. Compound 38 demonstrated antiviral activity in vitro and exhibited anti-COVID-19 effects by interfering with the binding of N proteins to RNA. This research underscores the potential of targeting the SARS-CoV-2 N protein for therapeutic intervention and illustrates the efficacy of deep learning model in the design of lead compounds. The application of these deep learning models represents a promising approach for accelerating the discovery and development of antiviral agents.

Development and characterization of monoclonal antibodies recognizing nucleocapsid protein of multiple SARS-CoV-2 variants.

Rapid antigen test (RAT) is widely used for SARS-CoV-2 infection diagnostics. However, test sensitivity has decreased recently due to the emergence of the Omicron variant and its sublineages. Here we developed a panel of SARS-CoV-2 nucleocapsid protein (NP) specific mouse monoclonal antibodies (mAbs) and assessed their sensitivity and specificity to important SARS-CoV-2 variants. We identified seven mAbs that exhibited strong reactivity to SARS-CoV-2 variants and recombinant NP (rNP) by Western immunoblot or ELISA. Their specificity to SARS-CoV-2 was confirmed by negative or low reactivity to rNPs from SARS-CoV-1, MERS, and common human coronaviruses (HCoV-HKU1, HCoV-CO43, HCoV-NL63, and HCoV-229E). These seven mAbs were further tested by immunoplaque assay against selected variants of concern (VOCs), including two Omicron sublineages, and five mAbs (F461G13, F461G7, F459G7, F457G3, and F461G6), showed strong reactions, warranting further suitability testing for the development of diagnostic assay.

Surface Plasmon Resonance Immunosensor for Direct Detection of Antibodies against SARS-CoV-2 Nucleocapsid Protein.

The strong immunogenicity of the SARS-CoV-2 nucleocapsid protein is widely recognized, and the detection of specific antibodies is critical for COVID-19 diagnostics in patients. This research proposed direct, label-free, and sensitive detection of antibodies against the SARS-CoV-2 nucleocapsid protein (anti-SCoV2-rN). Recombinant SARS-CoV-2 nucleocapsid protein (SCoV2-rN) was immobilized by carbodiimide chemistry on an SPR sensor chip coated with a self-assembled monolayer of 11-mercaptoundecanoic acid. When immobilized under optimal conditions, a SCoV2-rN surface mass concentration of 3.61 ± 0.52 ng/mm2 was achieved, maximizing the effectiveness of the immunosensor for the anti-SCoV2-rN determination. The calculated KD value of 6.49 × 10-8 ± 5.3 × 10-9 M confirmed the good affinity of the used monoclonal anti-SCoV2-rN antibodies. The linear range of the developed immunosensor was from 0.5 to 50 nM of anti-SCoV2-rN, where the limit of detection and the limit of quantification values were 0.057 and 0.19 nM, respectively. The immunosensor exhibited good reproducibility and specificity. In addition, the developed immunosensor is suitable for multiple anti-SCoV2-rN antibody detections.

Phase Separation of SARS-CoV-2 Nucleocapsid Protein with TDP-43 Is Dependent on C-Terminus Domains.

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid-liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.

Establishment and application of PDCoV antigen-specific DAS-ELISA detection method.

BACKGROUND: Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour. RESULTS: This study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method. CONCLUSION: The developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV.

The network interactions between the porcine deltacoronavirus nucleocapsid protein and host cellular proteins.

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.

Utility of plasma nucleocapsid protein in predicting severity and prognosis in severe COVID-19 patients with comorbidities.

OBJECTIVES: The COVID-19 pandemic poses ongoing challenges to global public health systems, emphasizing the critical necessity for efficient diagnostic and prognostic markers. This study evaluates the MAGLUMI® SARS-CoV-2 Ag N protein chemiluminescent immunoassay (MAG-CLIA) for its analytical performance and its role in predicting disease severity and prognosis among severe COVID-19 patients with comorbidities. METHODS: Analytical validation of plasma MAG-CLIA SARS-CoV-2 Ag N protein encompassed precision, interference, LoQ and linearity. Plasma N protein concentrations and other biomarkers were measured within 48 h of admission, tracked until discharge or death. The Mann-Whitney U test explored the association between plasma N protein and COVID-19 severity or prognosis. Longitudinal monitoring of plasma N protein dynamics was conducted in representative patients. RESULTS: MAG-CLIA demonstrated precise quantification of plasma N protein with a CV below 10 % and minimal interference. The LoQ was 0.88 ng/L, with a broad linear range. Plasma N protein showed high diagnostic accuracy for COVID-19, achieving 95.42 % specificity and 78.32 % sensitivity at 2.388 ng/L. Plasma N protein emerged as a valuable prognostic indicator, correlating with mechanical ventilation need and patient survival. Plasma N protein concentrations ≥ 424.3 ng/L (AUC 0.8102, sensitivity 78.38 %, specificity 85.48 %) were associated with poor prognosis in severe COVID-19 patients with comorbidities. CONCLUSIONS: MAG-CLIA's SARS-CoV-2 N protein detection in plasma demonstrates both analytical reliability and clinical relevance in our inaugural evaluation. As a promising prognostic biomarker for severe COVID-19 patients, it offers crucial insights into disease severity and progression, emphasizing the significance of early monitoring and intervention, especially for patients with comorbidities.

Comparative Proteomics and Interactome Analysis of the SARS-CoV-2 Nucleocapsid Protein in Human and Bat Cell Lines.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 and responsible for the global coronavirus pandemic which started in 2019. Despite exhaustive efforts to trace its origins, including potential links with pangolins and bats, the precise origins of the virus remain unclear. Bats have been recognized as natural hosts for various coronaviruses, including the Middle East respiratory coronavirus (MERS-CoV) and the SARS-CoV. This study presents a comparative analysis of the SARS-CoV-2 nucleocapsid protein (N) interactome in human and bat cell lines. We identified approximately 168 cellular proteins as interacting partners of SARS-CoV-2 N in human cells and 196 cellular proteins as interacting partners with this protein in bat cells. The results highlight pathways and events that are both common and unique to either bat or human cells. Understanding these interactions is crucial to comprehend the reasons behind the remarkable resilience of bats to viral infections. This study provides a foundation for a deeper understanding of host-virus interactions in different reservoirs.