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Due to the complex pathogenic and immune escape mechanisms of herpes simplex virus type 1 (HSV-1), especially the failure of induced immune responses to block the initial cell-to-cell transmission of the virus from skin cells to neurons, the body struggles to establish effective prevention and control methods, resulting in the failure of currently developed vaccines. Previous studies have highlighted the crucial roles of surface glycoproteins and nucleocapsid proteins in activating the body's immune defense system against HSV-1 infection. In this study, recombinant adenoviruses were used as vectors to generate adenoviruses carrying the nucleocapsid protein genes UL18 and UL25, as well as the surface glycoprotein gene gB. This approach aimed to mimic the protein expression process that occurs following viral infection of the host and to investigate the immune response characteristics induced by UL18, UL25, and gB proteins. The findings revealed that UL18, UL25, and gB proteins could all trigger the expression of genes associated with innate immune responses; however, the specific genes induced varied in type and level. Furthermore, all three proteins were capable of promoting the proliferation of CD8+ T cells in the lymph nodes. Notably, only UL18 and gB could elicit a Th1 cell immune response. Interestingly, among these proteins, only UL18 could also induce a relatively higher IL-4 level, indicating a Th2 cell immune response. In addition to cellular immunity, all three proteins stimulated the production of specific IgG antibodies. Notably, UL18 induced higher and more sustained levels of specific IgG antibodies in mice. By contrast, only glycoprotein gB induced lower levels of neutralizing antibodies in mice. Moreover, when these mice were challenged with HSV-1, the co-immunization with UL18 and gB provided better protection than gB alone. In conclusion, HSV-1 surface glycoproteins and nucleocapsid proteins exhibit differences in their ability to induce innate and adaptive immunity in the body, suggesting potential avenues for vaccine design by leveraging their complementary advantages. [ABSTRACT FROM AUTHOR]

The nucleocapsid (N) protein is one of the four structural proteins in SARS-CoV-2, playing key roles in viral assembly, immune evasion, and stability. One of its primary functions is to protect viral RNA by forming the nucleocapsid. However, the precise mechanisms by which the N protein interacts with viral RNA and assembles into a nucleocapsid remain unclear. Compared to other SARS-CoV-2 components, targeting the N protein has several advantages: it exhibits higher sequence conservation, lower mutation rates, and stronger immunogenicity, making it an attractive target for antiviral drug development and diagnostics. Therefore, a detailed understanding of the N protein's structure is essential for deciphering its role in viral assembly and developing effective therapeutics. In this study, we report the expression and purification of a soluble recombinant N protein, along with a 1.55 Å resolution crystal structure of its nucleic acid-binding domain (N-NTD) in complex with ssDNA. Our structure revealed new insights into the conformation and interaction of the flexible N-arm, which could aid in understanding nucleocapsid assembly. Additionally, we identified residues that are critical for ssDNA interaction. [ABSTRACT FROM AUTHOR]

We report the case of an 84‐year‐old male patient who was transferred to our hospital because of impaired consciousness and high fever, and died about 10 weeks after his fourth "coronavirus disease 2019" (COVID‐19) vaccination. Autopsy revealed acute ischemic change with microhemorrhage and perivascular T‐cell infiltration in the thalamus, pons, and cerebellum, which were considered to be related to neurological symptoms. There were dilatation of the right ventricle, accumulation of pleural effusion, and ascites, suggesting right heart failure. Although the patient had a negative COVID‐19 polymerase chain reaction test, immunohistochemical analysis for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) antigens (spike and nucleocapsid proteins) was performed to identify the cause of death. Surprisingly, only SARS‐CoV‐2 spike protein was detected in the thalamus, pons, and pituitary and adrenal glands. The presence of SARS‐CoV‐2 spike protein might have been due to vaccination rather than viral infection, because no SARS‐CoV‐2 nucleocapsid protein was detected. The spike protein in the central nervous system might have been related to the acute ischemic change, and that in the pituitary and adrenal glands may have been associated with right heart failure, possibly through dysfunction of the renin‐angiotensin‐aldosterone system. [ABSTRACT FROM AUTHOR]

Serological diagnostic tests are available that measure antibody levels against SARS-CoV-2 antigens. We utilized the Vaxarray Coronavirus (CoV) seroassay, which measures SARS-CoV-2 IgG antibodies against the full-length spike protein (FLS), receptor binding domain (RBD), and S2 extracellular domain (ECD). Previous serological studies have used reference values that have not been validated and require many samples. Here, we show statistically established reference values determined using the upper tail of the Student t-distribution method. The target population was any personnel age 18 years and older working on a U.S. Navy ship, and vaccinated with Wuhan variant. The relative fluorescence mean (RFM) reference values for the full-length spike protein, RBD, and S2 ECD were 17,731, 13,990 and 9096, respectively. By using generalized non-parametric regression and reference values for the RBD spike protein and S2 ECD of SARS-CoV-2, this study was able to distinguish vaccine-mediated immune responses from natural infections. We provide the method and statistical code as a resource to determine future reference values for other serological assays. [ABSTRACT FROM AUTHOR]

Background: SARS-CoV-2, a highly contagious coronavirus, is responsible for the global pandemic of COVID-19 in 2019. Currently, it remains uncertain whether SARS-CoV-2 possesses oncogenic or oncolytic potential in influencing tumor progression. Therefore, it is important to evaluate the clinical and functional role of SARS-CoV-2 on tumor progression. Methods: Here, we integrated bioinformatic analysis of COVID-19 RNA-seq data from the GEO database and performed functional studies to explore the regulatory role of SARS-CoV-2 in solid tumor progression, including lung, colon, kidney and liver cancer. Results: Our results demonstrate that infection with SARS-CoV-2 is associated with a decreased expression of genes associated with cancer proliferation and metastasis in lung tissues from patients diagnosed with COVID-19. Several cancer proliferation or metastasis related genes were frequently downregulated in SARS-CoV-2 infected intestinal organoids and human colon carcinoma cells. In vivo and in vitro studies revealed that SARS-CoV-2 nucleocapsid (N) protein inhibits colon and kidney tumor growth and metastasis through the N-terminal (NTD) and the C-terminal domain (CTD). The molecular mechanism indicates that the N protein of SARS-CoV-2 interacts with YBX1, resulting in the recruitment of PKM mRNA into stress granules mediated by G3BP1. This process ultimately destabilizes PKM expression and suppresses glycolysis. Conclusion: Our study reveals a new function of SARS-CoV-2 nucleocapsid protein on tumor progression. [ABSTRACT FROM AUTHOR]

Middle East Respiratory Syndrome (MERS) is a zoonoLc disease caused by MERS-CoV, a beta coronavirus with a mortality rate of approximately 35%. The disease exhibits a wide range of clinical symptoms, from mild respiratory issues to severe condiLons like mulL-organ failure and pneumonia. Person-to-person transmission has led to significant hospital and community outbreaks, underscoring the urgent need for effecLve infecLon control measures. This study explores MERS-CoV's epidemiology, pathogenesis, and transmission dynamics, aiming to enhance understanding of its replicaLon, spread, and control strategies due to limited pharmaceuLcal intervenLons. A comprehensive review of current literature was conducted, focusing on epidemiological data, geneLc characterisLcs, and transmission paZerns across affected regions, including the Middle East, Asia, Africa, and North America. Findings indicate that MERS-CoV originated from recombinaLon events in the spike protein of African dromedaries and spread to the Arabian Peninsula via camels. The virus affects not only humans but also domesLc animals like sheep, caZle, horses, and pigs, with global transmission facilitated by travelers, resulLng in outbreaks in Asia and North America. Despite extensive research, no effecLve vaccines, anLviral drugs, or immune therapies control MERS-CoV. The findings emphasize the high pandemic potenLal of MERS-CoV due to its mortality rate and lack of effecLve treatments, highlighLng the need for strict infecLon control and further research into viable therapeuLc opLons. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that mutations in the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may enhance viral replication by modulating the assembly process. However, the mechanisms governing the selective packaging of viral genomic RNA by the N protein, along with the assembly and budding processes, remain poorly understood. Utilizing a virus-like particles (VLPs) system, we have identified that the C-terminal domain (CTD) of the N protein is essential for its interaction with the membrane (M) protein during budding, crucial for binding and packaging genomic RNA. Notably, the isolated CTD lacks M protein interaction capacity and budding ability. Yet, upon fusion with the N-terminal domain (NTD) or the linker region (LKR), the resulting NTD/CTD and LKR/CTD acquire RNA-dependent interactions with the M protein and acquire budding capabilities. Furthermore, the presence of the C-tail is vital for efficient genomic RNA encapsidation by the N protein, possibly regulated by interactions with the M protein. Remarkably, the NTD of the N protein appears dispensable for virus particle assembly, offering the virus adaptive advantages. The emergence of N* (N=N209) in the SARS-CoV-2 B.1.1 lineage corroborates our findings and hints at the potential evolution of a more streamlined N protein by the SARS-CoV-2 virus to facilitate the assembly process. Comparable observations have been noted with the N proteins of SARS-CoV and HCoV-OC43 viruses. In essence, these findings propose that ß-coronaviruses may augment their replication by fine-tuning the assembly process. IMPORTANCE As a highly transmissible zoonotic virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve. Adaptive mutations in the nucleocapsid (N) protein highlight the critical role of N protein-based assembly in the virus's replication and evolutionary dynamics. However, the precise molecular mechanisms of N protein-mediated viral assembly remain inadequately understood. Our study elucidates the intricate interactions between the N protein, membrane (M) protein, and genomic RNA, revealing a C-terminal domain (CTD)-based assembly mechanism common among ß-coronaviruses. The appearance of the N* variant within the SARSCoV-2 B.1.1 lineage supports our conclusion that the N-terminal domain (NTD) of the N protein is not essential for viral assembly. This work not only enhances our understanding of coronavirus assembly mechanisms but also provides new insights for developing antiviral drugs targeting these conserved processes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for the calibration and quality assurance of inactivated SARS-CoV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)-C-terminal domain (CTD) across all SARS-CoV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprised of UV-inactivated SARS-CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (limit of detection: 1.72-8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a 20 amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust 'pan-variant' antigen-based assays for viruses prone to high mutational rates.

Abstract 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 integrated stress response, especially stress granules (SGs), contributes to host immunity. Typical G3BP1+ stress granules (tSGs) are usually formed after virus infection to restrain viral replication and stimulate innate immunity. Recently, several SG-like foci or atypical SGs (aSGs) with proviral function have been found during viral infection. We have shown that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein induces atypical N+/G3BP1+ foci (N+foci), leading to the inhibition of host immunity and facilitation of viral infection. However, the precise mechanism has not been well clarified yet. In this study, we showed that the SARS-CoV-2 N (SARS2-N) protein inhibits dsRNA-induced growth arrest and DNA damage-inducible 34 (GADD34) expression. Mechanistically, the SARS2-N protein promotes the interaction between GADD34 mRNA and G3BP1, sequestering GADD34 mRNA into the N+foci. Importantly, we found that GADD34 participates in IRF3 nuclear translocation through its KVRF motif and promotes the transcription of downstream interferon genes. The suppression of GADD34 expression by the SARS2-N protein impairs the nuclear localization of IRF3 and compromises the host's innate immune response, which facilitates viral replication. Taking these findings together, our study revealed a novel mechanism by which the SARS2-N protein antagonized the GADD34-mediated innate immune pathway via induction of N+foci. We think this is a critical strategy for viral pathogenesis and has potential therapeutic implications. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Crimean–Congo hemorrhagic fever virus (CCHFV) is an orthonairovirus from the Bunyavirales order that is widely distributed geographically and causes severe or fatal infections in humans. The viral genome consists of three segmented negative-sense RNA molecules. The CCHFV nucleocapsid protein (CCHFV NP) is encoded by the smallest segment of the virus. CCHFV NP, the primary function of which is the encapsidation of viral RNA molecules, plays a critical role in various mechanisms important for viral replication and pathogenesis. This review is an attempt to revisit the literature available on the highly immunogenic and highly conserved CCHFV NP, summarizing the multifunctional roles of this protein in the immunology of CCHFV. Specifically, the review addresses the impact of CCHFV NP on innate, humoral, and cellular immune responses, epitopes recognized by B and T cells that limit viral spread, and its role as a target for diagnostic tests and for vaccine design. Based on the extensive information generated by many research groups, it could be stated that NP constitutes a significant and critical player in the immunology of CCHFV. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The swine acute diarrhea syndrome coronavirus (SADS-CoV) has caused significant disruptions in porcine breeding and raised concerns about potential human infection. The nucleocapsid (N) protein of SADS-CoV plays a vital role in viral assembly and replication, but its structure and functions remain poorly understood. This study utilized biochemistry, X-ray crystallography, and immunization techniques to investigate the N protein’s structure and function in SADS-CoV. Our findings revealed distinct domains within the N protein, including an RNA-binding domain, two disordered domains, and a dimerization domain. Through biochemical assays, we confirmed that the N-terminal domain functions as an RNA-binding domain, and the C-terminal domain is involved in dimerization, with the crystal structure analysis providing visual evidence of dimer formation. Immunization experiments demonstrated that the disordered domain 2 elicited a significant antibody response. These identified domains and their interactions are crucial for viral assembly. This comprehensive understanding of the N protein in SADS-CoV enhances our knowledge of its assembly and replication mechanisms, enabling the development of targeted interventions and therapeutic strategies. IMPORTANCE SADS-CoV is a porcine coronavirus that originated from a bat HKU2-related coronavirus. It causes devastating swine diseases and poses a high risk of spillover to humans. The coronavirus N protein, as the most abundant viral protein in infected cells, likely plays a key role in viral assembly and replication. However, the structure and function of this protein remain unclear. Therefore, this study employed a combination of biochemistry and X-ray crystallography to uncover distinct structural domains in the N protein, including RNA-binding domains, two disordered domains, and dimerization domains. Additionally, we made the novel discovery that the disordered domain elicited a significant antibody response. These findings provide new insights into the structure and functions of the SADS-CoV N protein, which have important implications for future studies on SADS-CoV diagnosis, as well as the development of vaccines and anti-viral drugs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Antigen-specific T cells are an important part of antiviral responses, and modern influenza vaccines are designed to induce this mode of immunity. Live attenuated influenza vaccine (LAIV) is a potent inducer of T-cell immunity because of its ability to cause productive infection in the upper respiratory tract. Inactivated influenza vaccines (IIV) and novel vaccine candidates can also induce virus-specific T-cells when appropriate adjuvants are used. In this case, non-structural and intrinsic antigens of the master donor viruses, particularly nucleoprotein (NP), are the main targets for the development of T-cell immunity. The most commonly used donor strains for LAIVs and IIVs worldwide were derived from viruses isolated between 1933 and 1960. In this regard, the question of conservation of epitopes immunogenic for CD8⁺ T-lymphocytes (CTL-epitopes) in donor-derived NPs, i.e., the ability of cytotoxic T cells specific to the donor’s NP to recognize modern influenza A virus nucleoproteins, is relevant. The aim of the study was to evaluate the conservation of CTL-immunogenic NP epitopes of donors traditionally used to create LAIVs and IIVs. Materials and methods. Epitope NP analysis was performed for 1614 and 1767 strains of influenza A virus subtypes H1N1 and H3N2, respectively, which circulated in 2009–2023 (data from the NCBI Influenza Virus Database). Immune Epitope Database (IEDB, www.iedb.org), NetCTL’s built-in CTL-epitope prediction algorithm and NetChop proteolysis site predictor were used. CTL-epitopes were mapped to NPs of master donor viruses A/Leningrad/134/17/57 (H2N2), A/Ann Arbor/6/60 (H2N2), A/PR/8/34 (H1N1), and A/WSN/1933 (H1N1) using the CrustalO alignment algorithm in JalView 2.8.1 Software. The immunogenicity and conservation of selected epitopes were further evaluated using IEDB T-cell Immunogenicity Predictor and Epitope Conservancy Assay, respectively. Results. The majority of immunogenic CTL-epitopes of donor viruses proved to be non-conserved, i.e., not found in NPs of circulating influenza strains. Conversely, most CTL-immunogenic NP epitopes of modern viruses are absent in donor viruses and cannot be induced by vaccination with conventional vaccines. The data obtained indicate the need to actualize NP in vaccine composition by directed mutagenesis of the donor-derived NP gene or by introduction of the gene encoding NP of circulating influenza viruses into vaccine strains.

The nucleocapsid protein (NP) plays a crucial role in SARS-CoV-2 replication and is the most abundant structural protein with a long half-life. Despite its vital role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assembly and host inflammatory response, it remains an unexplored target for drug development. In this study, we identified a small-molecule compound (ciclopirox) that promotes NP degradation using an FDA-approved library and a drug-screening cell model. Ciclopirox significantly inhibited SARS-CoV-2 replication both in vitro and in vivo by inducing NP degradation. Ciclopirox induced abnormal NP aggregation through indirect interaction, leading to the formation of condensates with higher viscosity and lower mobility. These condensates were subsequently degraded via the autophagy-lysosomal pathway, ultimately resulting in a shortened NP half-life and reduced NP expression. Our results suggest that NP is a potential drug target, and that ciclopirox holds substantial promise for further development to combat SARS-CoV-2 replication.

Oral delivery of an inexpensive COVID-19 (coronavirus disease 2019) vaccine could dramatically improve immunization rates, especially in low- and middle-income countries. Previously, we described a potential universal COVID-19 vaccine, rLVS ΔcapB/MN, comprising a replicating bacterial vector, LVS (live vaccine strain) ΔcapB, expressing the highly conserved SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) membrane and nucleocapsid (N) proteins, which, when administered intradermally or intranasally, protects hamsters from severe COVID-19-like disease after high-dose SARS-CoV-2 respiratory challenge. Here, we show that oral administration of the vaccine also protects against high-dose SARS-CoV-2 respiratory challenge; its protection is comparable to that of intradermal, intranasal, or subcutaneous administration. Hamsters were protected against severe weight loss and lung pathology and had reduced oropharyngeal and lung virus titers. Protection against weight loss and histopathology by the vaccine, which in mice induces splenic and lung cell interferon gamma in response to N protein stimulation, was correlated in hamsters with pre-challenge serum anti-N TH1-biased IgG (IgG2/3). Thus, rLVS ΔcapB/MN has potential as an oral universal COVID-19 vaccine. IMPORTANCE The COVID-19 pandemic continues to rage into its fourth year worldwide. To protect the world's population most effectively from severe disease, hospitalization, and death, a vaccine is needed that is resistant to rapidly emerging viral variants of the causative agent SARS-CoV-2, inexpensive to manufacture, store, and transport, and easy to administer. Ideally, such a vaccine would be capable of oral administration, especially in resource-poor countries of the world where there are shortages of needles, syringes and trained personnel to administer injectable vaccines. Here, we show that oral administration of a bacterium-vectored vaccine meeting all these criteria protects naturally susceptible Syrian hamsters from severe COVID-19-like disease, including severe weight loss and lung pathology, after high-dose SARS-CoV-2 respiratory challenge. As the vaccine is based upon inducing immunity to highly conserved SARS-CoV-2 membrane and nucleocapsid proteins, as opposed to the rapidly mutating Spike protein, it should remain resistant to newly emerging SARS-CoV-2 variants.

Background: Knowledge about SARS-CoV-2 antibody dynamics in neonates and direct comparisons with maternal antibody responses are not well established. This study aimed to characterize and directly compare the maternal and infant antibody response in a national birth cohort from the Faroe Islands. Methods: The levels of immunoglobulins (Ig) targeting the receptor binding domain (RBD) of the spike protein and the nucleocapsid protein (N protein) of SARS-CoV-2 were investigated in maternal blood and umbilical cord blood from neonates. The study included 537 neonates and 565 mothers from the Faroe Islands, and follow-up samples were collected 12 months after birth. Multiple linear regression models were used to assess associations of maternal parameters with maternal and neonatal Ig levels and pregnancy outcomes. Results: The finding showed that neonates acquired varying levels of SARS-CoV-2 antibodies through transplacental transfer, and the levels were significantly influenced by the mother's vaccination and infection status. The study also found that maternal vaccination and the presence of SARS-CoV-2 antibodies targeting spike RBD were associated with gestational age and APGAR scores. Furthermore, the anti-RBD and -N protein-specific antibody response dynamics during 12 months after birth exhibited differences between mothers and children. RBD and N protein responses were maintained at follow-up in the mother's cohort, while only the N protein response was maintained at follow-up in the children's cohort. Conclusion: In conclusion, SARS-CoV-2-specific immune responses in newborns rely on maternal immunity, while the persistence of SARS-CoV-2-specific Igs appears to be differently regulated between mothers and children. The study provides new insights into the dynamics of SARS-CoV-2-specific immune responses in newborns and underscores the nuanced relationship between maternal factors and neonatal humoral responses. [ABSTRACT FROM AUTHOR]

Highly pathogenic coronaviruses have caused significant outbreaks in humans and animals, posing a serious threat to public health. The rapid global spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in millions of infections and deaths. However, the mechanisms through which coronaviruses evade a host's antiviral immune system are not well understood. Liquid–liquid phase separation (LLPS) is a recently discovered mechanism that can selectively isolate cellular components to regulate biological processes, including host antiviral innate immune signal transduction pathways. This review focuses on the mechanism of coronavirus-induced LLPS and strategies for utilizing LLPS to evade the host antiviral innate immune response, along with potential antiviral therapeutic drugs and methods. It aims to provide a more comprehensive understanding and novel insights for researchers studying LLPS induced by pandemic viruses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Puumala orthohantavirus (PUUV) is an emerging zoonotic virus endemic to Europe and Russia that causes nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome (HFRS). There are limited options for treatment and diagnosis of orthohantavirus infection, making the search for potential immunogenic candidates crucial. In the present work, various bioinformatics tools were employed to design conserved immunogenic peptides containing multiple epitopes of PUUV nucleocapsid protein. Eleven conserved peptides (90% conservancy) of the PUUV nucleocapsid protein were identified. Three conserved peptides containing multiple T and B cell epitopes were selected using a consensus epitope prediction algorithm. Molecular docking using the HPEP dock server demonstrated strong binding interactions between the epitopes and HLA molecules (ten alleles for each class I and II HLA). Moreover, an analysis of population coverage using the IEDB database revealed that the identified peptides have over 90% average population coverage across six continents. Molecular docking and simulation analysis reveal a stable interaction with peptide constructs of chosen immunogenic peptides and Toll-like receptor-4. These computational analyses demonstrate selected peptides' immunogenic potential, which needs to be validated in different experimental systems. [ABSTRACT FROM AUTHOR]

Porcine deltacoronavirus (PDCoV) is an enteric pathogenic coronavirus that causes acute and severe watery diarrhea in piglets and has the ability of cross-species transmission, posing a great threat to swine production and public health. The interferon (IFN)-mediated signal transduction represents an important component of virus-host interactions and plays an essential role in regulating viral infection. Previous studies have suggested that multifunctional viral proteins encoded by coronaviruses antagonize the production of IFN via various means. However, the function of these viral proteins in regulating IFN-mediated signaling pathways is largely unknown. In this study, we demonstrated that PDCoV and its encoded nucleocapsid (N) protein antagonize type I IFN-mediated JAK-STAT signaling pathway. We identified that PDCoV infection stimulated but delayed the production of IFN-stimulated genes (ISGs). In addition, PDCoV inhibited JAK-STAT signal transduction by targeting the nuclear translocation of STAT1 and ISGF3 formation. Further evidence showed that PDCoV N is the essential protein involved in the inhibition of type I IFN signaling by targeting STAT1 nuclear translocation via its C-terminal domain. Mechanistically, PDCoV N targets STAT1 by interacting with it and subsequently inhibiting its nuclear translocation. Furthermore, PDCoV N inhibits STAT1 nuclear translocation by specifically targeting KPNA2 degradation through the lysosomal pathway, thereby inhibiting the activation of downstream sensors in the JAK-STAT signaling pathway. Taken together, our results reveal a novel mechanism by which PDCoV N interferes with the host antiviral response. IMPORTANCE Porcine deltacoronavirus (PDCoV) is a novel enteropathogenic coronavirus that receives increased attention and seriously threatens the pig industry and public health. Understanding the underlying mechanism of PDCoV evading the host defense during infection is essential for developing targeted drugs and effective vaccines against PDCoV. This study demonstrated that PDCoV and its encoded nucleocapsid (N) protein antagonize type I interferon signaling by targeting STAT1, which is a crucial signal sensor in the JAK-STAT signaling pathway. Further experiments suggested that PDCoV N-mediated inhibition of the STAT1 nuclear translocation involves the degradation of KPNA2, and the lysosome plays a role in KPNA2 degradation. This study provides new insights into the regulation of PDCoV N in the JAK-STAT signaling pathway and reveals a novel mechanism by which PDCoV evades the host antiviral response. The novel findings may guide us to discover new therapeutic targets and develop live attenuated vaccines for PDCoV infection. [ABSTRACT FROM AUTHOR]

Introduction: Rift Valley fever virus (RVFV) belonging to the Phenuiviridae family is responsible for a zoonotic disease called Rift Valley fever (RVF). Currently, RVFV has spread from Africa to Asia, and due to its ability to cause high mortality rates, it has significantly impacted human health and economic development in many societies. Highly specific and sensitive systems for sero-diagnosis of RVFV infection are needed for clinical use. Method: BALB/c mice were immunized with recombinant RVFV nucleocapsid (rRVFV-N) protein and the spleen cells fused with SP2/0 myeloma cells to create hybridoma cell lines. The secreted monoclonal antibodies (MAbs) were purified and characterized. Enzyme-linked immunosorbent assay (ELISA) systems for the detection of IgG and IgM using the new MAbs were established and evaluated. Serum samples from 96 volunteers and 93 patients of suspected RVF from Kenya were tested compared with the ELISA systems based on inactivated viruses and the rabbit polyclonal antibody. Result: Three monoclonal antibodies against rRVFV-N protein were established. The performance of the MAb-based sandwich IgG ELISA and the IgM capture ELISA perfectly matched the ELISA systems using the inactivated virus or the polyclonal antibody. Conclusions: Recombinant RVFV-N protein-specific MAbs were developed and they offer useful tools for RVFV studies. The MAb-based ELISA systems for detecting IgG and IgM offer safe and useful options for diagnosing RVFV infections in humans. [ABSTRACT FROM AUTHOR]

This paper reports a rapid and sensitive sensor for the detection and quantification of the COVID-19 N-protein (N-PROT) via an electrochemical mechanism. Single-frequency electrochemical impedance spectroscopy was used as a transduction method for real-time measurement of the N-PROT in an immunosensor system based on gold-conjugate-modified carbon screen-printed electrodes (Cov-Ag-SPE). The system presents high selectivity attained through an optimal stimulation signal composed of a 0.0 V DC potential and 10 mV RMS−1 AC signal at 100 Hz over 300 s. The Cov-Ag-SPE showed a log response toward N-PROT detection at concentrations from 1.0 ng mL−1 to 10.0 μg mL−1, with a 0.977 correlation coefficient for the phase (θ) variation. An ML-based approach could be created using some aspects observed from the positive and negative samples; hence, it was possible to classify 252 samples, reaching 83.0, 96.2 and 91.3% sensitivity, specificity, and accuracy, respectively, with confidence intervals (CI) ranging from 73.0 to 100.0%. Because impedance spectroscopy measurements can be performed with low-cost portable instruments, the immunosensor proposed here can be applied in point-of-care diagnostics for mass testing, even in places with limited resources, as an alternative to the common diagnostics methods. [ABSTRACT FROM AUTHOR]

Corona Virus Disease 2019 (COVID-19) is a highly prevalent and potent infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2). Until now, the world is still endeavoring to develop new ways to diagnose and treat COVID-19. At present, the clinical prevention and treatment of COVID-19 mainly targets the spike protein on the surface of SRAS-CoV-2. However, with the continuous emergence of SARS-CoV-2 Variants of concern (VOC), targeting the spike protein therapy shows a high degree of limitation. The Nucleocapsid Protein (N protein) of SARS-CoV-2 is highly conserved in virus evolution and is involved in the key process of viral infection and assembly. It is the most expressed viral structural protein after SARS-CoV-2 infection in humans and has high immunogenicity. Therefore, N protein as the key factor of virus infection and replication in basic research and clinical application has great potential research value. This article reviews the research progress on the structure and biological function of SARS-CoV-2 N protein, the diagnosis and drug research of targeting N protein, in order to promote researchers' further understanding of SARS-CoV-2 N protein, and lay a theoretical foundation for the possible outbreak of new and sudden coronavirus infectious diseases in the future. [ABSTRACT FROM AUTHOR]

The nucleocapsid protein (NP) plays a crucial role in SARS-CoV-2 replication and is the most abundant structural protein with a long half-life. Despite its vital role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assembly and host inflammatory response, it remains an unexplored target for drug development. In this study, we identified a small-molecule compound (ciclopirox) that promotes NP degradation using an FDA-approved library and a drug-screening cell model. Ciclopirox significantly inhibited SARS-CoV-2 replication both in vitro and in vivo by inducing NP degradation. Ciclopirox induced abnormal NP aggregation through indirect interaction, leading to the formation of condensates with higher viscosity and lower mobility. These condensates were subsequently degraded via the autophagy-lysosomal pathway, ultimately resulting in a shortened NP half-life and reduced NP expression. Our results suggest that NP is a potential drug target, and that ciclopirox holds substantial promise for further development to combat SARS-CoV-2 replication. Ciclopirox exhibited potent anti-SARS-CoV-2 activity in vivo and in vitro by inducing NP abnormal aggregation and degrading NP condensates via autophagy–lysosome pathway. [Display omitted] [ABSTRACT FROM AUTHOR]

Fv-antibodies against the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were screened from an Fv-antibody library, and a one-step immunoassay was performed to detect SARS-CoV-2 using real viral samples. The Fv-antibody library was prepared using site-directed mutagenesis of the CDR3 region, which was composed of 11 amino acids. To screen the target Escherichia coli from the Fv-antibody library, the expressed probes [N-terminal domain (NTD) labeled with GFP and C-terminal domain (CTD) labeled with GFP] were reacted separately with the Fv-antibody library. After oligonucleotide sequencing, two clones for each probe were selected as the final clones. The screened Fv-antibodies with the binding affinity to NTD (or CTD) were expressed as soluble proteins, and the affinity constant (KD) was calculated to be 25.4 nM for NTD and 26.9 nM for CTD. The expressed Fv-antibodies were used for the one-step immunoassay based on switching-peptides, which were bound to the expressed Fv-antibodies. The one-step immunoassay based on Fv-antibodies could be used for the linear detection of SARS-CoV-2 NP, and the limit of detection (LOD) was estimated to be 9.6 nM (438 ng/mL) for Anti-NTD and 14.1 nM (639 ng/mL) for Anti-CTD. For the demonstration of one-step immunoassay for SARS-CoV-2, NATtrol™ SARS-CoV-2 real sample was used, and the LOD was estimated to be 29.7 copies/mL (Ct = 39.5) using Anti-NTD and 117.8 copies/mL (Ct = 38.0) using Anti-CTD. The measured LOD for the detection of SARS-CoV-2 using a one-step immunoassay based on the switching-peptide was considered feasible for the medical diagnosis of COVID-19. Finally, the interaction between the screened Fv-antibodies and SARS-CoV-2 NP was investigated using docking simulation. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to elucidate the factors associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that may initiate cytokine cascades and correlate the clinical characteristics of patients with coronavirus disease 2019 (COVID-19) with their serum cytokine profiles. Methods: Recombinant baculoviruses displaying SARS-CoV-2 spike or nucleocapsid protein were constructed and transfected into A549 cells and THP-1-derived macrophages, to determine which protein initiate cytokine release. SARS-CoV-2-specific antibody titers and cytokine profiles of patients with COVID-19 were determined, and the results were associated with their clinical characteristics, such as development of pneumonia or length of hospital stay. Results: The SARS-CoV-2 nucleocapsid protein, rather than the spike protein, triggers lung epithelial A549 cells to express IP-10, RANTES, IL-16, MIP-1α, basic FGF, eotaxin, IL-15, PDGF-BB, TRAIL, VEGF-A, and IL-5. Additionally, serum CTACK, basic FGF, GRO-α, IL-1α, IL-1RA, IL-2Rα, IL-9, IL-15, IL-16, IL-18, IP-10, M-CSF, MIF, MIG, RANTES, SCGF-β, SDF-1α, TNF-α, TNF-β, VEGF, PDGF-BB, TRAIL, β-NGF, eotaxin, GM-CSF, IFN-α2, INF-γ, and MCP-1 levels were considerably increased in patients with COVID-19. Among them, patients with pneumonia had higher serum IP-10 and M-CSF levels than patients without. Patients requiring less than 3 weeks to show negative COVID-19 tests after contracting COVID-19 had higher serum IP-10 levels than the remaining patients. Conclusion: Our study revealed that nucleocapsid protein, lung epithelial cells, and IP-10 may be potential targets for the development of new strategies to prevent, or control, severe COVID-19. [ABSTRACT FROM AUTHOR]

Rapid and precise detection of SARS‐CoV‐2 antibodies is paramount for effective outbreak monitoring and vaccine efficacy assessment. While existing approaches for antibody detection often rely on complex electrochemical immunosensing with nanomaterial functionalization targeting S‐protein antibodies, their limitations in sensitivity and complexity have hindered widespread application. Here, we present a simplistic immunosensing platform designed for the rapid, and precise detection of SARS‐CoV‐2 specific IgG and Nucleocapsid antibodies. Notably, this study marks only the second exploration of SARS‐CoV‐2 N‐protein antibody detection. The platform utilizes traditional self‐assembled monolayers to establish selective bio‐affinity between SARS‐CoV‐2 specific Nucleocapsid antibodies and a gold electrode functionalized with the N‐protein antigen. Interestingly, despite the absence of nanomaterial functionalization, the developed platform achieves sensitivity comparable to existing sensors across a wide detection range (0.025 to 1 ng/mL) with an impressive limit of detection (0.019 ng/mL). The simplicity of the approach, relying solely on immunocomplex reactions, underscores that effective binding efficiency may be achieved in the absence of complex functionalization and determines its affordability, specificity, and high sensitivity. By eliminating the need for additional functionalization steps, the platform offers a streamlined solution for SARS‐CoV‐2 antibody detection and demonstrates the possibility of N‐protein antibody detection as a promising avenue for widespread application in SARS‐CoV‐2 outbreak monitoring and vaccine efficacy assessment particularly in underdeveloped regions. [ABSTRACT FROM AUTHOR]

A high-sensitivity silicon microring (Si MRR) optical biosensor for detecting the nucleocapsid protein of SARS-CoV-2 is proposed and demonstrated. In the proposed biosensor, the surface of a Si MRR waveguide is modified with antibodies, and the target protein is detected by measuring a resonant wavelength shift of the MRR caused by the selective adsorption of the protein to the surface of the waveguide. A Si MRR is fabricated on a silicon-on-insulator substrate using a CMOS-compatible fabrication process. The quality factor of the MRR is approximately 20,000. The resonant wavelength shift of the MRR and the detection limit for the environmental refractive index change are evaluated to be 89 nm/refractive index unit (RIU) and 10−4 RIU, respectively. The sensing characteristics are examined using a polydimethylsiloxane flow channel after the surface of the Si MRR waveguide is modified with the IgG antibodies through the Si-tagged protein. First, the selective detection of the protein by the MRR sensor is experimentally demonstrated by the detection of bovine serum albumin and human serum albumin. Next, various concentrations of nucleocapsid protein solutions are measured by the MRR, in which the waveguide surface is modified with the IgG antibodies through the Si-tagged protein. Although the experimental results are very preliminary, they show that the proposed sensor has a potential nucleocapsid sensitivity in the order of 10 pg/mL, which is comparable to the sensitivity of current antigen tests. The detection time is less than 10 min, which is much shorter than those of other antigen tests. [ABSTRACT FROM AUTHOR]

With the continuous spread of new SARS-CoV-2 variants of concern (VOCs), the monitoring of diagnostic test performances is mandatory. We evaluated the changes in antigen diagnostic tests' (ADTs) accuracy along the Delta to Omicron VOCs transition, exploring the N protein mutations possibly affecting ADT sensitivity and assessing the best sampling site for the diagnosis of Omicron infections. In total, 5175 subjects were enrolled from 1 October 2021 to 15 July 2022. The inclusion criteria were SARS-CoV-2 ADT combined with a same-day RT-PCR swab test. For the sampling site analysis, 61 patients were prospectively recruited during the Omicron period for nasal and oral swab analyses by RT-PCR. Next-Generation Sequencing data were obtained to evaluate the different sublineages. Using RT-PCR as a reference, 387 subjects resulted in becoming infected and the overall sensitivity of the ADT decreased from 63% in the Delta period to 33% in the Omicron period. This decrease was highly statistically significant (p < 0.001), and no decrease in viral load was detected at the RNA level. The nasal site presented a significantly higher viral load than the oral site during the Omicron wave. The reduced detection rate of Omicron infections by ADT should be considered in the global testing strategy to preserve accurate diagnoses across the changing SARS-CoV-2 variants. [ABSTRACT FROM AUTHOR]

Abstract The emergence of corona virus disease 2019 (COVID-19), resulting from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has left an indelible mark on a global scale, causing countless infections and fatalities. This investigation delves into the role of the SARS-CoV-2 nucleocapsid (N) protein within the HEK293 cells, shedding light on its influence over apoptosis, interferon signaling, and cytokines production. The N gene was amplified, inserted into the pAdTrack-CMV vector, and then transfected to the HEK293 cells. Changes in the expression of IRF3, IRF7, IFN-β, BAK, BAX, and BCL-2 genes were evaluated. The levels of proinflammatory cytokines of IL-6, IL-12, IL-1β, and TNF-α were also determined. The N protein exhibited an anti-apoptotic effect by modulating critical genes associated with apoptosis, including BAK, BAX, and BCL-2. This effect potentially prolonged the survival of infected cells. The N protein also played a role in immune evasion by suppressing the interferon pathway, evidenced by the downregulation of essential interferon regulatory factors of IRF3 and IRF7, and IFN-β expression. The N protein expression led to a substantial increase in the production of proinflammatory cytokines of IL-6, IL-12, IL-1β, and TNF-α. The N protein emerged as a versatile factor and was exerted over apoptosis, interferon signaling, and cytokine production. These findings carry potential implications for the development of targeted therapies to combat COVID-19 and mitigate its global health impact.

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.

The rice stripe virus (RSV) is transmitted by the small brown planthopper Laodelphax striatellus in a persistent and circulative–propagative manner. During the last few decades, RSV has caused serious disease outbreaks in rice fields in China. The results of long-term coevolution have led to complex and diverse relationships between viruses and vector insects, and understanding these interactions is critical for the management of diseases and vector organisms. In this study, three groups of comparative experiments were performed to investigate the effects of RSV infection on the reproductive parameters, vitellogenin (Vg), and Vg receptor (VgR) expressions of L. striatellus. The results showed that RSV infection promoted the fecundity, hatchability, and Vg and VgR expressions of L. striatellus but had no effect on the preoviposition period and oviposition period. Furthermore, the RNA interference of the nucleocapsid protein (CP) gene and the injection of the CP antibody in RSV-viruliferous L. striatellus reduced the fecundity, hatchability, and Vg and VgR expressions, further strengthening the hypothesis that RSV may manipulate the reproductive behavior of insect vectors to promote its own transmission. The results of this study can further reveal the interaction mechanism of virus–vector insects in reproduction and virus transmission, and provide new insights for the control of insect-borne viruses. [ABSTRACT FROM AUTHOR]

As there are limited data on B-cell epitopes for the nucleocapsid protein in SARS-CoV-2, we sought to identify the immunodominant regions within the N protein, recognized by patients with varying severity of natural infection with the Wuhan strain (WT), delta, omicron, and in those who received the Sinopharm vaccines, which is an inactivated, whole virus vaccine. Using overlapping peptides representing the N protein, with an in-house ELISA, we mapped the immunodominant regions within the N protein, in seronegative (n = 30), WT infected (n = 30), delta infected (n = 30), omicron infected + vaccinated (n = 20) and Sinopharm (BBIBP-CorV) vaccinees (n = 30). We then investigated the sensitivity and specificity of these immunodominant regions and analyzed their conservation with other SARS-CoV-2 variants of concern, seasonal human coronaviruses, and bat Sarbecoviruses. We identified four immunodominant regions aa 29–52, aa 155–178, aa 274–297, and aa 365–388, which were highly conserved within SARS-CoV-2 and the bat coronaviruses. The magnitude of responses to these regions varied based on the infecting SARS-CoV-2 variants, >80% of individuals gave responses above the positive cut-off threshold to many of the four regions, with some differences with individuals who were infected with different VoCs. These regions were found to be 100% specific, as none of the seronegative individuals gave any responses. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines. We identified four immunodominant regions within SARS-CoV-2 N protein, aa 29–52, aa 155–178, aa 274–297, and aa 365–388, were highly conserved within SARS-CoV-2 and the bat coronaviruses. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines. Graphical Abstract [ABSTRACT FROM AUTHOR]

The coronavirus 2019 disease (COVID-19) course and serological statuses of patients with relapsing–remitting multiple sclerosis (RRMS), treated with disease-modifying therapies (DMTs) are generally parallel that of the general population. Over the pandemic's course, however, a notable increase in the number of RRMS patients who received vaccination against severe acute respiratory coronavirus 2 (SARS-CoV-2) and those who had COVID-19 (symptomatic and asymptomatic) was reported. This virus and/or vaccination likely influenced DMT-treated RRMS patients' serological statuses regarding the presence of SARS-CoV-2 antibodies and their quantitative expression. This investigation assesses the presence and levels of the antibody directed against the S1 protein receptor binding domain (SRBD) and against the N protein of SARS-CoV-2 in 38 DMT-treated RRMS patients. The findings indicate that people vaccinated against SARS-CoV-2 exhibited significantly higher levels of IgG antibodies against S1-RBD at both assessment points. Patients with a prior history of COVID-19 demonstrated statistically significant increases in anti-N antibodies at visit 1, whereas such statistical significance was not observed at visit 2. DMT-treated RRMS patients generated neutralizing antibodies following vaccination and/or COVID-19 infection. Nevertheless, it is noteworthy that antibody levels more accurately reflect the serological status and exhibit a stronger correlation with vaccination than just the presence of antibodies. [ABSTRACT FROM AUTHOR]

The Nucleocapsid Protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is not only the core structural protein required for viral packaging, but also participates in the regulation of viral replication, and its post‐translational modifications such as phosphorylation have been shown to be an important strategy for regulating virus proliferation. Our previous work identified NP could be ubiquitinated, as confirmed by two independent studies. But the function of NP ubiquitination is currently unknown. In this study, we first pinpointed TRIM6 as the E3 ubiquitin ligase responsible for NP ubiquitination, binding to NP's CTD via its RING and B‐box‐CCD domains. TRIM6 promotes the K29‐typed polyubiquitination of NP at K102, K347, and K361 residues, increasing its binding to viral genomic RNA. Consistently, functional experiments such as the use of the reverse genetic tool trVLP model and gene knockout of TRIM6 further confirmed that blocking the ubiquitination of NP by TRIM6 significantly inhibited the proliferation of SARS‐CoV‐2. Notably, the NP of coronavirus is relatively conserved, and the NP of SARS‐CoV can also be ubiquitinated by TRIM6, indicating that NP could be a broad‐spectrum anti‐coronavirus target. These findings shed light on the intricate interaction between SARS‐CoV‐2 and the host, potentially opening new opportunities for COVID‐19 therapeutic development. [ABSTRACT FROM AUTHOR]