Objective: To establish a rapid and specific quantitative real-time PCR (qPCR) method for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA (SgN) in patients with COVID-19 or environmental samples. Methods: The qPCR assay was established by designing specific primers and TaqMan probe based on the SARS-CoV-2 genomic sequence in Global Initiative of Sharing All Influenza Data (GISAID) database. The reaction conditions were optimized by using different annealing temperature, different primers and probe concentrations and the standard curve was established. Further, the specificity, sensitivity and repeatability were also assessed. The established SgN and genomic RNA (gRNA) qPCR assays were both applied to detect 21 environmental samples and 351 clinical samples containing 48 recovered patients. In the specimens with both positive gRNA and positive SgN, 25 specimens were inoculated on cells. Results: The primers and probes of SgN had good specificity for SARS-CoV-2. The minimum detection limit of the preliminarily established qPCR detection method for SgN was 1.5×10² copies/ml, with a coefficient of variation less than 1%. The positive rate of gRNA in 372 samples was 97.04% (361/372). The positive rates of SgN in positive environmental samples and positive clinical samples were 36.84% (7/19) and 49.42% (169/342), respectively. The positive rate and copy number of SgN in Wild strain were lower than those of SARS-CoV-2 Delta strain. Among the 25 SgN positive samples, 12 samples within 5 days of sampling time were all isolated with virus; 13 samples sampled for more than 12 days had no cytopathic effect. Conclusion: A qPCR method for the detection of SARS-CoV-2 SgN has been successfully established. The sensitivity, specificity and repeatability of this method are good.
Humans ; SARS-CoV-2/genetics* ; COVID-19/diagnosis* ; Subgenomic RNA ; Real-Time Polymerase Chain Reaction/methods* ; RNA, Viral/genetics* ; Sensitivity and Specificity ; Nucleocapsid/chemistry* ; COVID-19 Testing

Since the outbreak of corona virus disease 2019 (COVID-19), viral strains have mutated and evolved. Vaccine research is the most direct and effective way to control COVID-19. According to different production mechanisms, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines included inactivated virus vaccine, live attenuated vaccine, mRNA vaccine, DNA vaccine, viral vector vaccine, virus-like particle vaccine and protein subunit vaccine. Among them, viral protein subunit vaccine has a wide application prospect due to its high safety and effectiveness. Viral nucleocapsid protein has high immunogenicity and low variability which could be a new direction for vaccine production. We summarized the current development of vaccine research by reviewing the current progress, vaccine safety and vaccine immune efficiency. It is hoped that the proposed possible development strategies could provide a reference for epidemic prevention work in future.
Humans ; SARS-CoV-2/genetics* ; COVID-19/prevention & control* ; Protein Subunits ; Vaccines, DNA ; Nucleocapsid Proteins

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.
Ebolavirus/physiology* ; HEK293 Cells ; HeLa Cells ; Humans ; Nucleocapsid Proteins/metabolism* ; RNA, Viral/metabolism* ; Viral Matrix Proteins/metabolism* ; Virion/metabolism* ; Virus Assembly

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

We aim to screen out the active components that may have therapeutic effect on coronavirus disease 2019 (COVID-19) from the severe and critical cases' prescriptions in the "Coronavirus Disease 2019 Diagnosis and Treatment Plan (Trial Ninth Edition)" issued by the National Health Commission of the People's Republic of China and explain its mechanism through the interactions with proteins. The ETCM database and SwissADME database were used to screen the active components contained in 25 traditional Chinese medicines in 3 prescriptions, and the PDB database was used to obtain the crystal structures of 4 proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular docking was performed using Autodock Vina and molecular dynamics simulations were performed using GROMACS. Binding energy results showed that 44 active ingredients including xambioona, gancaonin L, cynaroside, and baicalin showed good binding affinity with multiple targets of SARS-CoV-2, while molecular dynamics simulations analysis showed that xambioona bound more tightly to the nucleocapsid protein of SARS-CoV-2 and exerted a potent inhibitory effect. Modern technical methods are used to study the active components of traditional Chinese medicine and show that xambioona is an effective inhibitor of SARS-CoV-2 nucleocapsid protein, which provides a theoretical basis for the development of new anti-SARS-CoV-2 drugs and their treatment methods.
Humans ; SARS-CoV-2 ; Molecular Docking Simulation ; Medicine, Chinese Traditional ; Molecular Dynamics Simulation ; Nucleocapsid Proteins ; Antiviral Agents/pharmacology* ; COVID-19 Drug Treatment

COVID-19 ; Coronavirus Nucleocapsid Proteins ; Humans ; Nucleocapsid Proteins ; Phosphoproteins ; SARS-CoV-2

The aim of this study was to estimate the seroprevalence of immunoglobulin M (IgM) and G (IgG) antibodies against SARS-CoV-2 in asymptomatic people in Wuhan. This was a cross-sectional study, which enrolled 18,712 asymptomatic participants from 154 work units in Wuhan. Pearson Chi-square test,
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Viral/blood* ; COVID-19/immunology* ; Carrier State/immunology* ; Child ; Child, Preschool ; China/epidemiology* ; Coronavirus Nucleocapsid Proteins/immunology* ; Cross-Sectional Studies ; Female ; Humans ; Immunoglobulin G/blood* ; Immunoglobulin M/blood* ; Male ; Middle Aged ; Occupations/classification* ; Phosphoproteins/immunology* ; SARS-CoV-2/immunology* ; Seroepidemiologic Studies ; Spike Glycoprotein, Coronavirus/immunology* ; Young Adult

Adolescent ; Adult ; Aged ; COVID-19/virology* ; COVID-19 Nucleic Acid Testing/methods* ; Child ; Coronavirus Nucleocapsid Proteins/genetics* ; Feces/virology* ; Female ; Humans ; Male ; Middle Aged ; Phosphoproteins/genetics* ; RNA, Viral/genetics* ; SARS-CoV-2/isolation & purification* ; Young Adult

To develop the large scale serological assay for severe fever with thrombocytopenia syndrome virus (SFTSV) infection, we evaluated two different enzyme-linked immunosorbent assay (ELISA) methods using nucleocapsid protein (NP) and Gn proteins of CB1 (genotype B) SFTSV strains. The NP-based ELISA tests showed more sensitive with broad cross-reactivity between two different genotype A and B strains compared with those of Gn-based ELISA tests. However, Gn-based ELISA showed more genotype specificity and specificity. These result suggested that NP-based ELISA test could be applicable for general sero-prevalence studies of SFTSV infections, while Gn-based ELISA could be applicable for a certain specific genotype sero-prevalence study.
Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Fever ; Genotype ; Nucleocapsid Proteins ; Sensitivity and Specificity ; Thrombocytopenia

Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most important infectious diseases causing serious economic loss in the swine industry worldwide. Due to its increasing genetic diversity, a rapid and accurate diagnosis is critical for PRRS control. The immunochromatographic strip test (ICST) is a rapid and convenient type of immunoassay. In this study, an on-site immunochromatographic assay-based diagnostic method was developed for detection of PRRS virus (PRRSV)-specific antibodies. The method utilized colloidal gold nanoparticle-labeled dual-type nucleocapsid proteins encoded by open reading frame 7. We evaluated 991 field samples from pig farms and 66 serum samples from experimentally PRRSV-inoculated pigs. Based on true PRRSV-specific antibody-positive or
Agriculture ; Antibodies ; Colloids ; Communicable Diseases ; Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Genetic Variation ; Gold Colloid ; Immunoassay ; Immunochromatography ; Immunoglobulin M ; Methods ; Nucleocapsid Proteins ; Open Reading Frames ; Porcine Reproductive and Respiratory Syndrome ; Porcine respiratory and reproductive syndrome virus ; Sensitivity and Specificity ; Swine