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

Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451-739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451-739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.
Ebolavirus ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Nucleocapsid ; chemistry ; genetics ; metabolism ; RNA, Viral ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Virus Assembly

Severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the Phlebovirus genus from the Bunyaviridae family endemic to China, is the causative agent of life-threatening severe fever with thrombocytopenia syndrome (SFTS), which features high fever and hemorrhage. Similar to other negative-sense RNA viruses, SFTSV encodes a nucleocapsid protein (NP) that is essential for viral replication. NP facilitates viral RNA encapsidation and is responsible for the formation of ribonucleoprotein complex. However, recent studies have indicated that NP from Phlebovirus members behaves in inhomogeneous oligomerization states. In the present study, we report the crystal structure of SFTSV NP at 2.8 Å resolution and demonstrate the mechanism by which it processes a ringshaped hexameric form to accomplish RNA encapsidation. Key residues essential for oligomerization are identified through mutational analysis and identified to have a significant impact on RNA binding, which suggests that correct formation of highly ordered oligomers is a critical step in RNA encapsidation. The findings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection.
Binding Sites ; Crystallography, X-Ray ; Mutation ; Nucleocapsid Proteins ; chemistry ; genetics ; metabolism ; Phlebovirus ; metabolism ; Protein Binding ; Protein Multimerization ; Protein Structure, Quaternary ; RNA, Viral ; metabolism ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

Nucleocapsid protein (NPs) of negative-sense single-stranded RNA (-ssRNA) viruses function in different stages of viral replication, transcription, and maturation. Structural investigations show that -ssRNA viruses that encode NPs preliminarily serve as structural building blocks that encapsidate and protect the viral genomic RNA and mediate the interaction between genomic RNA and RNA-dependent RNA polymerase. However, recent structural results have revealed other biological functions of -ssRNA viruses that extend our understanding of the versatile roles of virally encoded NPs.
Animals ; Capsid ; metabolism ; Humans ; Lassa virus ; chemistry ; physiology ; Nucleocapsid Proteins ; chemistry ; metabolism ; Orthobunyavirus ; chemistry ; physiology ; RNA Viruses ; chemistry ; physiology

Prokaryotic expression plasmids carrying N-terminal(1-163aa) and C-terminal(141-306aa) gene of HCoV-NL63 nucleocapsid protein were constructed with pET-30a(+) vector. Consequently, we prepared two purified proteins, Np and Cp, respectively, and established a Western blotting-based line assay (WBLA) for detection of antibodies against HCoV-NL63 using three purified proteins: Np , Cp and Nf, a full-length HCoV-NL63 nucleocapsid protein as previously reported. We detected anti-HCoV-NL63 antibodies among 50 sera samples collected from adult for health-examination by WBLA. The results showed that: 25 (50%), 27 (54%), 36 (72%) of 50 sera were indentified as anti-HCoV-NL63 antibody positive when the antigen was from Nf, Np and Cp, respectively. Among these sera with positive anti-HCoV-NL63 antibody,Cp showed highest antibody positive rate in WBLA,and consistent rates of detection were 64% between Nf and Np, 54% between Nf and Cp, 54% between Np and Cp. Our study provides the foundation for development of HCoV-NL63 serological detection reagents and an experimental tool for immunological research of HCoV-NL63 infection.
Adult ; Antibodies, Viral ; blood ; Blotting, Western ; Coronavirus ; chemistry ; immunology ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; isolation & purification ; Peptide Fragments ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Serologic Tests

Coronaviruses are the causative agent of respiratory and enteric diseases in animals and humans. One example is SARS, which caused a worldwide health threat in 2003. In coronaviruses, the structural protein N (nucleocapsid protein) associates with the viral RNA to form the filamentous nucleocapsid and plays a crucial role in genome replication and transcription. The structure of N-terminal domain of MHV N protein also implicated its specific affinity with transcriptional regulatory sequence (TRS) RNA. Here we report the crystal structures of the two proteolytically resistant N- (NTD) and C-terminal (CTD) domains of the N protein from murine hepatitis virus (MHV). The structure of NTD in two different crystal forms was solved to 1.5 Å. The higher resolution provides more detailed structural information than previous reports, showing that the NTD structure from MHV shares a similar overall and topology structure with that of SARS-CoV and IBV, but varies in its potential surface, which indicates a possible difference in RNA-binding module. The structure of CTD was solved to 2.0-Å resolution and revealed a tightly intertwined dimer. This is consistent with analytical ultracentrifugation experiments, suggesting a dimeric assembly of the N protein. The similarity between the structures of these two domains from SARS-CoV, IBV and MHV corroborates a conserved mechanism of nucleocapsid formation for coronaviruses.
Amino Acid Sequence ; Binding Sites ; Crystallography, X-Ray ; Molecular Sequence Data ; Murine hepatitis virus ; chemistry ; metabolism ; Nucleocapsid Proteins ; chemistry ; metabolism ; Phosphoproteins ; chemistry ; metabolism ; Protein Binding ; Protein Folding ; Protein Multimerization ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA ; metabolism ; Sequence Alignment

The N gene and genome promoter nucleotide sequence of a Chinese Peste des petits rumiants virus (PPRV) ("China/Tib/Gej/07-30") was firstly determined. The length of N gene was 1689 nucleotides with a single open reading frame (ORF). The nucleotide and deduced amino acid sequence was compared with the homologous region of other PPRV isolates. The nucleotide sequence of the "China/Tib/Gej/07-30" was 91.7%-97.6% identical to other PPRV isolates, while a homology of 94.9%-98.5% could be observed at the amino acids level. The N gene encoded a protein of 525 amino acids. Several sequence motifs were identified on the basis of conservation in the PPRVs and the morbilliviruses. The genome length of promoter region was 107 nucleotides with 91.8%-98.2% identity to other PPRV isolates. Phylogenetic analysis showed that the "China/Tib/Gej/07-30" belonged to the Asian lineage.
Amino Acid Sequence ; Animals ; Base Sequence ; China ; Female ; Genome, Viral ; Goat Diseases ; virology ; Goats ; Molecular Sequence Data ; Nucleocapsid Proteins ; chemistry ; genetics ; Peste-des-Petits-Ruminants ; virology ; Peste-des-petits-ruminants virus ; chemistry ; classification ; genetics ; isolation & purification ; Phylogeny ; Promoter Regions, Genetic ; Sequence Alignment ; Sequence Analysis

The aim of this study was to characterize the N and E protein encoding genes of a new human coronavirus (HCoV-NL63) which was identified from one of the clinical specimens (BJ8081) collected from a 12 years-old patient with acute respiratory infection in Beijing. The complete N and E gene sequences of HCoV-NL63 were amplified from clinical sample by RT-PCR, then were cloned into the pCF-T and pUCm-T vectors respectively and sequenced. The complete sequences of N and E genes were submitted to GenBank by Sequin and compared with N and E genes of prototype HCoV-NL63 and the other coronaviruses published in GenBank. The secondary structure and the characteristics of sample BJ8081 N and E proteins were predicted by bioinformatics. It was indicated that the N and E genes amplified from sample BJ8081 were 1134 bp and 234 bp in length and the predicted proteins including 377 amino acids and 77 amino acids, respectively. The data suggested that the region of amino acids 78-85 within N protein probably was the conserved region for all coronaviruses identified so far including HCoV-NL63. The region of amino acids 15-37 for E protein was probably the transmembrane domain. In conclusion, the recombinant plasmids pCF-T-8081 N and pUCm-T-8081 E were successfully constructed and sequenced, and the data predicted by bioinformatics are helpful for the further analysis of HCoV-NL63.
Amino Acid Sequence ; Child ; China ; Computational Biology ; methods ; Coronavirus ; classification ; genetics ; metabolism ; Coronavirus Infections ; virology ; Humans ; Molecular Sequence Data ; Nucleocapsid Proteins ; chemistry ; genetics ; metabolism ; Phylogeny ; Protein Structure, Secondary ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo obtain monoclonal antibodies (McAbs) against severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) nucleocapsid (N) protein to develop diagnostic test for SARS and study the pathogenesis of the disease.

METHODSBALB/c mice were immunized with purified N protein of SARS-CoV. Hybridoma cell lines secreting monoclonal antibodies against SARS-associated coronavirus nucleocapsid were established after cell fusion with mouse splenic cells and SP2/0 cells. The specificity of the McAbs obtained was examined by Western blot and indirect fluorescence assay. Epitopes reacted with the McAbs were preliminarily located through Western blot by expressing truncated N proteins.

RESULTSAfter cell fusion and three rounds of cell cloning, six hybridoma cell lines secreting monoclonal antibodies specifically against SARS-CoV nucleocapsid were obtained. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with nucleocapsid protein and SARS-CoV. Among the six McAbs, three recognize the epitopes located in the N-terminus of the protein, whereas the others reacted with those located in the C-terminus.

CONCLUSIONThe anti-SARS-CoV nucleocapsid McAbs were developed and these McAbs may be useful in the development of diagnosis assays and basic research of SARS.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Antibody Specificity ; Female ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; immunology ; isolation & purification ; SARS Virus ; chemistry ; immunology

The gene encoding the nucleocapsid (N) protein of vesicular stomatitis virus (VSV-NJ) was subcloned from pMD-VN5, and inserted into pBAD/Thio TOPO vector. The recombinant plasmid was identified by restriction analysis and PCR. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted N gene. The results of SDS-PAGE and Western immunoblotting revealed that the N protein was expressed in Escherichia coli LGM194 in a high level and the recombinant fusion protein, which contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. It had a molecular mass of approximately 63.5 kD and immunologically reactive activity. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of vesicular stomatitis using 186 serum samples from experimentally infected goats and guinea-pigs with VSV-NJ and VSV-IN, and from field origin and reference serum samples. The sensitivity and specificity of the ELISA were compared with those of the standard microtiter serum neutralization (MTSN) tests. The ELISA and MTSN test results were highly correlated for detection of VSV antibodies. The ELISA was as sensitive as the SN assay in detecting positive serum to VSV. The correlation between SN titers and ELISA titers was statistically significant. These data suggest that the recombinant fusion N protein of VSV could be used as a recombinant test antigen for the serodiagnosis of vesicular stomatitis. The ELISA based on the reconmbinant nucleocapsid protein may offer the best combination of rapidity, sensitivity, simplicity, economy, and laboratory biosafety of any of the methods yet developed for VSV serodiagnosis. This study lay on foundation for the development of the diagnosis methods in serology for VSV.
Amino Acid Sequence ; Animals ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Molecular Sequence Data ; Neutralization Tests ; Nucleocapsid Proteins ; chemistry ; genetics ; immunology ; isolation & purification ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Serologic Tests ; Vesicular stomatitis Indiana virus ; genetics ; Vesicular stomatitis New Jersey virus ; genetics