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

The genome of Hantaan virus, the prototype of the hantavirus genus, is composed of three segmented, single stranded negative sense RNA genome. The 5' and 3' termini of the Hantaan virus RNA genome contain noncoding regions (NCRs) that are highly conserved and complementary to form panhandle stuctures. There are some reports that these NCRs seems to control gene expression and viral replication in influenza virus and vesicular stomatitis virus. In this study, we examined whether NCRs in Hantaan virus play a role in expression of the viral nucleocapsid protein (Np) and foreign (luciferase) gene. The 5' and/or 3' NCR-deleted mutants were constructed and analysed. The Np expression of 5' NCR-deleted clone, it showed 40% reduction. To investigate the role of NCR in foreign gene expression, the clones which are replaced ORF of Hantaan viral Np gene with that of luciferase gene were constructed. The results were similar to those of the experiments using Np gene. These results suggest that 3' NCR is more important than 5' NCR in protein expression. To find out a critical region of 3' NCR in more important than 5' NCR in protein expression. To find out a critical region of 3' NCR in protein expression, several clones with a deleted part of 3' NCR were constructed and analyzed. The deletion of the conserved region in 3' NCR showed 20~30% decrease in Np expression. However there were no change in luciferase activities between clones with or without non-conserved region of 3' NCR. These results suggest that the 3' NCR of Hantaan virus S genome, especially conserved region in 3' NCR, plays and important role in the expression of Hantaan viral Np and foreign genes.
Animals ; Clone Cells ; Ecthyma, Contagious ; Gene Expression ; Genome* ; Hantaan virus ; Hantavirus ; Luciferases ; Nucleocapsid Proteins* ; Nucleocapsid* ; Orthomyxoviridae ; RNA ; Vesicular Stomatitis

Apoptosis, as a part of the natural defense mechanisms that protect against viral infection, plays a vital role in the pathogenic mechanisms. It also plays a key role in the pathogenesis of diseases including many viral diseases. Mechanisms of virus-induced apoptosis are not completely understood because of the complexity of the underlying biochemical cascades and all of the participating host factors. Mumps virus belongs to the genus Rubulavirus in the family Paramyxoviridae. It contains single stranded RNA genome with negative polarity. It was observed that mumps virus induced apoptosis in VeroE6 cells, and adsorption and penetration of mumps virus to cell membrane alone were not sufficient for the induction of cell death. When mumps virus was superinfected onto nucleocapsid protein (NP) expressing VeroE6 cells, cell viability and facterial titer were maintained until 13 and 12 day, respectively. The levels of p53, Bax, and Bcl-2 were increased in NP-expressing VeroE6 cells, and the increase in Bax, and Bcl-2 was outstanding. It was observed that NP protein did not directly affect the efficiency of the infection of mumps virus in NP-expressing VeroE6 cells. The levels of p53, and Bax were decreased in both mock-infected VeroE6 cells and NP-expressing VeroE6 cells infected with mumps virus. However, the Bcl-2 level was little affected by the virus infection.
Adsorption ; Apoptosis* ; Cell Death ; Cell Membrane ; Cell Survival ; Defense Mechanisms ; Genome ; Humans ; Mumps virus* ; Mumps* ; Nucleocapsid Proteins* ; Nucleocapsid* ; Paramyxoviridae ; RNA ; Rubulavirus ; Virus Diseases

OBJECTIVEThe purpose of this study is to express partial S gene of Hantavirus Z10.

METHODSThe 300 bp S gene of Z10 strain was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris and subcloned into pMD19-T. The SP300 gene was constructed into pPICZaA and sequenced. The recombinant pPICZaA-SP300 and pPICZaA-S300 was transformed into Pichia with LiCI.

RESULTSThe recombination Pichia were cultivate, and expressed the SP300 or S300 gene induced in Pichia by methanol.

CONCLUSIONThe nucleocapsid secreted from the Pichia can be detected by ELISA and WesternBlot.

Gene Expression ; Hantavirus ; genetics ; metabolism ; Nucleocapsid Proteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism

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

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

The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.
Cloning, Molecular ; Nucleocapsid Proteins ; biosynthesis ; genetics ; immunology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; SARS Virus ; genetics

Canine respiratory coronavirus (CRCoV) is commonly associated with canine kennel cough worldwide. Clinically infected dogs present coughing, sneezing, and nasal discharge. Severe infections may progress to pneumonia. Through serological surveys, CRCoV has been identified as a worldwide pathogen found in the respiratory tracts of dogs suffering from mild or severe respiratory disease. In this study, three dogs were obtained from a dog kennel. Over the previous 5 days, the dogs showed coughing, sneezing, and nasal discharge. To detect the etiologic pathogen, we performed multiplex RT-PCR (mRT-PCR) to amplify the genes encoding canine influenza virus matrix protein, canine distemper virus nucleocapsid protein, and CRCoV spike protein. Dot blotting was achieved with a CRCoV-specific probe. Nasal-secreting CRCoV was detected by the 442 bp CRCoV-positive PCR reaction in the nasal swabbing samples from dogs. Further, CRCoV-positive reactions by dot blot hybridization were detected in the nasal swabbing samples from dogs. In conclusion, we detected CRCoV in kenneled dogs with respiratory disease in Korea. Multiplex RT-PCR was able to detect successfully CRCoV infection in dogs. We suggest that mRT-PCR would be useful and effective for monitoring CRCoV infection in various kinds of dogs.
Animals ; Coronavirus* ; Cough ; Distemper Virus, Canine ; Dogs* ; Korea ; Nucleocapsid Proteins ; Orthomyxoviridae ; Pneumonia ; Polymerase Chain Reaction ; Respiratory System ; Sneezing

BACKGROUNDSeveral coronaviruses establish persistent infections in vitro and in vivo, however it is unknown whether persistence is a feature of the severe acute respiratory syndrome coronavirus (SARS-CoV) life cycle. This study was conducted to investigate viral persistence.

METHODSWe inoculated confluent monolayers of Vero cells with SARS-CoV at a multiplicity of infection of 0.1 TCID50 and passaged the remaining cells every 4 to 8 days for a total of 11 passages. Virus was titrated at each passage by limited dilution assay and nucleocapsid antigen was detected by Western blot and immunofluoresence assays. The presence of viral particles in passage 11 cells was assessed by electron microscopy. Changes in viral genomic sequences during persistent infection were examined by DNA sequencing.

RESULTSCytopathic effect was extensive after initial inoculation but diminished with serial passages. Infectious virus was detected after each passage and viral growth curves were identical for parental virus stock and virus obtained from passage 11 cells. Nucleocapsid antigen was detected in the majority of cells after initial inoculation but in only 10%-40% of cells at passages 2-11. Electron microscopy confirmed the presence of viral particles in passage 11 cells. Sequence analysis at passage 11 revealed fixed mutations in the spike (S) gene and ORFs 7a-8b but not in the nucleocapsid (N) gene.

CONCLUSIONSSARS-CoV can establish a persistent infection in vitro. The mechanism for viral persistence is consistent with the formation of a carrier culture whereby a limited number of cells are infected with each round of virus replication and release. Persistence is associated with selected mutations in the SARS-CoV genome. This model may provide insight into SARS-related lung pathology and mechanisms by which humans and animals can serve as reservoirs for infection.

Animals ; Antigens, Viral ; analysis ; Cercopithecus aethiops ; Microscopy, Electron ; Nucleocapsid Proteins ; analysis ; SARS Virus ; genetics ; growth & development ; Vero Cells

To clarify the pathogenesis of Duck enteritis virus (DEV), the cDNA library of duck's liver infected by DEV and a bait plasmid containing DEV nucleocapsid protein (NP) gene were constructed, then the receptor was screened from the cDNA library plasmid by the yeast two-hybrid system and verified by GST pull-down test. The results showed that the capacity of the primary cDNA library was 1 x 106 CFU with insertion size from 0.5 to 1 kb, and the bait plasmid of pGBKT7-NP showed no self-activation. The receptor reacting with DEV NP in duck liver was initially confirmed as the protein kinase C inhibitor (PKCI). These results provide new clues for further investigation on pathogenesis of DEV.
Alphaherpesvirinae ; pathogenicity ; Animals ; Ducks ; virology ; Gene Library ; Liver ; virology ; Nucleocapsid Proteins ; genetics ; Plasmids ; Receptors, Virus ; analysis ; Two-Hybrid System Techniques