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

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

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

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon (IFN) signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid (N) protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid (poly(I:C)) in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly(I:C)-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB (NF-κB) nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.
Active Transport, Cell Nucleus ; Animals ; Coronavirus Infections ; immunology ; veterinary ; virology ; Genes, Viral ; Host-Pathogen Interactions ; immunology ; Interferons ; antagonists & inhibitors ; biosynthesis ; genetics ; Interleukins ; antagonists & inhibitors ; biosynthesis ; genetics ; NF-kappa B ; metabolism ; Nucleocapsid Proteins ; genetics ; immunology ; physiology ; Porcine epidemic diarrhea virus ; genetics ; pathogenicity ; physiology ; Promoter Regions, Genetic ; Swine ; Swine Diseases ; immunology ; virology

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

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

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.
Antibodies, Viral/*immunology ; Blotting, Western ; Coronavirus 229E, Human/*immunology ; Coronavirus OC43, Human/*immunology ; Cross Reactions ; Humans ; Nucleocapsid Proteins/genetics/*immunology ; Recombinant Proteins/immunology ; SARS Virus/genetics/*immunology ; Severe Acute Respiratory Syndrome/diagnosis/*immunology

To evaluate the immune responses of recombinant Lactobacillus casei 393 expressing Porcine Epidemic Diarrhea Viral (PEDV) N protein as oral vaccine, n gene of PEDV was subcloned into the expression vector pPG-1, and then transformed into L. casei 393 by electroporation, resulting in recombinant strain pPG-1-n/L, casei 393. The recombinant strains were induced to express interest protein, which was detected by Western blotting, immunofluorescence microscopy and the whole bacteria ELISA. And then BALB/C mice were used as an animal model immunized with recombinant strains by oral administration, and the immune efficacy was analyzed. The recombinant PEDV N protein showed the antigenic specificity, and was located on the bacterial cell walls of pPG-1-n transformed L. casei. The results of ELISA showed that the mice immunized with recombinant strains could produce remarkable special sIgA level in the feces, and high level of anti-PEDV N protein IgG in the serum (P < 0.01), but the induced antibodies in serum did not demonstrated neutralizing effect. Statistical significant difference was observed among the spleen lymphocyte proliferation index (LPI) among the immunization groups of mice and control groups. And there was significant increase. of IFN-gamma and IL-4 contents in the supernatant of spleen cell culture in immunized group. In conclusion, the oral immunizations with recombinant L. casei 393 can induce significant specific mucosal PEDV N-specific IgA response as well as serum IgG responses, and can evoke both mucosal immune and system immune responses.
Administration, Oral ; Animals ; Antibody Formation ; Coronavirus Infections ; prevention & control ; Female ; Immunity, Mucosal ; immunology ; Lactobacillus casei ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; biosynthesis ; genetics ; immunology ; Porcine epidemic diarrhea virus ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Viral Vaccines ; administration & dosage ; immunology

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 prepare and characterize monoclonal antibodies (mAbs) against the recombinant nucleocapsid (N) protein of 3 human coronaviruses SARS-CoV, 229E and OC43 and study the antigenic relationship between the 3 N proteins.

METHODSBALB/c mice were immunized with the recombinant N proteins of SARS-CoV, 229E and OC43 to obtain the mAbs by means of hybridoma. Screening and identification of the mAbs were performed using indirect enzyme-linked immunosorbent assay (ELISA), Western blotting and indirect immunofluorescence assay. Cross-reactivity between the N proteins of the 3 coronaviruses was analyzed with the prepared mAbs.

RESULTSThe mAbs against the recombinant N proteins of SARS-CoV, 229E and OC43 were obtained, which reacted specifically with the corresponding viral N protein as shown by indirect ELISA, Western blotting and indirect immunofluorescence assay. No cross-reactivity was found between the 3 N proteins.

CONCLUSIONThe prepared mAbs against the recombinant N proteins may provide valuable assistance in studying antigenic relationships of N proteins between the 3 human coronaviruses.

Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Coronavirus 229E, Human ; genetics ; immunology ; Coronavirus OC43, Human ; genetics ; immunology ; Cross Reactions ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Fluorescent Antibody Technique, Indirect ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; genetics ; immunology ; Recombinant Proteins ; immunology ; SARS Virus ; genetics ; immunology