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

Adolescent ; Adult ; Animals ; Bites and Stings ; Dog Diseases ; virology ; Dogs ; Female ; Humans ; Male ; Middle Aged ; Nucleocapsid Proteins ; genetics ; Phylogeny ; Post-Exposure Prophylaxis ; Rabies ; prevention & control ; veterinary ; virology ; Rabies Vaccines ; immunology ; Young Adult

This article aimed to study the antigenicity of nucleocapsid proteins (NPs) in six pathogenic phleboviruses and to provide theoretical evidence for the development of serological diagnostic reagents. NPs of six pathogenic phleboviruses were expressed and purified using a prokaryotic expression system and rabbits were immunized with individual recombinant NPs. Cross-reactions among NPs and rabbit sera were determined by both indirect ELISA and Western blotting analyses, and the sera titer was determined by indirect ELISA. Furthermore, sera from SFTS patients were also detected by each recombinant NP as a coating antigen using indirect ELISA. The cross-reactions and the sera titer were subsequently determined. Both the concentration and purity of recombinant NPs of six pathogenic phleboviruses met the standards for immunization and detection. The results of indirect ELISA and Western blotting showed that each anti-phlebovirus NP rabbit immune serum had potential serological cross-reactivity with the other five virus NP antigens. Furthermore, the sera from SFTS patients also had cross-reactivity with the other five NP antigens to a certain extent. Our preliminary study evaluated the antigenicity and immune reactivity of six pathogenic phleboviruses NPs and laid the foundation for the development of diagnostic reagents.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Cross Reactions ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; Phlebotomus Fever ; diagnosis ; immunology ; virology ; Phlebovirus ; classification ; genetics ; immunology ; isolation & purification ; Rabbits

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

To evaluate the effectiveness of rabies vaccination, we developed the SPA-ELISA method to detect the antibodies against rabies virus (RV) using the main antigenic determinant of nucleoprotein (RV N1) as antigen. The complete Nucleoprotein (N) gene and the partial N1 gene (1 000-1 353 bp) of RV Flury LEP strain were amplified using RT-PCR and PCR approaches. The two fragments were inserted into pGEX-6P-1 respectively. Then we transformed the recombinant plasmids into Escherichia coli BL21(DE3) strain and expressed them by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). SDS-PAGE analysis showed that both of the two recombinant proteins were presented as inclusion bodies. Compared with the complete nucleoprotein, the partial protein (RV N1) was expressed at a much higher level in E. coli BL21(DE3). The antigenic specificity of the partial N1 protein was confirmed by Western blotting. By coating the plates with purified RV N1 as an antigen, an SPA-ELISA method for the detection of the antibodies against RV was established. By optimizing this method, the optimal concentration of RV N1 coating the ELISA plate was 2 mg/L. The optimal concentration of serum samples and SPA-HRP was 1:100 and 1:4 000 respectively. Compared with a commercially available ELISA kit coating RV as antigen, the coincidence rate of SPA-ELISA was 94.1%. Our results show that the developed SPA-ELISA based on the RV N1 was useful for the detection of the antibodies against RV in the sera of dogs.
Animals ; Antibodies, Viral ; analysis ; immunology ; Dogs ; Enzyme-Linked Immunosorbent Assay ; methods ; Epitopes ; immunology ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Nucleocapsid Proteins ; immunology ; Rabies virus ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Staphylococcal Protein A

Despite global efforts to control porcine reproductive and respiratory syndrome virus (PRRSV) infection, the virus continues to cause economic problems in the swine industry worldwide. In this study, we attempted to generate and characterize a panel of stable BHK cell lines that constitutively express the nucleocapsid (N) protein of type 1 or type 2 PRRSV. The established BHK cell lines were found to react well with N-specific antibodies as well as the hyperimmune serum of pigs raised against each genotype of PRRSV. Taken together, the data implicate a potential usefulness for the newly generated stable cell lines as a diagnostic reagent for PRRSV serology.
Animals ; Antibodies, Viral/analysis/immunology ; Blotting, Western/veterinary ; Cell Line ; Cricetinae ; Female ; Genotype ; Nucleocapsid Proteins/genetics/*immunology ; Porcine Reproductive and Respiratory Syndrome/diagnosis/*immunology ; Porcine respiratory and reproductive syndrome virus/genetics/*immunology ; Swine ; Transfection/veterinary

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

To characterize nucleocapsid (N) protein genes of human respiratory syncytial viruses isolated from children in Beijing and express the N genes in E. coli,seven HRSV strains (three subtype A and four subtype B) were isolated from clinical samples of infants and children with acute respiratory infections and visited the Children's Hospital affiliated to Capital Institute of Pediatrics in Beijing during the period of Jan. 2006 to Mar. Full length of N genes from seven HRSV strains were amplified by reverse-transcription PCR (RT-PCR). The seven PCR amplicons were sequenced after cloning into pUCm-T and the sequences were compared with the N genes from HRSVs in GenBank. N gene was amplified from recombinant plasmid pUCm-N9968 by PCR and then sub-cloned into the prokaryotic expression vector pET30a(+) after digestion with EcoR I and Xho I . The pET30a-N9968 was transformed into E. coli BL21 (DE3) and expressed by inducing with IPTG. Target protein was characterized by SDS-PAGE electrophoresis and Western blot. The amplified N genes were 1 176 bp in length and the deduced N proteins were 391 amino acids in length. The nucleotide identities of N genes among these seven strains were 85.4%-99.7% and the de-duced amino acid similarities were 95.4%-100%. The recombinant plasmid pET30a-N9968 had correct open reading frame confirmed by dual-enzyme digestion and sequence analysis. The fusion protein 6 x HisN was produced after inducing by 1 mmol/L IPTG at 37 degrees C. A unique protein band with molecular weight 49 kD was characterized by SDS-PAGE and purified by Ni2+ affinity chromatography column. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein had specific binding reaction to specific monoclonal antibody and human sera, indicating that the expressed protein is of specific antigenicity.
Base Sequence ; Child ; Escherichia coli ; genetics ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; Respiratory Syncytial Virus, Human ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

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