Objective The study aims to investigate the immunological functions of the nucleocapsid (N) protein of the novel coronavirus Omicron (BA.1, BA.2) and evaluate the differences among different N proteins of mutant strains in immunogenicity. Methods By aligning sequences, the mutation sites of the Omicron (BA.1, BA.2) N protein relative to prototype strain of the novel coronavirus (Wuhan-Hu-1) were determined. The pET-28a-N-Wuhan-Hu-1 plasmid was used as template to construct pET-28a-BA.1/BA.2-N through single point mutation or homologous recombination. The three kinds of N protein were expressed in prokaryotic system, purified through Ni-NTA affinity chromatography, and then immunized into mice. The titer and reactivity of the polyclonal antibody, as well as the expression level of IL-1β and IFN-γ in mouse spleen cells, were detected using indirect ELISA and Western blot assay. Results The constructed prokaryotic expression plasmids were successfully used to express the Wuhan-Hu-1 N, BA.1 N, and BA.2 N proteins in E.coli BL21(DE3) at 37 DegreesCelsius for 4 hours. The indirect ELISA test showed that the titers of polyclonal antibody prepared by three N proteins were all 1:51 200. All three N proteins can increase the expression of IFN-γ and IL-1β cytokines, but the effect of Omicron N protein in activing two cytokines was more obvious than that of Wuhan-Hu-1 N protein. Conclusion The study obtained three new coronavirus N proteins and polyclonal antibodies, and confirmed that mutations in the amino acid sites of the N protein can affect its immunogenicity. This provides a basis for developing rapid diagnostic methods targeting N protein of different novel coronavirus variants.
Animals ; Mice ; SARS-CoV-2/genetics* ; Coronavirus Nucleocapsid Proteins/immunology* ; Nucleocapsid Proteins/isolation & purification* ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Mice, Inbred BALB C ; Interferon-gamma/metabolism* ; Interleukin-1beta/metabolism* ; Female ; Escherichia coli/metabolism* ; Mutation ; Humans

This study aims to establish an antibody detection method for porcine deltacoronavirus (PDCoV). The recombinant proteins PDCoV-N1 and PDCoV-N2 were expressed via the prokaryotic plasmid pColdII harboring the N gene sequence of the PDCoV strain CH/XJYN/2016. The reactivity and specificity of PDCoV-N1 and PDCoV-N2 with anti-PEDV sera were analyzed after the recombinant proteins were analyzed by SDS-PAGE and purified by the Ni-NTA Superflow Cartridge. Meanwhile, Western blotting and indirect immunofluorescence assay were carried out separately to validate the recombinant proteins PDCoV-N1 and PDCoV-N2. Finally, we established an indirect ELISA method based on the recombinant protein PDCoV-N2 after optimizing the conditions and tested the sensitivity, specificity, and reproducibility of the method. Then, the established method was employed to examine 102 clinical serum samples. The recombinant protein PDCoV-N2 showed low cross-reactivity with anti-PEDV sera. The optimal conditions of the indirect ELISA method based on PDCoV-N2 were as follows: the antigen coating concentration of 1.25 μg/mL and coating at 37 ℃ for 1 h; blocking by BSA overnight at 4 ℃; serum sample dilution at 1:50 and incubation at 37 ℃ for 1 h; secondary antibody dilution at 1:80 000 and incubation at 37 ℃ for 1 h; color development with TMB chromogenic solution at 37 ℃ for 10 min. The S/P value ≥ 0.45, ≤0.38, and between 0.45 and 0.38 indicated that the test sample was positive, negative, and suspicious, respectively. The testing results of the antisera against porcine epidemic diarrhea virus (PEDV), porcine circovirus 2 (PCV2), transmissible gastroenteritis virus (TGEV), foot-and-mouth disease virus (FMDV), and African swine fever virus (ASFV) showed that the S/P values were all less than 0.38. The testing results of the 800-fold diluted anti-PDCoV sera were still positive. The results of the inter- and intra-batch tests showed that the coefficients of variation of this method were less than 10%. Clinical serum sample test results showed the coincidence rate between this method and neutralization test was 94.12%. In this study, an ELISA method for the detection of anti-PDCoV antibodies was successfully established based on the truncated N protein of PDCoV. This method is sensitive, specific, stable, and reproducible, serving as a new method for the clinical diagnosis of PDCoV.
Animals ; Enzyme-Linked Immunosorbent Assay/methods* ; Swine ; Antibodies, Viral/blood* ; Recombinant Proteins/genetics* ; Deltacoronavirus/isolation & purification* ; Coronavirus Infections/virology* ; Swine Diseases/diagnosis* ; Coronavirus Nucleocapsid Proteins ; Sensitivity and Specificity

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

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

OBJECTIVETo investigate the virulence characteristics of two fixed strains (CTN and aG) and a street strain (HN10) of rabies viruses isolated in China.

METHODSICR mice of different age groups were inoculated with CTN, aG and HN10 rabies virus strains via the intracracerebral (i.c.) or intramuscular (i.m.) routes, and observed for 20 days.

RESULTSThe CTN strain was pathogenic to 7-day-old suckling mice that received i.c. inoculations and 3-day-old suckling mice that received i.m. inoculations. The aG strain was pathogenic to 4-week-old mice that received i.c. inoculations and 7-day-old suckling mice that received i.m. inoculations. The HN10 strain was pathogenic to mice of all age groups via both inoculation routes. In moribund mice, the viruses had spread to most regions of the brain. The CTN and HN10 strains had similar dissemination patterns in the brain; both viral antigens could be found in the dentate gyrus (DG), whereas few viral antigens were present in the DG from specimens that had been infected with the aG strain.

CONCLUSIONA comprehensive sequence analysis of the G protein suggested that differences in gene sequences may be responsible for producing strain-specific differences in pathogenicity and distribution in the brain.

Animals ; Antigens, Viral ; immunology ; Brain ; immunology ; virology ; China ; Mice ; Mice, Inbred ICR ; Nucleocapsid Proteins ; immunology ; Rabies ; virology ; Rabies virus ; immunology ; isolation & purification ; pathogenicity

This study measured the clinical prevalence of peste des petits ruminants (PPR) among sheep and goats in India between 2003 and 2009 by analyzing clinical samples from suspected cases of PPR that were submitted to the Rinderpest and Allied Disease Laboratory, Division of Virology, IVRI, Mukteswar for PPR diagnosis. PPR outbreaks were confirmed by detecting PPR virus (PPRV)-specific antigen in the clinical samples. Clinical samples (blood, nasal swabs, spleen, lymph node, kidney, liver, intestine, and pooled tissue materials) were taken from a total of 592 sheep and 912 goats in different states of India and screened for the presence of PPRV antigen using a monoclonal antibody-based sandwich ELISA kit. A total of 20, 38, and 11 laboratory-confirmed PPR outbreaks occurred among sheep, goat, and combined sheep and goat populations, respectively. Our findings provide evidence of widespread PPR endemicity in India. The underlying reasons could be variations in husbandry practices in different geographical regions, agro-climatic conditions, and livestock migration. Furthermore, decrease in the number of PPR outbreaks over time might be due to the effectiveness of current live PPR vaccines and timely vaccination of target species. Vaccination against PPR has been practiced in India since 2002 to control this disease.
Animals ; Antibodies, Monoclonal/immunology ; Antigens, Viral/*blood ; Disease Outbreaks/*veterinary ; Enzyme-Linked Immunosorbent Assay/veterinary ; Goat Diseases/*epidemiology/immunology/prevention & control ; Goats ; India/epidemiology ; Nucleocapsid Proteins/immunology ; Peste-des-Petits-Ruminants/epidemiology/immunology/prevention & control/*veterinary ; Peste-des-petits-ruminants virus/*immunology/isolation & purification ; Prevalence ; Risk Factors ; Seasons ; Sheep ; Sheep Diseases/*epidemiology/immunology/prevention & control ; Vaccination/veterinary ; Viral Vaccines/*immunology/therapeutic use

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

OBJECTIVETo find out the status of human metapneumovirus (hMPV) infection in children under 14 years old with acute respiratory tract infections (ARI) in Guangzhou, analyze the epidemiology and clinical characteristics among the hMPV-infected children, and provide some basis for research of hMPV.

METHODSAll 521 throat and pharyngeal swabs were collected among the children with acute respiratory tract infections in outpatient departments and those admitted to the wards from September 2006 to August 2008. Then total nucleic acid was extracted from respiratory specimens. The 213 nucleosides of nucleoprotein gene were detected by RT-PCR and 16 strong positive samples were picked to compare with the sequence of hMPV in GenBank after the sequence of the amplification products were determined. Then applied statistical analysis to the data of the collected patients.

RESULTSAll 521 samples were detected by RT-PCR, and confirmed that N gene was positive in 39 samples with a detection rate of 7.49%, and the peak time was in October and April. The 16 amplification products were compared by using the analysis of gene sequence. The nucleocapsid protein (N) gene similarity to BJ1897 of Beijing was up to 99%, and to AY550156 of Thailand was up to 97%, genotype B was the most common genotype.

CONCLUSIONThere existed hMPV infection in children acute respiratory system diseases in Guangzhou areas, in which the children under the age of 6 years were accounted for the main group, however there was no difference in gender. The main symptoms of the patients with hMPV infection were high fever and cough symptom of catarrh. Co-infections other than respiratory virus with hMPV were detected as 41.03% of positive samples.

Adolescent ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Male ; Metapneumovirus ; genetics ; isolation & purification ; Molecular Sequence Data ; Nucleocapsid Proteins ; genetics ; RNA, Viral ; genetics ; Respiratory Tract Infections ; epidemiology ; virology

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

OBJECTIVETo detect serve acute respiratory syndrome-associated coronavirus (SARS-CoV) and SARS-like-CoV in fruit bats captured in Guangzhou and its vicinity.

METHODSTotally 927 bats of 9 species (Cynopterus sphinx, Rousettus leschenaulti, Miniopterus schreibersi, Hipposideros pratti, Rhinolophusasinicus, Scotophilusakuhlii, Hipposideros Pomona, Rhinolophus affinis, and Rhinolophus pusillus) captured in Guangzhou and its vicinity from September 2004 to November 2005 were available for this investigation, from which 3,043 samples (813 throat swasb, 524 sera, 853 lung tissues and 853 colorectal tissue specimens) were obtained. SARS-Cov and SARS-like-CoV were detected in these specimens using diagnostic kit for novel coronavirus N protein (ELISA), SARS-CoV Virus RNA detection kit, fluorescence PCR, Genchip, RT-PCR and cell isolation culture methods.

RESULTS AND CONCLUSIONNo SARS-CoV and SARS-like-CoV were detected in the 3043 samples, indicating the current absence of SARS-CoV and SARS-like-CoV in the bats captured in Guangzhou and its vicinity.

Animals ; China ; epidemiology ; Chiroptera ; virology ; Disease Vectors ; Enzyme-Linked Immunosorbent Assay ; Humans ; Nucleocapsid Proteins ; metabolism ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; isolation & purification ; metabolism ; Severe Acute Respiratory Syndrome ; epidemiology ; transmission ; virology