Twenty-four isolates of Newcastle disease virus (NDV) prevailing during 1997 -- 2005 in China were collected. These isolates were purified by CEF plaque assay and replicated in SPF chicken embryos. The hemagglutinin-neuraminidase (HN) genes of these viruses were cloned and sequenced. The HN gene sequences of thirty-six NDV reference strains in GenBank were also used in this study. The amino acid homologing of these viruses were compared and analyzed. The correlations among different fragments of HN gene were also analyzed. The results indicated that the homology of Chinese field NDV strains was 94.4%-99.4%, but 86.9%-89% compared with LaSota and Clone30, 87.9%-89.9% to F48E9, and 87.2%-96.2% to foreign NDV strains. There had the nearest distances among Chinese NDV isolates as compared with that of the LaSota, Clone30 and F48E9 by the phylogenetic tree. However, the distances of seven foreign NDV isolates were very close to Chinese NDV isolates as compared with these of the other foreign NDV isolates. We also found that all the Chinese field isolates were devoid of glycosylation site in position 538 -- 540. There were good correlations between different length amino acid fragments and the genomes of HN, especially the 5'-terminus first 80aa.
Animals ; Chick Embryo ; Chickens ; China ; HN Protein ; genetics ; Newcastle disease virus ; classification ; genetics ; Phylogeny

The goal of this study is to research the genetic characteristics and relationship between HN and P genes of NDV. The nucleotide sequence and deduced amino acid sequence were analyzed for the Hemagglutinin-neuramindase (HN) and Phosphoprotein (P) gene of twelve field isolates of Newcastle disease virus (NDV) during 1997-2005 in China. The HN and P gene sequences of fifteen NDV reference strains from GenBank were also used in this study. The molecular evolution distance of nucleotides and amino acids were calculated by MEGA 4.0 software, and analysis of variance and correlations were analyzed by SPSS11.0 software among different length sequences of the HN gene or P gene. The nucleotide and amino acids correlation of HN and P gene were analyzed respectively. The correlation of evolution distance and isolation year were also calculated. The results indicated that there were difference and good correlation of nucleotide and amino acid among different length sequences of the HN gene or P gene. These results revealed that the HN and P gene of NDV have the different response to selective pressure to adopt to landscape and closely relationship on heredity mutations. Nucleotide variations of HN and P gene have relationship with isolation year of strains.
Evolution, Molecular ; Genetic Variation ; HN Protein ; genetics ; Newcastle disease virus ; classification ; genetics ; Phosphoproteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log₂, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Animals ; Antibodies, Viral ; Chickens ; HN Protein/metabolism* ; Newcastle Disease/prevention & control* ; Newcastle disease virus/metabolism* ; Oryza/genetics*

To determine the functions of N-carbohydrate chains in human parainfluenza virus type 3 hemagglutinin-neuraminidase(HN) protein, a PCR-based site-directed mutagenesis method was used to obtain N-glycan mutants. Protein electrophoresis rate, cell surface expression,receptor binding activity, neuraminidase activity and cell fusion promotion activity were determined. The HN proteins of single mutants (G1, G2, and G4) and multiple mutants (G12, G14, G24 and G124) migrated faster than the wild-type (wt) HN protein on polyacrylamide gels, while G3-mutated protein and wt HN protein migrated at the same position. There was no statistic difference in cell surface expression and neuraminidase activity between wt and each mutant HN protein (P>0.05), but receptor binding activity and cell fusion promotion activity of each mutant protein was reduced to significant extent (P<0.05). G1, G2 and G4 mutants exhibited re duced receptor binding activity, which was 83.94%, 76.45% and 55.32% of the wt level, respectively. G1, G2 and G4-mutated proteins also showed reductions in fusion promotion activity, which was 80.84%, 77.83% and 64.16%, respectively. Multiple mutants with G12-, G14-, G24- and G124- substitutions could further reduce receptor binding activities, 33.07%, 20.67%, 19.96% and 15.11% of the wt HN level, respectively. G12, G14, G24 and G124 mutants exhibited levels of fusion promotion activity that were only 46.360, 12.04%, 13.43% and 4.05% of the wt amount, respectively. As N-glycans of hPIV3 HN protein play an important role in receptor binding activity and cell fusion promotion activity of HN protein. We propose that the loss of N-glycans change the conformation or orientation of globular domain that is responsible for receptor binding and lower receptor binding activity and cell fusion promotion activi ty.
Glycosylation ; HN Protein ; chemistry ; genetics ; metabolism ; Humans ; Mutation ; Parainfluenza Virus 3, Human ; chemistry ; enzymology ; genetics ; physiology ; Protein Binding ; Receptors, Virus ; metabolism ; Respirovirus Infections ; metabolism ; virology ; Virus Internalization

Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutinin-neuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2(13) HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.
Avulavirus* ; Baculoviridae* ; Blotting, Western ; Clone Cells ; Cross Reactions ; Diagnosis ; Ducks ; Glycosylation ; Hemagglutination ; HN Protein ; Immune Sera ; Insects ; N-Acetylneuraminic Acid ; Serologic Tests ; Serotyping ; Spodoptera

A neutralization-resistant mutant of Newcastle disease virus (NDV) Kr005 strain belonging to class II genotype VII was generated using a neutralizing monoclonal antibody and its biological effects were assessed. The mutant showed single amino acid substitution (E to K) at position 347 of the hemagglutinin-neuraminidase (HN) protein (E347K mutant). The E347K mutant exhibited marked rounding of the cells and few syncytia in infected chicken embryofibroblast (CEF) cells. The hemadsorption and neuraminidase activities of the E347K mutant of the wild-type virus were 118% and 166%, respectively. The mutant produced a rapid elution pattern whereas the wild type had a slow elution pattern. Growth kinetics studies showed that the E347K mutant produced an 80-times higher yield of extracellular virus in CEF cells compared with the wild-type virus. The time-course virus titer showed a marked increase in mutant-infected cells from 6 h to 12 h post infection (pi), which was consistent with the titer pattern time-course for NA activity. The E347K mutant virus showed a slight decrease in virulence compared to the wild-type virus, but there was no change in pathotype when measured by in vivo pathogenicity testing. These results suggest that an E347K mutation in HN protein might be associated with increased NA activity and subsequent enhancement of virus release from infected cells without change in viral pathotype.
Amino Acid Substitution ; Animals ; Chickens ; Genotype ; Giant Cells ; Hemadsorption ; HN Protein ; Kinetics ; Neuraminidase ; Newcastle Disease ; Newcastle disease virus ; Sprains and Strains ; Viral Load ; Virus Release ; Viruses

The prokaryotic expression plasmid pQE30-HN of hemagglutinin-neuraminidase (HN) protein gene of bovine parainfluenza virus type 3 (BPIV3) strain HJ-1 was expressed by IPTG induction in E. coli XL1Blue. The recombinant HN protein(rHN) was purified by electroeluting method, and used as coated antigen. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody valence of BPIV3. The best working conditions of ELISA were as follows: the antigen concentration was 6 microg/mL; the serum dilution was 1:50; the blocking reagent was 5% skimmed milk; the blocking time was 60 min at 37 degrees C; the second antibody concentration was 1:10 000; The cut-off value was 0.30. The method revealed a good specificity, no cross-reaction to the positive sera of BCV, IBRV or BRSV was observed. We applied the method to detect 323 serum samples of dairy cow in Heilongjiang Province, the seropositivity rate of BPIV3 was about 58%. The indirect ELISA established provided a technological basis for the development of ELISA kit.
Animals ; Antibodies, Viral ; blood ; Blotting, Western ; Cattle ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; Female ; HN Protein ; genetics ; Parainfluenza Virus 3, Bovine ; genetics ; immunology

PURPOSE: Most chemical transfection reagents are ineffective for the transfection of cells in suspension, such as leukemic cell and stem cell lineages. We developed two different types of viroplexes, cationic Sendai F/HN viroplexes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN viroplexes (PCSVs) for the efficient transfection of T-leukemic cells. MATERIALS AND METHODS: The viroplex systems were prepared by reconstitution of fusogenic Sendai F/HN proteins in DMKE (O,O'-dimyristyl-N-lysyl glutamate) cationic liposomes. The viroplexes were further optimized for plasmid DNA and siRNA delivery to suspension cells. The particle size and surface charge of the viroplexes were analyzed with a zeta-sizer. Transfection of plasmid DNA (pDNA) and small interfering RNA (siRNA) by CSVs or PCSV was evaluated by measurement of transgene expression, confocal microscopy, FACS, and RT-PCR. RESULTS: The optimized CSVs and PCSVs exhibited enhanced gene and siRNA delivery in the tested suspension cell lines (Jurkat cells and CEM cells), compared with conventional cationic liposomes. In the case of pDNA transfection, the CSVs and PCSVs show at least 10-fold and 100-fold higher transgene expression compared with DMKE lipoplexes (or lipofectamine 2000), respectively. The CSVs showed more effective siRNA delivery to the suspension cells than cationic liposomes, as assessed by confocal microscopy, FACS, and RT-PCR. The effective transfection by the CSVs and PCSVs is presumably due to fusogenic activity of F/HN proteins resulting in facilitated internalization of pDNA and siRNA. CONCLUSION: This study suggests that Sendai F/HN viroplexes can be widely applicable for the transfection of pDNA and siRNA to suspension cell lines.
Cell Line, Tumor ; HN Protein/genetics ; Humans ; Jurkat Cells ; RNA, Small Interfering ; Sendai virus/genetics ; Transfection/*methods ; Viral Fusion Proteins/genetics ; Virosomes

To analyze the genetic characterization of epidemic mumps virus strains in Liaoning Province and provide the basis for mumps control. A total of 32 mumps viruses strains were isolated during 2008-2104. The fragment of SH genes and HN genes were amplified by RT-PCR, the PCR products were sequenced and analyzed. Basing on the 316 nucleotides of SH gene, The phylogenetic analyses were processed with the data of WHO mumps reference strains downloaded from GenBank and 32 mumps viruses strains. It showed that the 31 mumps virus strains belong to F genotype except MuVi/Liaoning. CHN/16.11 which was G genotype . Comparing to the A reference strains (Jeryl-Lynn and S-79), F genotype MuV were mutated on 12 amino acids sites and 27 amino acids siteson on HN gene. F genotype MuV added one N-glycosylation site in 464th-466th amino acids. The antigenic sites on HN were mutated on 121th, 123th, 279th, 287th, 336th, 356th and 442th. Maybe, it will influence the MuV antigenic.
Base Sequence ; China ; Genotype ; HN Protein ; chemistry ; genetics ; Humans ; Molecular Sequence Data ; Mumps ; virology ; Mumps virus ; chemistry ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p < 0.01) in chickens inoculated with C79-13ΔcrpΔasd (pYA-HN) than with C79-13ΔcrpΔasd (pYA) but were not significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.
Animals ; Antibodies ; Bacteria ; Chickens ; Hemagglutination ; HN Protein ; Homologous Recombination ; Immunoglobulin A, Secretory ; Immunoglobulin G ; Lymphocytes ; Methods ; Newcastle disease virus ; Newcastle Disease ; Plasmids ; Poultry ; Salmonella ; Suicide ; Vaccines