A total of 18 Newcastle disease virus (NDV) isolates that were recovered from 1949 through 1997 were characterized and pathotyped. All viruses were highly virulent as determined by intracerebral pathogenicity indices > or = 1.81 in day-old. These pathotypes are typical for viscerotropic velogenic NDV (VVNDV) pathotype viruses. Some differences were observed for the chicken red blood cell elution rate and thermostability of the hemagglutinin at 56degrees C. Three antigenic groups were identified by a hemagglutination-inhibition assay using NDV monoclonal antibodies. And the predominant gross lesions were as follows: discharge from the nasal cavity, tracheal mucus, petechial hemorrhage in the heart fat, kidney urates and hemorrhage with or without necrosis in the gastrointestinal tract. Severe hemorrhagic or necrotic lesions were also noted in the lymphoid organs and were localized primarily in the spleen and cecal tonsil. However, differences in the occurrence and frequency of the gross lesions were observed between the virus strains. Among them, NDV strains that induced neurological symptoms belonged only to genotype VI. This strain had spread throughout Korea during the late 1980s to the 1990s, which suggests that specific VVNDVs genotypes might result in neurological symptoms.
Animals ; Antibodies, Monoclonal ; Avulavirus ; Chickens ; Erythrocytes ; Gastrointestinal Tract ; Genotype ; Heart ; Hemagglutinins ; Hemorrhage ; Kidney ; Korea ; Mucus ; Nasal Cavity ; Necrosis ; Newcastle Disease ; Newcastle disease virus ; Palatine Tonsil ; Spleen ; Sprains and Strains ; Viruses

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 hybridoma cell line 1G4A7 secreting monoclonal antibody (McAb) specific to hemagglutinin of avian paramyxovirus type 2 (APMV-2) was developed by fusing the spleen cells of APMV-2 immunized BAlb/c mice with SP2/0 myeloma cells. The immunoglobulin subclass of 1G4A7 was IgG1 with light chain kappa and the affinity constant against APMV-2 was 1.02 X 10(10). Identified by HI and indirect ELISA, the McAb titers in ascities were 10 log 2 and 1 : 10(6) respectively. The McAb did not cross react with the common avian viruses, showing good specificity. There existed obvious differences in antigenitic relationship among APMV-2 viruses analyzed by HI and indirect ELISA using McAb 1G4A7.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Avulavirus ; immunology ; Enzyme-Linked Immunosorbent Assay ; Hemagglutination Inhibition Tests ; Hemagglutinins, Viral ; immunology ; Mice ; Mice, Inbred BALB C

No abstract available.
Animals ; Baculoviridae* ; Newcastle disease virus* ; Newcastle Disease*

Recent advances in reverse genetics techniques make it possible to manipulate the genome of RNA viruses such as Newcastle disease virus (NDV). Several NDV vaccine strains have been used as vaccine vectors in poultry, mammals, and humans to express antigens of different pathogens. The safety, immunogenicity, and protective efficacy of these NDV-vectored vaccines have been evaluated in pre-clinical and clinical studies. The vaccines are safe in mammals, humans, and poultry. Bivalent NDV-vectored vaccines against pathogens of economic importance to the poultry industry have been developed. These bivalent vaccines confer solid protective immunity against NDV and other foreign antigens. In most cases, NDV-vectored vaccines induce strong local and systemic immune responses against the target foreign antigen. This review summarizes the development of NDV-vectored vaccines and their potential use as a base for designing other effective vaccines for veterinary and human use.
Animals ; Genome ; Humans ; Mammals ; Newcastle disease virus* ; Newcastle Disease* ; Poultry ; Reverse Genetics ; RNA Viruses ; Vaccines*

A Newcastle disease virus (NDV) isolate designated IBS002 was isolated from a commercial broiler farm in Malaysia. The virus was characterised as a virulent strain based on the multiple basic amino acid motif of the fusion (F) cleavage site 112RRRKGF117 and length of the C-terminus extension of the hemagglutinin-neuraminidase (HN) gene. Furthermore, IBS002 was classified as a velogenic NDV with mean death time (MDT) of 51.2 h and intracerebral pathogenicity index (ICPI) of 1.76. A genetic distance analysis based on the full-length F and HN genes showed that both velogenic viruses used in this study, genotype VII NDV isolate IBS002 and genotype VIII NDV isolate AF2240-I, had high genetic variations with genotype II LaSota vaccine. In this study, the protection efficacy of the recombinant genotype VII NDV inactivated vaccine was also evaluated when added to an existing commercial vaccination program against challenge with velogenic NDV IBS002 and NDV AF2240-I in commercial broilers. The results indicated that both LaSota and recombinant genotype VII vaccines offered full protection against challenge with AF2240-I. However, the LaSota vaccine only conferred partial protection against IBS002. In addition, significantly reduced viral shedding was observed in the recombinant genotype VII-vaccinated chickens compared to LaSota-vaccinated chickens.
Amino Acids, Basic ; Animals ; Chickens* ; Genetic Variation ; Genotype* ; Malaysia ; Newcastle disease virus* ; Newcastle Disease* ; Vaccination ; Vaccines* ; Virulence ; Virus Shedding

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*

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

Rabies remains an important worldwide health problem. Newcastle disease virus (NDV) was developed as a vaccine vector in animals by using a reverse genetics approach. Previously, our group generated a recombinant NDV (LaSota strain) expressing the complete rabies virus G protein (RVG), named rL-RVG. In this study, we constructed the variant rL-RVGTM, which expresses a chimeric rabies virus G protein (RVGTM) containing the ectodomain of RVG and the transmembrane domain (TM) and a cytoplasmic tail (CT) from the NDV fusion glycoprotein to study the function of RVG's TM and CT. The RVGTM did not detectably incorporate into NDV virions, though it was abundantly expressed at the surface of infected BHK-21 cells. Both rL-RVG and rL-RVGTM induced similar levels of NDV virus-neutralizing antibody (VNA) after initial and secondary vaccination in mice, whereas rabies VNA induction by rL-RVGTM was markedly lower than that induced by rL-RVG. Though rL-RVG could spread from cell to cell like that in rabies virus, rL-RVGTM lost this ability and spread in a manner similar to the parental NDV. Our data suggest that the TM and CT of RVG are essential for its incorporation into NDV virions and for spreading of the recombinant virus from the initially infected cells to surrounding cells.
Animals ; Antibody Formation ; Cytoplasm* ; Glycoproteins* ; GTP-Binding Proteins ; Humans ; Mice ; Newcastle disease virus* ; Newcastle Disease* ; Parents ; Rabies virus ; Rabies* ; Reverse Genetics ; Tail* ; Vaccination ; Virion*

Elution time of velogenic, mesogenic and lentogenic strains of Newcastle disease virus was determined. The differences in their elution time were also calculated. Four samples, each of a velogenic strain (VGF2), a mesogenic strain (Komarov) and a lentogenic strain (LaSota) were used for hemagglutination test with 0.6% chicken red blood cells. The time it took for wells of the end hemagglutination points (highest dilution that gave agglutination) to elute was recorded as elution time for each sample. The mean elution time of the three strains of Newcastle disease virus differed significantly (p < 0.05). The velogenic strain gave the highest mean elution time of 118 min, followed by the mesogenic strain with 59 min and the lentogenic strain with 25 min. Based on this result it appears that elution time could form a basis for rough characterization of isolates of Newcastle disease virus into the three major strains.
Animals ; Chickens/blood ; Erythrocytes/*virology ; Hemagglutination Tests ; *Hemagglutination, Viral ; Newcastle disease virus/isolation&purification/*pathogenicity