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PubMed1984

[Isolation, identification and typing of Newcastle disease virus isolates in a chick embryo cell culture].

Routine methods of investigation were employed in parallel experiments for the isolation (in chick embryos), identification (serologically via HI), and typing (MDT, ICPI, IVPI, and via cloacal tests) of local isolates and standard strains of the Newcastle disease virus (NDV). The results were the same as those obtained by the single-phase complex investigation after the plaque technique method in a monolayer cell culture of chick embryo fibroblasts under agar cover. The cultures were inoculated with tissue suspension centrifugate in multiple dilutions of trachea and lung, spleen and liver, and brain of experimentally infected birds, using local isolates (13 in all) and standard strains of NDV (6 of the various types) as well as of birds that died of Newcastle disease (3 cases). The complex cell culture technique of investigation included plaque-forming under agar (and under agarose as well) following the direct inoculation with the infectious tissue suspensions in the presence of a normal (known negative), resp., specific hyperimmune serum against Newcastle disease. It also included a plaque-neutralization (plaque reduction) test and tests for hemadsorption and elution of erythrocytes--all made use of in a single planned programme of investigation. This technique, which is a modification of the one suggested by Beard et al. (1970) and known as single-phase complex plaque technique, provides the simultaneous isolation and identification of NDV isolates, which may well be coupled with some of the routinely practiced methods for definitive isolate typing (e. g., with the cloacal test for velogenic, and with MDT for lento- and mesogenic isolates). Thus, time can be saved, and labour and means are reduced, i. e., the laboratory diagnostics of ND may be perfected.

Animals↗
PubMedDate not supplied

Protection of chickens from Newcastle and Marek's diseases with a recombinant herpesvirus of turkeys vaccine expressing the Newcastle disease virus fusion protein.

Recombinant strains of herpesvirus of turkeys (HVT) were constructed that contain either the fusion protein gene or the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) inserted into a nonessential gene of HVT. Expression of the NDV antigens was regulated from a strong promoter element derived from the Rous sarcoma virus long terminal repeat. Recombinant HVT strains were stable and fully infectious in cell culture and in chickens. Chickens receiving a single intra-abdominal inoculation at 1 day of age with recombinant HVT expressing the NDV fusion protein had an immunological response and were protected (> 90%) against lethal intramuscular challenge at 28 days of age with the neurotropic velogenic NDV strain Texas GB. Recombinant HVT expressing the NDV hemagglutinin-neuraminidase provided partial protection (47%) against the same challenge. Chickens vaccinated with recombinant HVT vaccines had low levels of protection against NDV replication in the trachea when challenged ocularly. Recombinant HVT vaccines and the parent HVT strain provided similar levels of protection to chickens challenged with the very virulent RB1B strain of Marek's disease virus, indicating that insertion of foreign sequences into the HVT genome did not compromise the ability of HVT to protect against Marek's disease.

Animals↗
PubMedDate not supplied

Differentiation of exotic strains of Newcastle disease virus by oligonucleotide fingerprinting.

The oligonucleotide fingerprints of five of six strains of velogenic-viscerotropic Newcastle disease (VVND) virus were readily distinguishable, even though the strains could not be differentiated by a series of in vivo and in vitro procedures. Two of the VVND virus strains tested (10333-0 and 8231412) showed marked similarity to the prototype strain (MIL-0). Two of the remaining three strains (80-27489 and 80-28634) had identical oligonucleotide fingerprints, even though they had a different isolation history and exhibited small differences in their virulence for chickens. Fingerprints of the sixth VVND strain, 80-12927, demonstrated some similarities when it was compared with the prototype virus fingerprint, but it was quite different from strains 80-27489, 10333-0 and 8231412.

Newcastle disease virus↗
PubMed2004

Regions on nucleocapsid protein of Newcastle disease virus that interact with its phosphoprotein.

The nucleocapsid (NP) and phospho-(P) proteins of paramyxoviruses are involved in transcription and replication of the viral genome. An in vitro protein binding assay was used to investigate the regions on NP protein that interact with the P protein of Newcastle disease virus (NDV). Truncated NP mutants were first immobilised on a solid phase and then interacted with radio-labelled [(35)S]-P protein synthesised in rabbit reticulocyte. The interaction affinity was quantitated by measuring the radioactivity that was retained on the solid phase. Using this approach, a highly interactive region was identified to be resided at the first 25 amino acids of NP N-terminus. The interaction between these two proteins remained strong even with the removal of 114 amino acids from the C-terminal end of NP. However, it is possible that the 49 amino acids at the C-terminal end might have another contact region for P protein, which is not as critical as the N-terminal end. The interaction regions mapped in this study are significantly different from the other two paramyxoviruses: Sendai and measles viruses in which the C-termini of their NP proteins play an important role in binding to the P.

Mutation↗
PubMed1976

Inhibition of Newcastle disease virus replication by 6-azauridine. I. Inefficacy of purified uridine kinase, effect of adenosine-5'-triphosphate.

As distinct from cell-free extracts prepared from tumour cells, partially purified uridine kinase prepared from the same cells was not effective in 6-azauridine (AzUrd) inhibition of Newcastle disease virus (NDV) replication. This showed that uridine kinase was not the effective component of cell-free extracts. Adenosine-5'-triphosphate (ATP) was found to exert a synergic effect in combination with AzUrd in the inhibition of NDV replication.

Adenosine Triphosphate↗
PubMed2004

Biological significance of the second receptor binding site of Newcastle disease virus hemagglutinin-neuraminidase protein.

The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein responsible for attachment to receptors containing sialic acid, neuraminidase (NA) activity, and the promotion of membrane fusion, which is induced by the fusion protein. Analysis of the three-dimensional structure of Newcastle disease virus (NDV) HN protein revealed the presence of a large pocket, which mediates both receptor binding and NA activities. Recently, a second sialic acid binding site on HN was revealed by cocrystallization of the HN with a thiosialoside Neu5Ac-2-S-alpha(2,6)Gal1OMe, suggesting that NDV HN contains an additional sialic acid binding site. To evaluate the role of the second binding site on the life cycle of NDV, we rescued mutant viruses whose HNs were mutated at Arg516, a key residue that is involved in the second binding site. Loss of the second binding site on mutant HNs was confirmed by the hemagglutination inhibition test, which uses an inhibitor designed to block the NA active site. Characterization of the biological activities of HN showed that the mutation at Arg516 had no effect on NA activity. However, the fusion promotion activity of HN was substantially reduced by the mutation. Furthermore, the mutations at Arg516 slowed the growth rate of virus in tissue culture cells. These results suggest that the second binding site facilitates virus infection and growth by enhancing the fusion promotion activity of the HN.

Binding Sites↗
PubMed1987

Vaccination of cyclophosphamide-treated chickens against Newcastle disease virus infection.

Newly-hatched chickens were treated with 3 mg of cyclophosphamide (CY) per day for 4 consecutive days. At 2 weeks of age, these chickens, together with a group of untreated controls, were vaccinated intranasally or subcutaneously with the La Sota strain of Newcastle disease virus (NDV). All chickens were challenged intranasally with the GB strain of NDV 2 weeks later. CY-treated, intranasally vaccinated chickens were highly resistant to NDV challenge, yet none of the chickens produced any detectable humoral antibodies to NDV; antibodies to NDV were detectable in the tracheal washings, however.

Animals↗
PubMed2006

Expression of the fusion glycoprotein of Newcastle disease virus in transgenic rice and its immunogenicity in mice.

Transgenic plant has become an attractive bioreactor to produce high-value medical peptides and proteins in biomedical research. In present study, two expression cassettes, pUNDVF and pGNDVF containing the fusion protein gene of Newcastle disease virus (NDV F) under the control of maize ubiquitin (Ubi) promoter or rice glutelin (Gt1) promoter, respectively, were constructed, and introduced into rice (Oryzy sativa L.) by Agrobacterium-mediated transformation. A total of 12 independent transgenic rice lines were regenerated, and the result from PCR analysis indicated that the T-DNA region containing the NDV F chimeric gene had been integrated into the genome of transgenic rice plants. ELISA and Western-blot analyses revealed that the NDV F protein could be expressed and accumulated in both leaf and seed tissue of several transgenic rice plants. Moreover, the immunogenicity of expressed proteins was tested in a mouse model and the results showed that specific antibodies were elicited in mice immunized intraperitoneally with crude protein extracts from transgenic rice plants. It implied the potential of using transgenic rice-based expression systems as supplementary bioreactor for NDV engineering subunit vaccine.

Animals↗
PubMed1990

Modification of tumor cells by a low dose of Newcastle disease virus. III. Potentiation of tumor-specific cytolytic T cell activity via induction of interferon-alpha/beta.

To investigate possibilities of augmenting tumor-specific immune responses against the highly metastatic murine lymphoma ESb, we tested the effects of the interferon inducer newcastle disease virus (NDV) or of interferon-alpha/beta as costimulator in mixed lymphocyte-tumor cell cultures (MLTC) on the tumor-specific cytolytic T cell (CTL) response. Both approaches, namely stimulation of ESb immune spleen cells with NDV-modified stimulator cells or with ESb stimulator cells and exogenous IFN-alpha/beta, led to a selective potentiation of tumor-specific CTL activity. The potent activation of tumor-specific CTL precursor (CTLP) required the simultaneous presence of the specific ESb tumor antigen--possibly to mediate a signal via the corresponding T cell receptor--and costimulators--possibly to mediate second activation signals. Increased CTL activity required only very low amounts of NDV or IFN-alpha/beta. The generation of CTL activity in the MLTC cultures could be blocked by antisera to IFN-alpha/beta, not, however by control sera. Similar effects were observed in vivo, suggesting that IFN-alpha/beta not only caused an increase in CTL activity, but was essential for the generation of CTL activity. The reduction of the generation of CTL by antiserum to IFN-alpha/beta could be overcome by excess interferon, especially when using ESb-NDV as stimulator cells.

Animals↗
PubMed1987

Translation and membrane insertion of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses is likely in the unusual class of glycoproteins with the amino terminus cytoplasmic and the carboxy terminus lumenal or external to the cell. The properties of the membrane insertion of the HN protein of Newcastle disease virus, a prototype paramyxovirus, were explored in wheat germ extracts containing microsomal membranes. HN protein was inserted into membranes cotranslationally, resulting in a glycosylated protein completely resistant to trypsin and proteinase K digestion. No detectable posttranslation insertion occurred. Insertion required signal recognition particle. Signal recognition particle in the absence of membranes inhibited HN protein synthesis. Comparisons of the trypsin digestion products of the HN protein made in the cell-free system with newly synthesized HN protein from infected cells showed that the cell-free product was in a conformation different from that of the pulse-labeled protein in infected cells. First, trypsin digestion of intact membranes from infected cells reduced the size of the 74,000-dalton HN protein by approximately 1,000 daltons, whereas trypsin digestion of HN protein made in the cell-free system had no effect on the size of the protein. Second, trypsin digestion of Triton X-100-permeabilized membranes isolated from infected cells resulted in a 67,000-dalton trypsin resistant HN protein fragment. A trypsin-resistant core of comparable size was not present in the digestion products of in-vitro-synthesized HN protein. Evidence is presented that the newly synthesized HN protein in infected cels contain intramolecular disulfide bonds not present in the cell-free product.

Animals↗
PubMed1999

Pathotyping isolates of Newcastle disease virus using antipeptide antibodies to pathotype-specific regions of their fusion and hemagglutinin-neuraminidase proteins.

Antipeptide antibodies have been evaluated for their abilities to predict the characteristics of the cleavage motifs of the fusion protein precursors (F0) of 25 isolates of Newcastle disease virus (NDV) with a range of virulences, grouped into 12 sets according to their monoclonal antibody reactivities. A Western blot format was used to show that antisera to synthetic peptides representing sequences at the C-termini of the F2-polypeptides of defined pathotypes of NDV usually distinguish between pathotypes on the basis of their Fo cleavage sequences. However, exceptions were found with three groups of virulent isolates. Protein sequencing and mass spectral analysis of the F2-polypeptide of isolate Texas GB from one of these groups, identified an anomalous cleavage/activation process which removed the amino acids required for recognition by the antisera. This probably also explained the lack of reactivity of the Roakin isolate and low reactivity of the Komarov isolate from this group. The other exceptions involved isolates in groups with cleavage region variations from the usual motif of virulent isolates or isolates with undefined cleavage motifs. Antipeptide antisera were also raised to sections of the 45 residue C-terminal extension the hemagglutinin-neuraminidase precursor (HN0) encoded by the genes of some avirulent isolates. Western blot analysis showed that positive reactions with antibodies to peptides based on sequences between residues 577 and 613 of the HN0 was evidence for the presence of an avirulent isolate but did not exclude the presence of other pathotypes. Antisera designed to target residues 569-577 detected HN0 extensions of 6 residues on isolates known to encode such extensions. These antisera also enabled differentiation of isolates with HN0 extensions of 6 residues from those with no extension, however, it was not possible to determine the virulence of isolates based on reaction with these antisera.

Amino Acid Sequence↗
PubMed1992

Detection of haemagglutination inhibition antibodies against Newcastle disease virus in unvaccinated indigenous chickens in Maiduguri, Borno State, Nigeria.

An examination of 200 serum samples from unvaccinated indigenous (local) chickens in Maiduguri, Borno State (Nigeria) using the haemagglutination inhibition (HI) test showed 73 sera to be positive and 127 to be negative for antibodies against Newcastle disease virus. The highest antibody titre observed was 1:128. The prevalence rate was higher (46.9%) in adult chickens than in young chickens of less than 12 weeks (23%). Presence of HI antibodies in unvaccinated indigenous chickens indicates that these birds had contracted infection and recovered thereafter.

Age Factors↗
PubMed2001

Structural and functional relationship between the receptor recognition and neuraminidase activities of the Newcastle disease virus hemagglutinin-neuraminidase protein: receptor recognition is dependent on neuraminidase activity.

The terminal globular domain of the paramyxovirus hemagglutinin-neuraminidase (HN) glycoprotein spike has a number of conserved residues that are predicted to form its neuraminidase (NA) active site, by analogy to the influenza virus neuraminidase protein. We have performed a site-directed mutational analysis of the role of these residues in the functional activity of the Newcastle disease virus (NDV) HN protein. Substitutions for several of these residues result in a protein lacking both detectable NA and receptor recognition activity. Contribution of NA activity, either exogenously or by coexpression with another HN protein, partially rescues the receptor recognition activity of these proteins, indicating that the receptor recognition deficiencies of the mutated HN proteins result from their lack of detectable NA activity. In addition to providing support for the homology-based predictions for the structure of HN, these findings argue that (i) the HN residues that mediate its NA activity are not critical to its attachment function and (ii) NA activity is required for the protein to mediate binding to receptors.

Amino Acid Substitution↗
PubMed2006

Expression of the Newcastle disease virus fusion protein in transgenic maize and immunological studies.

Transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins, including antibodies, antigens and hormones. Here, we report the expression of the fusion (F) gene of the Newcastle disease virus (NDV) in transgenic maize plants. The expression of the transgene, driven by the maize ubiquitin promoter, caused accumulation of the F protein in maize kernels. The presence of the transgene was verified by Southern and western blots. Feeding chickens with kernels containing the F protein induced the production of antibodies, which conferred protection against a viral challenge. This protection was comparable to that conferred by a commercial vaccine. Possible uses of this plant-based F protein as a potential mucosal vaccine are discussed.

Animals↗