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RNA-dependent DNA polymerase activity was found in peparations of a mutant of Newcastle disease virus. The enzyme activity was not found in wild-type virus preparations.
A system for detection and strain differentiation of Newcastle disease virus (NDV) by reverse transcription of polymerase chain reaction (RT-PCR) (isolation of RNA, choice of primers for nested PCR, and purification of PCR products) and sequencing is developed and optimized. A nucleotide sequence of gene F site, coding for the F2/F1 cleavage site of F0 fusion protein and including several hypervariable regions, is determined for 10 Russian strains and vaccine strains. The data indicate a replacement of NDV populations in Russia and a rapid evolution of the virus. The origin of pathogenic NDV strains which have been circulating up to the present time is still unknown.
Normal mouse bone marrow cells were exposed to encephalomyocarditis virus (EMC), reovirus type 3 (REO3), influenza virus (FLU), and Newcastle disease virus (NDV) then assayed for granulocyte-macrophage precursor cells by the technique of colony formation in agar. Exposure to EMC, REO3, and FLU caused a slight but variable loss of colony-forming potential, whereas exposure to NDV caused a very marked loss. NDV acted directly on the cells, not indirectly through release of colony-inhibiting factors or destruction of colony-stimulating factor. Experiments with NDV inactivated by heat, ether, or ultraviolet irradiation indicated that colony inhibition was associated with fully infective virus, even though some of the inactivated preparations had retained full hemagglutinin, neuraminidase, or hemolytic activity.
For the construction of transfer vector pFGHN1175-1, the gene encoding haemagglutinin-neuraminidase(HN) glycoprotein of newcastle disease virus(NDV) strain F48E8 was removed from plasmid pGEMHN, and inserted into the HindIII site of insertion vector pFG1175-1, downstream of P7.5 promotor. Chicken embryo fibroblast (CEF) cell cultures which had been infected with fowlpox virus (FPV) Chinese vaccine strain 282E4 for 3-4 hours were transfected with pFGHN1175-1 plasmid DNA by liposomal transfection. Recombinant FPV with blue plaques were selected and purified 3 times in CEF cell cultures overlaid agarose containing X-gal. PCR analysis and DNA dot-blotting hybridization assay indicated that the HN gene had inserted into the FPV genomic DNA. The expression of the NDV HN gene in recombinant FPV was confirmed by indirect immunofluorescence assay with specific monoclonal antibody.
Nucleocapsid protein (NP) gene of Newcastle disease virus (NDV) strains mainly isolated in Japan from 1930 to 2001 was genetically characterized. By deduced amino acid sequence comparison, the N-terminal region (from 1 to 401 residues) of the NP protein was found to be highly conserved, while the C-terminal region was highly variable among the NDV isolates. A phylogenetic tree construct based on the nucleotide sequence of the complete NP gene revealed that the old (prior to 1970s) and the new (after 1980s) isolates could be classified into two major different groups, i.e., a group comprising virulent strains, and another group composed of avirulent strains. By restriction enzyme analysis using Pst I, none of the virulent strains were cleaved, while avirulent strains were cleaved. The results may be useful for simple primary screening test for differentiating NDV isolates.
We have completely sequenced the genome of Newcastle disease virus (NDV) vaccine strain LaSota. The sequences of the 3'- and 5'-terminal ends of the RNA genome were determined by sequencing cDNA fragments generated by rapid amplification of cDNA ends. The entire genomic sequence, which was established by sequencing cDNA fragments generated by high-fidelity RT-PCR, consists of 15186 nt. Comparison of the 5'-terminal sequence of NDV LaSota with the 5'-terminal sequences of ten members of the Paramyxovirinae showed that NDV LaSota has an unusually long 5' untranscribed region. Comparison of the entire genomic sequences showed that NDV is only distantly related to the other members of the genus Rubulavirus, to which NDV has been assigned. In this paper we present data which suggest that NDV should not be classified in the genus Rubulavirus, but instead should be considered as a member of a new genus within the subfamily Paramyxovirinae.
The hemagglutinin-neuraminidase (HN) gene from the Beaudette C strain of Newcastle disease virus (NDV) has been expressed in a recombinant fowlpox virus vector. The HN gene, under the control of the vaccinia p7.5 promoter, was inserted into a nonessential gene in the terminal inverted repeats of fowlpox virus. Expression was demonstrated in tissue culture, a protein of the correct size for fully glycosylated HN protein being recognized by an HN-specific monoclonal antibody on Western blots. When the recombinant fowlpox virus was inoculated into chickens by intravenous or wing-web routes, antibody which recognizes HN from purified NDV virions was produced. Protective immunity to NDV was generated in the chickens; at the highest dose of vaccine 100% of the chickens tested were protected against challenge with a virulent strain of NDV.
Avridine, a lipoidal amine with interferon-inducing and adjuvant properties, was an effective adjuvant for Newcastle disease antigen (NDA) in chickens. Eleven vaccine lots were evaluated: 2 commercial water-in-oil vaccines, 4 experimental oil emulsion vaccines, 4 avridine-containing vaccines, and a control lot of nonadjuvanted antigen. Avridine significantly enhanced the immunologic responses of chickens against NDA. Chickens vaccinated with the avridine-containing vaccines had significantly higher antibody titers (hemagglutination inhibition) than did chickens vaccinated with the commercial vaccines. Experimental oil emulsion vaccines prepared from the same antigens as avridine-adjuvanted vaccines induced higher hemagglutination inhibition antibody titers after primary but not after booster vaccination. Use of avridine as an adjuvant for NDA in vaccines for chickens induced immunologic protection rates similar to those induced by oil emulsion vaccines, without causing the reactogenic and tissue residue problems associated with the use of oil vaccines in chickens.
Molecular cloning of most if not all of the genome of an avirulent strain D26 of Newcastle disease virus (NDV) was carried out. cDNA clones were aligned by mutual hybridization and restriction map analysis. The nucleotide sequence of 3672 bases which completed the partial sequence of P gene reported in our previous paper (Ishida, N. et al., 1986, Nucleic Acids Res. 14, 6551-6564), and also covered M and F genes, was determined. Each gene contained one long open reading frame which could code for polypeptides of 395, 364, and 553 amino acid residues, respectively. The deduced amino acid sequences of P and M gene products showed little homology to those of other paramyxoviruses. In contrast, comparison of the amino acid sequence of the F gene product revealed highly conserved regions including the amino terminal sequence of the F1 portion following the putative processing site. There was only one basic amino acid residue at the putative processing site, which would explain the low virulence of this strain.
On the base of obtaining the full length genome sequence of a Newcastle disease virus (NDV) isolated from goose, the minigenome was constructed by replacing all the encoding region with the reporter gene of enhanced green fluorescent protein (eGFP), except the virus regulating sequences relating to replication, transcription and packing of virus genome. The reporter gene could be expressed after it was transfected into the HEp-2 cells infected with helper virus of NDV. This result indicated that the minigenome could be translated by the NDV NP, P and L proteins provided by helper virus. Furthermore, the support plasmids expressing NDV NP, P and L protein were constructed respectively and the function of these plasmids was identified using the minigenome. Additionally, the virus rescue system was optimized by changing the infection dose of the recombinant vaccinia virus expressing T7 RNA polymerase. The work mentioned above will accelerate greatly the rescue of NDV and other relative research.
A large proportion of the Newcastle disease virus (NDV) matrix (M) protein is found in the nuclei of infected chicken embryo cells. Kinetic analysis indicated that much of the M protein enters the nucleus early in infection, concentrating in discrete regions of the nucleus and remaining there throughout infection. The M protein was found in localized regions of the nuclei of a variety of cell lines infected with NDV. Immunostaining for both M protein and nucleolar antigens indicated that most of these regions represent nucleoli. Moreover, this nucleolar localization of the M protein was observed in chicken embryo cells infected with 11 different strains of NDV. Only the M protein of strain HP displayed a modified pattern, concentrating in the nucleolus early in infection but in the cytoplasm late in infection. M protein transiently expressed in COS-1 cells also localized to the nucleus and nucleolus, indicating that the M protein does not require other NDV proteins for this localization.
We have recently reported the X-ray crystal structure of a fragment of the fusion protein (F) of Newcastle disease virus (NDV). This work describes the methodology involved in the production and crystallization of that protein in recombinant form. The full-length cDNA of NDV-F was cloned and the ectodomain expressed in both CHO-K1 and Lec-3.2.8.1 cells. The recombinant protein, secreted as a single-chain polypeptide F0', was purified using a c-myc antibody affinity column followed by gel filtration chromatography. Electron microscopic imaging showed the F0' product to consist of unaggregated club-shaped particles. Trypsin treatment of F0' could be used to produce disulfide-linked F2 and F1' chains. However, imaging revealed extensive rosette-like aggregation of the trypsin-treated material, indicative of a conformational change. Only the non-trypsin-treated product was thus suitable for crystallization and two crystal forms were obtained, diffracting to ca. 3.5 and 4.0 A, respectively. Both crystal forms were used in the structure determination.
Effects of tumor stimulator cell modification by infection with Newcastle Disease Virus (NDV) are described as analysed in vitro in mixed lymphocyte tumor cell cultures (MLTC). Direct antitumor effects were seen with human melanoma or colon-carcinoma cells in a dose- and time-dependent manner when using live but not UV inactivated virus. When T cell stimulation was measured by [3H]-thymidine uptake, NDV infected tumor stimulator cells did not show an augmentation but rather an inhibitory effect in comparison to non-infected stimulator cells. Virus infected tumor stimulator cells were, however, capable of augmenting the induction of tumor specific cytotoxic T cells in MLTC-CML assays when using murine ESb lymphoma immune cells and syngeneic NDV modified ESb cells as stimulators. A CML stimulatory effect was also shown for NDV modified third party cells and thereof derived conditioned medium. These effects are most likely explained by interferon- which is induced in tumor cells by NDV infection and by interferon-á which is induced in responder cells when stimulated with NDV infected stimulator cells.
Differential effect of various metabolic inhibitors on the development of hemadsorption activity and virus formation in cells infected with Newcastle disease virus (NDV) was investigated. It was found that, in BHK-21 cells infected with NDV, cycloheximide did not prevent the development of hemadsorption activity, whereas protein synthesis and virus formation by the cell were rapidly inhibited by the drug. When the drug was added to the culture at 4.5 h after infection or later, hemadsorption activity of the cell continued to develop normally for about 1 h. Similar increase in hemadsorption activity was found in cells which were treated with anti-NDV serum (to neutralize their hemadsorption activity) and then washed and incubated with cycloheximide. However, when cells were treated with the drug early in the infection (1.5 or 3.0 h), they did not show any detectable hemadsorption reaction throughout the infection. In contrast to cycloheximide, iodoacetate added to the culture together with sodium azide inhibited completely both the development of hemadsorption activity and the formation of progeny virus. These results suggest that the change of cell surface to become hemadsorptive may depend upon the energy generating system but not upon de novo synthesis of protein, whereas production of infectious virus may require continuous synthesis of protein.
The role of cotranslational disulfide bond formation in the folding pathway of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was explored. Electrophoresis of pulse-labeled HN protein in the presence or absence of reducing agent showed that, characteristic of many glycoproteins, the nascent HN protein contains intramolecular disulfide bonds. As reported by Braakman et al. (EMBO J. 11, 1717-1722, 1992), incubation of cells in dithiothreitol (DTT) blocked the formation of these bonds. Removal of DTT after a pulse-label allowed for the subsequent formation of intramolecular disulfide bonds and folding of the molecule as assayed by the appearance of conformationally sensitive antigenic sites and by the formation of disulfide-linked dimers. However, the t1/2 for the formation of a conformationally sensitive antigenic site after synthesis in the presence of DTT was over twice that of the control. Furthermore, the order of appearance of the antigenic sites was different from the control, suggesting that inhibition of cotranslational disulfide bond formation altered the folding pathway of the protein. Similar results were obtained in a cell-free system containing membranes. The HN protein forced to form intramolecular disulfide bonds posttranslationally had no detectable neuraminidase or cell attachment activity, suggesting that the protein had an abnormal conformation.
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The Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein mediates attachment to cellular receptors. The fusion (F) protein promotes viral entry and spread. However, fusion is dependent on a virus-specific interaction between the two proteins that can be detected at the cell surface by a coimmunoprecipitation assay. A point mutation of I175E in the neuraminidase (NA) active site converts the HN of the Australia-Victoria isolate of the virus to a form that can interact with the F protein despite negligible receptor recognition and fusion-promoting activities. Thus, I175E-HN could represent a fusion intermediate in which HN and F are associated and primed for the promotion of fusion. Both the attachment and fusion-promoting activities of this mutant HN protein can be rescued either by NA activity contributed by another HN protein or by a set of four substitutions at the dimer interface. These substitutions were identified by the evaluation of chimeras composed of segments from HN proteins derived from two different NDV strains. These findings suggest that the I175E substitution converts HN to an F-interactive form, but it is one for which receptor binding is still required for fusion promotion. The data also indicate that the integrity of the HN dimer interface is critical to its receptor recognition activity.
A broiler chicken population was divergently selected for high or low early immune responses to Escherichia coli and to Newcastle disease virus (NDV) vaccines. Four selection cycles were performed in one replicate, and a single cycle in a second replicate. Selection was based on sire-family averages of a titer index (TI) calculated as the mean titer of antibodies produced by offspring vaccinated with either E. coli or NDV at 18 or 10 days of age, respectively. After the first selection cycle, TI of the early-high (EH) line were 22 and 38% greater than those of the early-low (EL) line in Replicates A and B, respectively. After four selection cycles, the average immune response to E. coli and NDV of Line EH exceeded that of Line EL by 68%. Viability was greater in the EH than in the EL line. Realized family heritabilities were .72 and .67 in Replicates A and B, respectively and the levels of response to the two antigens were not genetically correlated. The immune response of the EH line developed earlier than that in the EL line as shown by fewer nonresponders against E. coli and the higher response of this line against the two antigens at young ages. Mortality after challenge at 18 days of age and general mortality from hatching to 20 wk of age was lower in Line EH than in Line EL. Body weights at 7 wk were higher in EH than EL chicks.