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An outbreak with mortality and acute nervous signs in ducks is reported. A haemagglutinating virus was recovered from dead and sick ducks that was neutralised by Newcastle disease antiserum. The virus was a thermostable paramyxovirus that was classified as a mesogenic Newcastle disease virus. The virus induced the infection in young ducklings following intramuscular inoculation.
A region constituting 88% of the matrix gene of Newcastle disease virus vaccine strains LaSota and B-1 was amplified by reverse transcription-polymerase chain reaction. Amplified products of LaSota and B-1 strains derived from vaccine serials of different companies were digested with restriction enzymes MboI and HinfI. Strain characteristic cleavage site maps were obtained that allowed for a reliable and rapid differentiation between strains LaSota and B-1.
Two kinds of RNA capable of producing interferon in a heterologous cell system occur in the cells producing interferon in response to induction by Newcastle disease virus. One of them has messenger activity for interferon and is presented by a single-stranded structure with sedimentation constant of 10-22 S. This RNA appears to belong to cellular messenger RNA. The other is capable of inducing interferon production by heterogenous cells, resistant to treatment with RN-ase and has sedimentation constant greater than 35 S. In our opinion, this RNA is the primary replicative RNA of the virus-inducer.
Cell cultures and chicken embryos were treated with maximum tolerant concentrations of different compounds and infected with Newcastle disease virus simultaneously or 24 hours after the compounds were introduced. The similar results were obtained in both cases. It was found that Reglone inhibited Carbaryl increased virus multiplication. The study on dynamics of virus multiplication indicates that only in the case of IPO and Carbaryl their stimulatory effect on virus at the final stage was preceded by its inhibition.
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A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR downward arrowL) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR downward arrowF). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.
Monoclonal antibodies were used to isolate nonneutralizable antigenic variants in the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus. It had been found that a large percentage of virus retains infectivity despite binding neutralizing antibody. This high persistent fraction of nonneutralized virus precluded the isolation of variants by the standard treatment with antibody alone. Rabbit anti-mouse immunoglobulin was used to reduce the percentage of virus that remains infectious despite the presence of bound antibody. This procedure made possible the isolation of variants of two distinct types: classical variants, not neutralized because they do not bind the antibody used to select them; and unique variants that, although still capable of binding the selecting antibody, are only slightly neutralized. The general applicability of this method for the isolation of antigenic variants in nonneutralizing epitopes is also discussed.
We have evaluated a diagnostic system based on the loop-mediated isothermal amplification (LAMP) assay for the rapid, simple, and sensitive detection of Newcastle disease virus (NDV) directly from culture isolates as well as clinical samples. By using one set of specific primers targeting the fusion protein gene, the LAMP assay rapidly amplified the target gene within 2 h, requiring only a regular laboratory water bath or heat block for reaction. The results obtained from testing the genomes of 38 NDV strains, other different viruses, and clinical samples of experimentally infected chickens showed that LAMP was as sensitive and specific as nested PCR. All LAMP-positive samples were positive by nested PCR. The LAMP assay is faster than nested PCR, cost-effective, and easy to perform. Our results clearly demonstrate that the LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV infection.
Uncooked white rice and cooked white rice were tested as vehicles for the V4 strain of oral Newcastle disease vaccine. The results of feeding experiments were evaluated by the measurement of haemagglutination inhibition antibodies against Newcastle disease virus. Little of the virus applied to uncooked white rice could be recovered, even immediately after mixing, whereas when the virus was applied to cooked white rice most of it could be recovered. In 4 separate experiments, chickens failed to respond serologically to vaccine supplied on uncooked white rice. In all of 4 experiments with cooked white rice, there were serological responses in vaccinated chickens, from 45% to 100% of the chickens developing titres sufficiently high to indicate protection against challenge with virulent virus. Development of haemagglutination inhibition antibodies in some control chickens indicated the ability of the vaccine virus for lateral spread or persistence in the environment.
The amino acid sequence at the F2/F1 cleavage site of the F0 fusion protein of 17 strains of Newcastle disease virus (NDV) was deduced from sequencing a 32 nucleotide area of the genome by reverse transcription and polymerase chain reaction (PCR) techniques. With the addition of sequences at the same area previously published for 9 other viruses comparisons were made of a total of 26 NDV strains and isolates (11 of low virulence, 15 of high virulence or mesogenic) covering ten antigenic groups determined by reactions with monoclonal antibodies. All the virulent viruses and the mesogenic strain Komarov showed the amino acid sequence 112R/K-R-Q-K/R-R116 for the C-terminus of the F2 protein and phenylalanine (F) at the N-terminus of the F1 protein, residue 117. The mesogenic isolate of the antigenic variant NDV responsible for the recent panzootic in racing pigeons, often termed "pigeon paramyxovirus type 1", examined in this study had the sequence 112G-R-Q-K-R-F117. The deduced amino acid sequence in the corresponding region of all viruses of low virulence was 112G/E-K/R-Q-G/E-R-L117. The virulent virus, PMV-1/chicken/Ireland/34/90 (34/90), which had a close antigenic relationship to a group of avirulent viruses, three of which were examined in the present study as representatives of the monoclonal antibody group H, showed between 4-6 nucleotide differences from these viruses in the 32 nucleotide region studied. These resulted in differences in the deduced amino acid sequence at residue 112 E-->K, 115 E-->K and 117-->F, giving 34/90 a typical virulent virus motif at the cleavage site. Despite the extremely small portion of the genome studied there were several areas which appeared characteristic for 34/90 and the three group H viruses of low virulence, which suggests that they may have arisen from the same gene pool.
Hybridisation of PCR fragments with fluorogenic probes specific for pathotype allowed an estimation of pathogenicity of Newcastle disease virus (NDV) isolates using a modified TaqMan procedure. Six probes were used, designed to recognise nucleotide sequences in the fusion protein gene sequence corresponding to the precursor protein F0 cleavage site of both virulent and avirulent viruses. Forty-three of the 45 isolates tested, including 18 examined in a blind study were pathotyped successfully and rapidly, with close correlation between cleavage site nucleotide sequences, TaqMan results and intracerebral pathogenicity index (ICPI) values. One isolate, which could not be pathotyped by nucleotide sequencing, was shown using the TaqMan system to be a mixture of virulent and avirulent NDV. The results of this study suggest that using this modified TaqMan protocol, the likely virulence of most ND isolates can be determined rapidly and reproducibly.
A recombinant baculovirus containing a cDNA clone encoding the nucleocapsid (NP) protein of Newcastle disease virus (strain Ulster 2C) has been used to infect insect cells (Spodoptera frugiperda). High levels of overexpressed NP protein were observed, comprising up to 40% of total cellular protein, which were subsequently shown to be antigenic. Nucleoprotein derived from the crude soluble lysate of infected insect cells has been used in an indirect ELISA to detect the presence of anti-NDV antibodies in a cohort of chicken sera. Data produced from these tests indicated a good correlation between ELISA titre and haemagglutination inhibition test data. The test was not affected by interference from background cellular proteins nor by cross-reactivity with non-NDV poultry pathogens. Additionally, the test did not generate false-positive readings.
The progression of tracheal lesions induced by vaccination of day-old specific pathogen-free chicks with the La Sota strain of Newcastle disease virus (NDV) was examined by relating surface changes as observed by scanning electron microscopy with subcellular changes seen by transmission electron microscopy. NDV infection resulted in hypertrophy of goblet cells, their rupture, and the formation of excess mucus. Activation of goblet cells peaked within 4 days postvaccination. Afterward, the activation levels gradually decreased. At the level of the ciliated cells, a marked increase in the proportion of nonciliated to ciliated cells and later an almost complete deciliation of the tracheal surface were observed because a simple squamous to cuboidal epithelium replaced the original pseudostratified epithelium. Fifteen days postvaccination, all epithelial damage was restored. Because the observed vaccination-induced lesions are detrimental to epithelial integrity and function as a barrier against invading microorganisms, they might explain at the ultrastructural level the secondary complications of vaccination with the La Sota strain against NDV.
The transcriptive complex, the nucleocapsid with the viral RNA-synthesizing activity, of Newcastle disease virus (NDV) contains three protein components, the major structural subunit (NP) and two associated proteins (P and L) involved in the RNA synthesis. We studied the pathway of these proteins from synthesis to assembly into the complex by pulse-chase labeling of infected cells followed by detergent extraction of the cells to separate soluble and cytoskeletal fractions. Most molecules of NP and P (and probably L) became associated with the cytoskeletal framework immediately after their synthesis. Most of the remaining molecules were initially found in the soluble fraction, but joined the cytoskeletal framework within several minutes. Once attached, none of the proteins left the cytoskeleton, and it was here that they assembled with 50 S viral RNA into nucleocapsids. The nucleocapsids thus formed remained bound to the cytoskeletal framework and were found by in situ autoradiography to exhibit viral RNA synthesis on the framework. These results suggested that the cytoskeletal framework could actively participate in the structural and functional assembly of NDV transcriptive complex.
Sequence analysis and comparison of the fusion glycoprotein genes of 11 Newcastle disease virus (NDV) isolates indicated a high degree of functional and structural constraint exerted on the change of the glycoprotein. However, synonymous nucleotide substitutions occurred frequently throughout the coding region. Facilitated by an analysis of synonymous difference (Ks) in pairwise strain comparison, we defined the branching orders of the strains and identified three distinct evolutionary lineages correlating with the virulence as expressed by mean death time (MDT) for chick embryo. The typically virulent strains with MDT of about 50 hr were associated with one lineage, while the typically nonvirulent strains with MDT of infinity were of another lineage. The third lineage consisted of both virulent and avirulent strains whose MDTs lay on a continuum from 50 to 120 hr. Synonymous substitutions were found to occur with almost the same rates in the adjacent hemagglutinin-neuraminidase and membrane protein genes as in the fusion protein gene, and the branching orders based upon the Ks for these genes were essentially identical to those derived from the fusion protein gene. Therefore, no gene exchange by recombination seems to have occurred to generate the strains of distinct lineages. Rather, the different strains appear to have evolved through various degrees of accumulation of point mutations. Besides these evolutionary features, the present study strongly supports the importance of the previously identified signals for gene expression and for the proteolytic activation of the gene product.
The effect of artificial air-ionization on air-borne transmission of Newcastle disease virus (NDV) infection in chickens was studied in an isolated system consisting of two side-by-side cages with solid walls and a wire-gauze roof. During a 3-week observation period more than 90% of the uninoculated indicator chickens, housed in one of the cages, contracted the virus shed to the air by the NDV-inoculated, diseased birds in the neighbouring cage. This air-borne transmission of NDV was completely prevented by increasing the ion concentration in the test room by a constant negative corona discharge above the wire-gauze roof. On the other hand, spreading of the infection within a group of chickens housed in a single cage was not affected by air ionization. These and other results suggest that artificial air-ionization may protect animals from certain air-borne infections by interfering with microbial aerosol formation and/or by facilitating their decay.
A panel of monoclonal antibodies raised against the hemagglutinin-neuraminidase glycoprotein (HN) of the Australia-Victoria strain of Newcastle disease virus has been used to compare that strain and eight other strains of the virus. The ability of the antibodies to neutralize infectivity, inhibit hemagglutination and neuraminidase, and bind to purified virions in solid-phase radioimmunoassays was determined for each strain. Of the four antigenic sites delineated by these antibodies on the HN of the homologous strain, site 1 (that with the greatest neutralizing susceptibility), is apparently conserved in all the strains tested as revealed by neutralization assays. The least neutralizing site, number 4, is also conserved in most of the strains tested. Site 2, which lies at or near the neuraminidase site, appears to be conserved in the avirulent strains but not in the virulent strains. An antibody to site 3 is unable to bind to a significant extent to any of the heterologous strains tested, and thus recognizes a strain-specific epitope. Inhibition of hemagglutination and neuraminidase by antibodies to each site were also examined and the results suggest that antibodies to sites 1 and 2 may distinguish virulent and avirulent strains at least with respect to these functions.
In chick embryo fibroblasts and in mouse fibroblasts of established L line, grown in the presence of IDU, the multiplication of Newcastle Disease virus was better and the CPE more distinct than in control cell cultures. In calf kidney cells only the CPE was enhanced. The most pronounced influence of IDU on virus multiplication was found after adding the analogue in maximum tolerated dose to the medium at the moment of cell culture preparation. The growth curve of virus in chick embryo fibroblasts grown in the presence of IDU shows the most distinct influence of this analogue with regard to LaSota strain. Already two days after inoculation with a smaller dose the titer was 2 log higher than in control cultures and the maximum difference of 3 log was found on the 5th day. In reference to Roakin strain the influence of IDU was weaker--maximum difference of 1,2 log on the fifth day after inoculation. Also the difference between the titers of virus in L cells grown in the presence of IDU and in the control ones, inoculated with LaSota strain was greater (about 1,5 lg during 2-8 days) than in the case of Roakin strain. In chick embryos treated with IDU intraallantoically at 24 and 48 hours before the inoculation with LaSota strain (only this was used) the enhancement of virus multiplication was much poorer than in chick embryo fibroblasts.