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Newcastle disease virus: the effect of monoclonal antibody in the overlay on virus penetration and the immunoselection of variants.

Monoclonal antibody to the haemagglutinin-neuraminidase or the fusion glycoprotein of Newcastle disease virus blocked virus penetration which otherwise occurred within 90 s of the temperature being increased from 4 degrees C to 37 degrees C. These antibodies regularly immunoselected variant plaques only when incorporated into the agarose overlay. Variant viruses then formed plaques whereas the growth of non-neutralized normal virus was suppressed.

Animals↗

Local immunity against Newcastle disease virus in the newly hatched chicken's respiratory tract.

Inoculation of 200 mean egg infectious doses (EID(50)) of lentogenic Newcastle disease virus strain B1 (NDV-B1) into the air sac of 4-day-old specific-pathogen-free chicks provided significant protection against challenge of the air sac with 100 chicken mean lethal doses (LD(50)) of velogenic NDV-H but no protection against reinfection when the challenge was by the eye. Conversely, inoculation of the eye with 200 EID(50) of NDV-B1 provided significant protection against challenge of the eye but not of the air sac with 100 chicken LD(50) of NDV-H. Birds that received both antiserum and intraocular immunization were subsequently protected against both eye and air-sac challenge. On the other hand, birds that received antiserum and air-sac immunization were protected only against air-sac challenge but not against ocular challenge. Low levels of passively administered antibody did not prevent infection of the eye or air sac but greatly reduced the mortality rate after inoculation of either the vaccine or the challenge viruses. Passively administered antibody also suppressed hemagglutination-inhibiting and virus-neutralizing antibody formation stimulated by air-sac infection but not antibody formation stimulated by ocular infection. These data are consistent with the hypothesis that local immunity is responsible for prevention of infection, since birds were immune to reinfection at one site and simultaneously susceptible at the other site of infection.

Air Sacs↗

[Characteristics of field isolates of Newcastle disease virus isolated in the course of outbreaks in the poultry plant in the Leningrad region in 2000].

A field isolate of Newcastle disease virus (NDV) was isolated in the Russko-Vysotskaya poultry farm, Leningrad region. Within four days after infection, the isolate caused 100% mortality in 60-day-old susceptible chickens. The HA titer of the allantoic fluid samples collected after one passage in SPF-chicken embryos was 1:512, and it reacted only with the NDV specific antiserum in HI test. Intracerebral pathogenicity index and mean embryo death time were 1.97 and 49 hours, respectively. The isolate has the amino acid sequence of the protease cleavage site of the fusion protein F0 (112R-R-Q-R-R-F117), which is similar to that in the velogenic strains of NDV. Therefore, it was concluded that the virus isolated in this work was an ethiological agent of the ND outbreak in this poultry farm.

Animals↗

Cleavage of mouse ribosomal RNAs by the endonuclease associated with Newcastle disease virus.

Mouse ribosomal ribonucleic acids (rRNAs) are specifically cleaved to polynucleotides of lower molecular mass by the endonuclease associated with Newcastle disease virus (NDV). The 28 S RNA yielded a fragment of about Mr = 1.7 X 10(5) which is resistant to the endonuclease. We assume that one molecule of 28 S RNA contains one such resistant region. 18 S RNA does not possess resistant regions of this character and it is cleaved by the endonuclease to smaller molecules.

Animals↗

Mutated form of the Newcastle disease virus hemagglutinin-neuraminidase interacts with the homologous fusion protein despite deficiencies in both receptor recognition and fusion promotion.

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.

Amino Acid Sequence↗

[Development of an enzyme immunoassay for the detection of antibodies from hens and mice against Newcastle disease virus].

The development of an enzyme-immuno-assay (EIA) for detection of avian and mouse antibodies against the Newcastle-Disease Virus is shown. The antigen preparation was the centre of the experiments. The basic assay programme and selected test steps were optimized. The established assay is part of the EIA-control system for a SPF (specific-pathogen-free)-flock and also the essential screening method for the production of anti-NDV-monoclonal antibodies.

Animals↗

Determination of organ tropism of Newcastle disease virus (strain I-2) by virus isolation and reverse transcription-polymerase chain reaction.

The vaccines I-2 and V4 are avirulent strains of Newcastle disease virus. Organ tropism of strain V4 has been determined and the virus has a predilection for the digestive tract. Tropism of strain I-2 has not yet been determined. The objective of this study was to determine the distribution of strain I-2 in various body organs and fluids following vaccination in comparison with V4. Four-week-old chickens were vaccinated by eye drop separately with these two avirulent strains. Virus isolation and the reverse transcription-polymerase chain reaction technique were employed to detect I-2 and V4 viruses in various tissues and body fluids for 7 days following vaccination. Tissues from the respiratory tract showed earlier positive signals than tissues from other organs for chickens vaccinated with strain I-2. Conversely, tissues from mainly digestive tract produced earlier positive signals than from respiratory tract and other organs from chickens vaccinated with strain V4. In early infection, strain I-2 had preferential predilection for the respiratory tract and strain V4 for the digestive tract. Later after vaccination, other organs showed positive results from chickens vaccinated with both I-2 and V4 strains. The differences in organ tropism observed in this study suggest that I-2 may perform better than V4 as a live vaccine strain.

Animals↗

Temperature-sensitive and other mutants of the Essex 70 strain of Newcastle disease virus as vaccines.

The most reliable method of distinguishing strains of low virulence amongst a collection of stock strains of Newcastle disease virus (NDV) was by their failure to produce a good c.p.e. in monolayers of baby hamster kidney cells and of chick embryo cells. This method was of no help in identifying avirulent mutants that emerged in the Essex 70 strain of NDV following ultraviolet light, nitrous acid, hydroxylamine or N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment. A marked reduction in the ability to kill developing chick embryos at 41 degrees C was a much more reliable indicator. Several of these temperature-sensitive mutants, most of which had been isolated from NTG-treated virus, were non-lethal for young chicks but they did have a depressive effect on their growth rate. The immunity produced by three of these mutants in chicks free of NDV antibody, but not in chicks possessing appreciable amounts of antibody, was probably even better than that produced by Hitchner B1 strain. All three mutants reverted to virulence during passage in chicks, although in no case were the revertants as virulent as the original Essex 70 strain. The virulent revertants obtained from one of the mutants had lost their temperature-sensitivity and proliferated in large numbers in the tissues of infected chicks. Those obtained from the other two had either not lost, or only partly lost, their temperature-sensitivity; they were found only in low concentrations in the tissues of infected chicks, their concentrations being little different from that found in the tissues of chicks infected with the mutants from which they were derived.

Animals↗

Cell-free coupling of Newcastle disease virus RNA transcription, translation and Co-translational processing.

A cell-free coupled system for transcription, translation and glycoprotein processing of the Newcastle disease virus genome is described. The system consists of a rabbit reticulocyte lysate preincubated with micrococcal nuclease and of detergent-disrupted purified Newcastle disease virions. [35S]methionine incorporation was linear for 2 h. Polypeptides NP and M, the presumably unglycosylated analogues of glycoproteins HN and possibly F, were identified as translation products. When in vitro synthesis was carried out in the presence of dog pancreas microsomes the HN analogue (pre-HN) was converted to an 80K (approx.) protein which comigrated on polyacrylamide gels with HN synthesized in vivo and which, except for a small fragment, was protected from proteolytic degradation. In immunoprecipitation studies, antiserum against HN purified from virions reacted with both the processed and the unprocessed form of HN synthesized in vitro.

Animals↗

[Thermostability of granulated viral vaccines against avian Newcastle disease virus].

Stability infectivity of viral vaccines against Newcastle disease (ND) from strains La-Sota and Bor-74 VGNKI, prepared by using the method of granulation and spray-coating in fluidizated bed, was studied during the storage of vaccines at different temperatures. The infectious activity of the vaccines was found to remain stable for 6 months at 4 and 8 degrees C and for 2 weeks at (25 +/- 1) degree C. An analysis of the findings showed that an improved content of the preparation contributed to an essentially increased ND virus thermal stability in the granulated lactose-containing vaccine.

Animals↗

Novel peptides that inhibit the propagation of Newcastle disease virus.

A disulfide constrained random heptapeptide library displayed on filamentous bacteriophage M13 was applied to select specific ligands that interact with Newcastle disease virus (NDV). A fusion phage carrying the amino acid sequence TLTTKLY was selected from the panning procedure. An antibody competition assay showed that the selected phage was capable of competing with the polyclonal antibodies raised against NDV for binding sites on the virus. Determination of the binding affinity of this phage with NDV by an equilibrium binding assay in solution revealed two different dissociation constants, suggesting that there could be two distinct binding sites for the phage on NDV. Synthetic peptides with the sequence CTLTTKLYC, either in linear or cyclic conformations inhibited the binding of phage bearing the same sequence to NDV. These peptides also inhibited the hemolytic activity of the virus as well as its propagation in embryonated chicken eggs.

Animals↗

Molecular characterization of the nucleocapsid protein gene of Newcastle disease virus strains in Japan and development of a restriction enzyme-based rapid identifying method.

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.

Amino Acid Sequence↗

Complete nucleotide sequence of Newcastle disease virus: evidence for the existence of a new genus within the subfamily Paramyxovirinae.

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.

Base Sequence↗

Molecular cloning and nucleotide sequence of P, M and F genes of Newcastle disease virus avirulent strain D26.

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.

Base Sequence↗

Nuclear entry and nucleolar localization of the Newcastle disease virus (NDV) matrix protein occur early in infection and do not require other NDV proteins.

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.

Animals↗

Molecular evolution of the Newcastle disease virus matrix protein gene and phylogenetic relationships among the paramyxoviridae.

Matrix (M) gene sequences for recent field isolates and older reference Newcastle disease viruses (NDV) were examined to determine phylogenetic relationships and population trends among these viruses. Overall, the M gene has a majority of synonymous nucleotide sequence substitutions occurring among NDV isolates. However, several predicted amino acid changes in the M protein of specific NDV isolates have occurred that correlate to phylogenetic relationships. Nucleotide substitutions in these codons have a greater number of nonsynonymous base changes. The NDV isolates arising since the 1970s belong to a population of viruses that expanded worldwide at an exponential rate. These viruses may have their origins in free-living birds, are present worldwide, and continue to circulate causing disease in poultry. A specific NDV lineage composed of virulent isolates obtained in the US prior to 1970 appears to no longer exists among free-living birds or commercial poultry. However, "vaccine-like" viruses are common in the US and continue to circulate among commercial poultry. Based on M protein amino acid sequences, NDV separates as a clade most closely related to morbilliviruses and not with their current designated category, the rubulaviruses among the Paramyxoviridae. Consequently, avian paramyxoviruses should have their own taxonomic subfamily among the Paramyxovirinae.

Animals↗