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Latex immunoassay for rapid detection of Newcastle disease virus.

A rapid test has been developed based on the technique of latex immunoassay for the detection of Newcastle disease virus from suspected tissue suspensions. The latex particles were sensitised with globulins and were used for antigen detection. Of the 258 samples tested, 165 samples were positive by this kit which was compared for its efficacy with the standard OIE approved haemagglutination (HA) and haemagglutination inhibition (HI) tests. No significant difference (P > 0.05) was observed between the tests. The sensitivity and specificity of the developed test was 94.19% and 87.63% respectively.

Animals↗

Sequence of 2,617 nucleotides from the 3' end of Newcastle disease virus genome RNA and the predicted amino acid sequence of viral NP protein.

DNA fragments complementary to the Newcastle disease virus genome (strain D26) were cloned and sequenced. The sequence of 2,617 nucleotides from the 3' end of the genome was determined and an open reading frame (OP-1) consisting of 1,467 nucleotides, most likely encoding NP protein, was found in this region. This was followed by a second unfinished open reading frame (OP-2) of at least 729 nucleotides which continued beyond the 2,617th nucleotide. Another relatively short (312 nucleotides long) open reading frame (OP-2') was found overlapping with OP-2, but its significance is still unclear. The amino acid sequence deduced from the nucleotide sequence of OP-1 showed a moderate homology to that of the NP protein of Sendai virus in the central portion of the peptide. The leader sequence of 53 nucleotides was also identified. The 5' end of mRNAs synthesized in the infected cells was analyzed and found to be m7GpppA, suggesting that the transcription of viral mRNAs starts with A, but not with G residue.

Amino Acid Sequence↗

Repair replication of HeLa cell deoxyribonucleic acid in cells infected with Newcastle disease virus or mengovirus or treated with puromycin.

We examined repair replication of HeLa cell deoxyribonucleic acid (DNA) in cells infected with mengovirus or Newcastle disease virus or treated with puromycin. Cellular DNA was damaged by ultraviolet light and then pulse-labeled with (3)H-thymidine. Autoradiographic analysis of non-S-phase DNA synthesis (repair replication) showed that there was no inhibition of this process at a time when overall cellular DNA synthesis was severely inhibited by either virus infection or puromycin treatment.

Autoradiography↗

Persistent Newcastle disease virus infection in embryonic chicken tracheal organ cultures.

The persistent infection of embryonic chicken tracheal organ cultures with Newcastle disease virus (NDV) is described. Tracheal explants remained morphologically intact and were able to support the replication of NDV for 6 months. Peak titers of released virus occurred at 1 week postinfection, whereas maximal immunofluorescence was not observed until 30 days postinfection. The inoculum titer was not critical, and viral persistence resulted with either of two strains of NDV tested. Serum was not required in the medium for explant viability or to maintain the persistent infection. The presence of a contaminating virus morphologically resembling a leukovirus neither altered the course of infection nor affected the survivability of explants. Although interferon was not detected in the culture medium, persistently infected explants were resistant to heterologous viral challenge, and a similar resistant state could be induced in uninfected explants with exogenous interferon or ultraviolet light-inactivated NDV. No evidence was found to implicate antibody as a regulatory factor in the establishment or maintenance of persistence. The results from electron microscopy and immunofluorescence suggest the cells of the subepithelial connective tissue as the site of NDV persistence.

Animals↗

Isolation and characterization of mouse FM3A cell mutants which are devoid of Newcastle disease virus receptors.

A method was developed to select host cell mutants which did not permit the replication of Newcastle disease virus (NDV), and 14 isolates of NDV-nonpermissive mutants of mouse FM3A cells were obtained. All these isolates were judged to be deficient in NDV receptors, since their ability to adsorb 3H-labeled NDV virions was markedly decreased. They were tested for genetic complementation in pairs by cell fusion and shown to fall into a single recessive complementation group, which was designated as Had-1. Vesicular stomatitis virus was able to replicate in this mutant to produce infectious progeny, but the glycoprotein of the released virion was abnormal in size, suggesting a defective processing of the asparagine-linked carbohydrate chains in the mutant cell. The Had-1 mutant was resistant to wheat germ agglutinin, but sensitive to a Griffonia simplicifolia lectin, GS-II, which recognizes terminal N-acetylglucosamine residues. The altered sensitivity to these plant lectins compared with that of the parental FM3A cells indicates that sialylated sugar chains on the cell surface are almost absent from the Had-1 cells, thereby rendering the cells NDV receptor deficient.

Animals↗

Postoperative active specific immunization in colorectal cancer patients with virus-modified autologous tumor-cell vaccine. First clinical results with tumor-cell vaccines modified with live but avirulent Newcastle disease virus.

Sixteen patients with colorectal carcinoma Dukes' Stage B2, C, or D were treated with an autologous virus-modified tumor-cell vaccine after potential curative tumor resection (R0-Resection). An inoculum of 1 X 10(7) cells incubated with 32 hemagglutination units of nonirradiated Newcastle disease virus (NDV) was given intracutaneously up to four times at 10-day intervals. The delayed-type hypersensitivity (DTH) skin reaction was measured. The vaccination was well tolerated. In 11 of 16 patients an increasing reactivity against the vaccine was observed during the vaccination procedure. A challenge test using autologous tumor cells without NDV after the vaccination cycle revealed a specific antitumor sensibilization in 12 patients. The DTH response was not due to bacterial contamination or sensibility to the virus. Histologic examination of the vaccination site showed a dense infiltration of predominantly helper T-lymphocytes. We conclude that in most of the patients treated active, specific immunization led to a specific antitumor sensitivity.

Adult↗

Retroviral expressed hemagglutinin-neuraminidase protein protects chickens from Newcastle disease virus induced disease.

The hemagglutinin-neuraminidase (HN) gene and the phosphoprotein (P) gene of Newcastle disease virus (NDV) were inserted into a replication competent avian leukosis virus vector. The expression of the HN gene from this vector in chick embryo cells has been previously reported. The P gene is also expressed from this vector in chick embryo cells. The retroviruses were used to immunize 4-week-old chickens. Birds receiving the virus containing the HN gene developed low levels of serum HI titers and NDV neutralization titers. Upon challenge, all birds vaccinated with the HN gene containing virus were protected from disease but not viral infection and replication. In contrast, birds immunized with the P gene containing retrovirus developed more severe clinical signs of disease earlier than birds receiving no immunization or retrovirus alone. The results obtained with the HN gene may have potential application to reducing disease due to NDV genetically engineered vaccines.

Animals↗

Haemagglutination inhibition antibodies, rectal temperature and total protein of chickens infected with a local Nigerian isolate of velogenic Newcastle disease virus.

Changes in values of haemagglutination inhibition (HI) antibody titre, rectal temperature (RT) and total protein (TP) were determined for Shaver Brown chickens infected with Newcastle disease virus (NDV) Kudu 113. The infected chickens came down with Newcastle disease by day 3 post infection (PI). The major clinical signs were depression, greenish diarrhoea, paralysis of legs and wings, opisthotonus and torticolis. Mortality and morbidity were 52% and 1000%, respectively. There were haemorrhagic lesions in the wall of the intestine, proventricular mucosa and caecal tonsils. There were necrosis and mononuclear cell infiltration of the liver, kidney and spleen. There was a significant increase in daily mean HI antibody titres from days 3 to 9 PI. Similarly, significant rise in daily mean RTs were noticed in the infected chickens from days 1 to 13 PI. On the other hand, there was a decrease in daily mean TP concentrations of infected chickens, beginning from day 3 PI, and the lowest concentration of 2.60 +/- 0.15 g/dl was obtained by days 7 and 11 PI. The values of HI, RT and TP for the control chickens were relatively constant during the experiment. The correlation coefficient (r) between HI and RT was positive and highly significant (r = 0.725, p<0.001), while the relationship between HI and TP was negative but highly significant (r = -0.712, p<0.001). It was concluded that NDV Kudu 113 induced increases in values of HI and RT, which occurred concurrently with a decrease in TP concentrations of infected chickens.

Animals↗

Accidental persistent infection of cell lines by Newcastle disease virus, showing three unusual features--defective neuraminidase, temperature sensitivity and intranuclear inclusions.

A persistent, defective infection by an unknown strain of Newcastle disease virus (NDV) appeared accidentally in established lines of pig, ox and sheep kidney cells. Virus particles released from the persistently infected cells were not infectious and were deficient in neuraminidase activity. Synthesis of some of the virus-specified proteins in the persistently infected cells was temperature-sensitive. Co-cultivation of mixed populations of carrier cells and healthy chick embryo cells induced cell fusion with the formation of multinucleate heterokaryons and intra-nuclear inclusions. The development of inclusions in the chicken nuclei was not accompanied by 'rescue' of infectious NDV.

Animals↗

Methanol-precipitated, detergent-treated Newcastle disease virus as an agar-gel precipitation test antigen.

Methanol-precipitated, detergent-treated (Triton X-100) Newcastle disease virus (NDV) was found suitable as an agar gel precipitation test (AGPT) antigen. One hundred and twenty sera were tested by the haemagglutination-inhibition (HI) test and AGPT against NDV. There was a very significant increase in the proportion of AGPT positive samples with increase in HI titre. Hence AGPT can be recommended as a field based test for seromonitoring following vaccinations against ND where laboratory facilities are inadequate.

Agar↗

Effect of fusion protein cleavage site mutations on virulence of Newcastle disease virus: non-virulent cleavage site mutants revert to virulence after one passage in chicken brain.

Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence of the fusion (F0) protein cleavage site. Full-length NDV cDNA clone pNDFL was used to generate infectious NDV with defined mutations in the F0 cleavage site (RRQRR downward arrow L, GRQGR downward arrow F, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F). All the mutants were viable and the mutations were maintained after virus propagation in embryonated eggs. The mutants showed single-cell infections on chicken embryo fibroblasts, which suggested that they were non-virulent. However, virulence tests in 1-day-old chickens resulted in an intracerebral pathogenicity index (ICPI) between 0 and 1.3. Moreover, virulent virus was isolated from chickens that had died in the virulence tests. Subsequent sequence analysis showed that the mutants RRQRR downward arrow L, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F gave rise to the appearance of revertants containing the virulent cleavage site RRQ(K/R)R downward arrow F and an ICPI of 1.4 or higher. This indicated that reversion to virulence was caused by alteration of the amino acid sequence of the F0 cleavage site from a non-virulent to a virulent type. Furthermore, the ICPI of the revertants was higher than that of cDNA-derived strain NDFLtag, which has the same cleavage site, RRQRR downward arrow F (ICPI=1.3). NDFLtag(Pass), which was isolated from dead chickens after intracerebral inoculation of NDFLtag, also showed an increase in the ICPI from 1.3 to 1.5. This study proves that reversion to virulence occurs within non-virulent NDV populations and that the virulence may increase after one passage in chicken brain.

Animals↗

Apoptosis as a cause of death in chicken embryos inoculated with Newcastle disease virus.

Eleven-day-old chicken embryos were inoculated by the allantoic route with the GB strain of Newcastle disease virus (NDV). At 0, 24, 36 and 42 h post-inoculation (p.i.), the brain and heart tissues were harvested for DNA extraction, and the thymus and the brain were fixed and processed for light and electron microscopy. At 42 h p.i., most of the embryos had died; however, no histopathological changes could be seen in the embryos at any stage of infection. DNA extracted from the brain cells started showing fragmentation at 24 h p.i., and from the heart muscle cells at 36 h p.i. Electron microscopy of the brain and thymus showed condensation of the nuclear chromatin, apoptotic bodies, various forms of crescent formation and some evidence of necrosis. Fragmentation of cellular DNA, crescent formation and apoptotic bodies are the typical signs of cells undergoing apoptosis. We suggest that apoptosis of the heart and brain is probably a cause of death of chicken embryos in acute Newcastle disease infection.

Animals↗

Identification and characterization of peptides binding to newcastle disease virus by phage display.

Three individual peptide sequences, EVSHPKVG, WVTTSNQW, and SGGSNRSP, which have potentials to bind to Newcastle disease virus (NDV), were identified by the biopanning method using phage display technology. The binding specificities of these peptides presented on phages were confirmed by ELISA competition assay using chicken anti-NDV antiserum. The synthetic peptides designed based on these results partially neutralized the infection of NDV in vitro. The peptide-motives identified here have the potential to lead to the identification of novel molecules that inhibit the NDV infection independent of the immune system.

Amino Acid Sequence↗

Antibody response to Newcastle disease virus (NDV) of recombinant fowlpox virus (FPV) expressing a hemagglutinin-neuraminidase of NDV into chickens in the presence of antibody to NDV or FPV.

Antibody response of recombinant fowlpox virus (FPV) was studied in chickens inoculated with the virus in the presence or absence of antibodies against Newcastle disease virus (NDV) or FPV. In the case of NDV, high hemagglutination-inhibition titers to NDV were obtained when the antibody was present. No immune response to NDV was observed in the chickens previously vaccinated with FPV.

Animals↗