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Precursor protein for Newcastle disease virus.

The course of viral protein synthesis during infection of chicken embryo fibroblasts with Newcastle disease virus (NDV) L. Kansas has been followed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Of the three major virion polypeptide molecular weight classes, I (78,400 daltons), II (53,500 daltons), and III (37,600 daltons), only II, having the same electrophoretic mobility as nucleocapsid polypeptide, appears to be the cleavage product of a precursor polypeptide PII (64,800 daltons) detected in NDV-infected cells after brief labeling with radioactive amino acids. Nucleocapsids were isolated from NDV-infected cells which had been pulse-labeled with radioactive amino acids or pulse-labeled and further incubated with unlabeled amino acids. Gel electrophoretic analysis of proteins derived from nucleocapsids showed that an increase in the period of incubation with unlabeled amino acids resulted in an increase in the amount of radioactivity in nucleocapsid protein. Polypeptide PII was not detected as a transient component of the isolated nucleocapsid fraction. These results are consistent with two interpretations. The product of PII cleavage is (i) nucleocapsid polypeptide, or (ii) a nonvirion or minor envelope polypeptide having the same electrophoretic mobility as nucleocapsid polypeptide.

Animals↗

Topological and operational delineation of antigenic sites on the HN glycoprotein of Newcastle disease virus and their structural requirements.

Monoclonal antibodies to the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus have identified four antigenic sites on the glycoprotein, which are topologically and operationally discriminated from one another. To define the metabolisms and cellular compartments required for formation of the individual antigenic sites, a panel of monoclonal antibodies were examined for their reactivity with the nascent and variously processed forms of the antigen molecules in combination with the use of inhibitors of glycosylation (tunicamycin and N-methyl-1-deoxynojirimycin) and glycoprotein transport (carbonyl cyanide m-chlorophenylhydrazone and monensin). Reactivity was also examined with the antigen molecules deglycosylated by endoglycosidase F and with the antigen molecules reduced by 2-mercaptoethanol. The results taken together suggest that posttranslational organization of the glycoprotein is important for all four of the antigenic sites. At the same time, there appeared to be marked site-specific requirements with respect to glycosylation and disulfide bond formation. However, all of these metabolic requirements were found to be provided within the rough endoplasmic reticulum, and no further processing of the antigen molecules appeared to be necessary for the formation of any of the antigenic sites.

Amino Acid Sequence↗

Efficacy in chickens of a herpesvirus of turkeys recombinant vaccine containing the fusion gene of Newcastle disease virus: onset of protection and effect of maternal antibodies.

The onset of protection against Newcastle disease and the effect of maternal antibodies to Newcastle disease virus (NDV) and Marek's disease virus (MDV) on vaccine efficacy were determined following vaccination of chickens with a recombinant herpesvirus of turkeys (HVT) vaccine expressing the fusion (F) glycoprotein gene of NDV. Onset of protection following intra-abdominal administration of the recombinant HVT/F vaccine at 1 day of age and subsequent ocular challenge with the neurotropic velogenic Texas GB strain of NDV was determined to occur between days 14 and 21 post-vaccination (PV). Vaccination with the Hitchner B1 strain of NDV resulted in protection by day 6 PV, and vaccination with an inactivated NDV oil-emulsion vaccine induced protection by day 14 PV. One-day-old broiler-type chickens with maternal antibodies to both NDV and MDV and 1-day-old specific-pathogen-free (SPF) white leghorn chickens lacking maternal antibodies were vaccinated with the recombinant HVT/F vaccine or with control vaccines, challenged intra-abdominally with the very virulent RB1B strain of MDV on day 8 PV, and challenged with the Texas GB strain of NDV on day 29 PV. The HVT/F and NDV strain Hitchner B1 vaccines provided 73% and 80% protection, respectively, against NDV in broilers, whereas both vaccines resulted in 100% protection in SPF leghorns.

Animals↗

Variability of antigenic epitopes of the fusion protein of Newcastle disease virus.

Using a panel of 10 monoclonal antibodies (Mab) against fusion (F) protein of Newcastle disease virus (NDV), strain Australia-Victoria, three non-overlapping antigenic sites (F1, F2 and F3) and one site partially overlapping with the sites F1 and F2 (F1.2) have been identified. The sites F2 and F3 are clusters that each include four antigenic epitopes. The antigenic stability of the above epitopes was estimated by comparison of the binding capacity of the corresponding Mabs towards 63 NDV strains isolated in different years and places from various avian species. The results demonstrated high variability of the site F1.2 and of all the four epitopes of the site F2. At the same time, the only epitope of the site F1 can be defined as highly conservative: the corresponding Mab gave positive binding with 60 from 63 NDV strains, one from the four epitopes pertaining to site F3 was the most conservative--the corresponding Mab reacted with all the 63 strains used in the studies, while the other three Mabs showed rather low stability--the corresponding Mabs reacted with 34-39 NDV strains. Thus, as opposed to the published data asserting antigenic stability of the F protein contrary to the high variability of the haemagglutinin-neuraminidase (HN) protein, our results have revealed a number of variable epitopes on the F protein. This demonstrates an evolutionary changeability of the F protein, which is of importance from the theoretical (viral antigenic evolution) as well as practical point of view.

Antibodies, Monoclonal↗

Interferon production by inactivated Newcastle disease virus in cell cultures and in mice.

Youngner, Julius S. (University of Pittsburgh, Pittsburgh, Pa.), Anne W. Scott, Jules V. Hallum, and Warren R. Stinebring. Interferon production by inactivated Newcastle disease virus in cell cultures and in mice. J. Bacteriol. 92:862-868. 1966.-A comparison was made of the effects of ultraviolet (UV) irradiation or heating at 56 C on the interferon-stimulating capacity of Newcastle disease virus in primary chick embryo fibroblast (CE) cultures, in L-cell cultures, and in the intact mouse. The data obtained indicated the critical importance of the host cell system used for interferon production. Virus inactivated by UV irradiation, as well as infective virus, was an effective stimulus of interferon production in L cells and in mice, and this property persisted on continued irradiation. In contrast, in CE cell cultures, infective virus produced no interferon, whereas UV-irradiated virus produced maximal interferon titers when all infectivity was destroyed; continued irradiation resulted in a rapid loss of the interferon-stimulating capacity of the virus. Virus inactivated at 56 C did not produce interferon in CE or L-cell cultures. In the intact mouse, on the other hand, heat-inactivated virus was capable of stimulating the release of significant levels of circulating interferon.

Animals↗

Comparison of Newcastle disease viruses isolated from cormorants in Canada and the USA in 1975, 1990 and 1992.

Seventeen Newcastle disease virus (NDV) isolates obtained from cormorants, turkeys, a pelican, and a gull in Canada and the USA collected in 1975, 1990 and 1992 were analyzed for relatedness by monoclonal antibody profiling. In addition, nucleotide sequence analysis was performed in two areas of the fusion (F) gene for 5 of the isolates. No difference in the antigenicity of these 17 viruses, as determined by monoclonal antibody binding patterns, was seen. The amino acid sequences obtained via nucleotide sequencing at the cleavage site of the F protein showed that all the isolates tested had two pairs of basic amino acids immediately upstream of the cleavage site, and a phenylalanine residue at the N-terminus of the F1 protein, which is consistent with velogenic NDV. The deduced amino acid sequence obtained at the cleavage site of the F protein from 6 of the isolates was virtually identical regardless of the species, year of isolation, or location. However, the 1975 cormorant isolate showed marked differences from the 1990-1992 isolates in the nucleotide and deduced amino acid sequence of the F gene signal region. These data indicate that the 1990 and 1992 outbreaks were caused by the same epizootic virus and further suggest that the population of NDV in these wild birds may be very stable. The belief that the velogenic NDV circulating in cormorants in 1992 was transmitted into the free-ranging turkey flocks located near the cormorants in North Dakota is supported by the present study in which no distinction could be made between the viruses isolated from turkeys or wild birds.

Amino Acid Sequence↗

A preliminary study of the role of ducks in the transmission of Newcastle disease virus to in-contact rural free-range chickens.

The role of ducks in the transmission of Newcastle disease virus (NDV) to free-range village chicken was investigated experimentally. Newcastle disease (ND) seronegative ducklings reared in a pen were infected oronasally with velogenic NDV of intracerebral pathogenicity index (ICPI) 1.8 isolated from outbreaks in village chickens in Uganda. A first group of 3-week-old ND seronegative chicks was mixed with the ducks and they were kept together for 7 days. Both ducks and chicks were observed for ND clinical signs and any mortality, and they were bled and their sera were tested for ND antibodies by haemagglutination inhibition (HI) test. The chicks were removed, euthanized and examined for any ND lesions, while the ducks were transferred to a fresh pen and a second group of chicks was introduced and observed and treated as above. The ducks and the chicks tested positive for ND antibodies 7 days post infection and contact, respectively, but showed no clinical signs, post-mortem lesions or mortality. The mean ND antibody titre of the second group of chicks was lower than for the first group. This study has shown that although ducks can be infected with velogenic NDV, they do not show clinical signs but are able to transmit NDV to in-contact chicks. Further investigations are needed of the lack of clinical signs in the in-contact chicks and how long the ducks remain infective.

Animals↗

Elevated virulence of Newcastle disease virus strains following serial passages in kidney cells in vitro.

Serial passage of two lentogenic Newcastle disease virus (NDV) strains in kidney cell lines increased virulence and changed viral biological properties. In two cell lines (BHK and MDBK), elevation in virulence was demonstrated by plaque formation under an overlay with no additives, decrease in mean death times, elevated intracerebral pathogenicity index, and cytopathic effect in chicken embryo fibroblasts. Some other markers not directly correlated to virulence, such as heat inactivation of hemagglutinin and neuraminidase, were not influenced by passage in kidney cells. In addition, all strains were slow eluters. This observation emphasizes the importance of preventing the virus from reaching the viscera.

Animals↗

Effect of butylated hydroxytoluene on Newcastle disease virus.

A study was done to examine the effects of butylated hydroxytoluene (BHT) on purified Newcastle disease virus (NDV). Treatment of the virus with 50 microgram of BHT/ml caused 92% inactivation of the virion infectivity. Virion adsorption to chicken-embryonated cells was inhibited 32% and synthesis of intracellular hemagglutinin was inhibited 29%. Electron microscopy of the BHT-treated virions revealed virion envelope damage. Chicken-embryonated cells treated with 25 microgram of BHT/ml before NDV infection demonstrated 65% inhibition of NDV progeny production.

Butylated Hydroxytoluene↗

Enhancement of haemolysis by Newcastle disease virus (NDV) after pre-treatment with heterophile antibody and complement.

Pre-treatment of Newcastle disease virus (NDV) with fresh human plasma enhances its haemolytic (HL) capacity by several factors. The effect is due to complement activation by the heterophile anti-chick antibody present in human plasma. All the adult human plasmas tested were effective, also 91/100 human cord blood sera. The antibody was mainly of the IgM class. The enhanced HL was due to integration and transference of the complement 'holed' virus envelope membrane and subsequent leakage of haemoglobin. High concentration of activated complement destroys the integrity of the virus enevelope. Treatment of chick erythrocytes and fibroblasts with human plasma also produced lysis of the cells.

Animals↗

Comparison of serological tests for antibodies against Newcastle disease virus and infectious bronchitis virus using ImmunoComb solid-phase immunoassay, a commercial enzyme-linked immunosorbent assay, and the hemagglutination-inhibition assay.

COMBSCORES determined using the ImmunoComb solid-phase immunoassay were compared with hemagglutination-inhibition (HI) titers specific for Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) and with mean enzyme-linked immunosorbent assay (ELISA) titers determined using Agritech Systems, Inc., ELISA. COMBSCORES for NDV and IBV increased proportionately in a stepwise manner as HI titers increased. The ImmunoComb solid-phase immunoassay was ablt to produce endpoint titers on sera with NDV-HI titers of 0 through 320 and IBV-HI titers of 0 through 1024 without reaching the maximum S-value. The ImmunoComb showed good correlation with the HI assay and the Agritech ELISA and should prove to be a useful tool for serological profiling, either alone or in conjunction with the HI test or commercial ELISA.

Animals↗

Newcastle disease virus contains a linkage-specific glycoprotein sialidase. Application to the localization of sialic acid residues in N-linked oligosaccharides of alpha 1-acid glycoprotein.

Newcastle disease virus sialidase was found to exhibit strict specificity for hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage contained in glycoprotein oligosaccharides both N-linked to asparagine and O-linked to threonine or serine under conditions that left oligosaccharides containing the NeuAc alpha 2 leads to 2 leads to 6Gal and NeuAc alpha 2 leads to 6GallNAc linkages intact. This was determined, in part, by examining the viral sialidase for its ability to hydrolyze glycoprotein oligosaccharides derivatized with purified sialyltransferases to contain the [14C]NeuAc alpha 2 leads to 3Gal, [14C]NeuAc alpha 2 leads to 6GalNAc, and [14C]NeuAc alpha 2 leads to 6Gal linkages. The viral sialidase was also tested for hydrolysis of the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages on the N-linked oligosaccharides of alpha 1-acid glycoprotein. Selective hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage was shown by periodate oxidation and by 500-MHz 1H-NMR spectroscopy of native and sialidase-treated glycopeptides. The NMR spectra, together with composition data, further indicated that the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages were localized to specific branches of the major tri- and tetraantennary oligosaccharides of alpha 1-acid glycoprotein. The results indicate that the Newcastle disease virus sialidase can initiate the selective degradation of N-linked oligosaccharide branches containing the NeuAc alpha 2 leads to 3Gal linkage.

Carbohydrate Conformation↗

Interactions between viscerotropic velogenic Newcastle diseases virus and pet birds of six species. I. Clinical and serologic responses, and viral excretion.

Clinical and serologic responses to a psittacine isolate of viscerotropic velogenic Newcastle disease virus (VVNDV) were evaluated in pet birds of six species: budgerigar, yellow-headed Amazon parrot, halfmoon conure, lesser hill mynah, black-headed nun, canary. The clinical response was most marked in the budgerigars, parrots, and conures, and only minimal in the nuns. Between post-exposure days (PED) 3 and 5 some birds developed ruffled plumage, conjunctivitis, and central nervous system dysfunction: ataxia, wing tremors, paralysis of the extremities, and tremors of the head accompanied by nodding and jerking. Mortality by PED 203 was 55% (29/52) in the halfmoon conures, 22% (23/105) in budgerigars, 29% (12/42) in parrots, and 21% (15/71) in nuns. The only clinical signs in canaries and mynahs were progressive death losses, respectively 25% (33/132) and 21% (10/48). The visceral lesions common in chickens with VVNDV were not observed in these six species. Canaries rapidly eliminated Newcastle disease virus (NDV), whereas it was detected for protracted periods in the oral and cloacal secretions of the other five species (for more than a year in parrots). Serologic evaluation by the hemagglutination-inhibition and neutralization tests also indicated prolonged NDV infections in 5 of the 6 species. The seroconversion rate observed in canaries was minimal (13%).

Animals↗