Electron micrographs of Newcastle disease virus preparation from chick embryos following infection by virus propagated in the short-tailed shrew (Blarina brevicauda).
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The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion.
The amino acid sequence at the F2/F1 cleavage site was determined for 15 strains of the so-called pigeon PMV-1 (PPMV-1) variant of Newcastle disease virus (NDV) which showed close antigenic identity, determined by their reactions with a panel of 28 monoclonal antibodies, but considerable variation in their pathogenicity for chickens. Thirteen of the isolates possessed the motif 112G-R-Q-K-R-F117. This motif was seen for one virus which had initially low pathogenicity and remained unaltered when virulence of the virus for chickens was increased by bird to bird passage. The two other viruses had the sequence 112R-R-Q-K-R-F117 at the cleavage site which is more typical of virulent viruses, however, pathogenicity index tests indicated that these isolates were of moderate and low pathogenicity. The nucleotide sequence coding for the HN/HN0 extension region was determined for two of the PPMV-1 isolates. In both cases a stop codon was present indicating that the product for these viruses would be HN571. We conclude that the wide variation in pathogenicity of the variant PPMV-1 for chickens is not related to variation in the amino acid motif at the F2/F1 cleavage site nor due to production of HN0 which may also influence pathogenicity. The high virulence of some of the viruses examined confirms that a double pair of basic amino acids in the region of the F2/F1 cleavage site is not necessary for the full expression of virulence.
To evaluate the usefulness of a DNA vaccine for chickens, we constructed a plasmid vector expressing the Newcastle disease virus F protein (NDV-F) under the control of the human cytomegalovirus immediate early enhancer and chicken beta-actin gene promoter. One-week-old chickens injected intramusculary with the circular plasmid DNA did not produce significant levels of antibody against NDV-F. However, two of five birds injected with the linearized plasmid DNA produced high levels of the antibody. Moreover, four of five birds injected with a mixture of the linearized-plasmid and Lipofectin produced the antibody efficiently. At 9 weeks post-injection, chickens were challenged with the velogenic NDV Sato strain. These chickens that had the antibody against NDV-F were protected from lethal NDV challenge. These results demonstrate that the DNA vaccine conferred efficient protection against the disease.
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In the rabbit, ovulation and conception occur during an 8-10 hour period immediately following mating ("reflex ovulation"). We report here that live Newcastle disease virus (NDV; an avian paramyxovirus) injected into rabbits immediately following mating resulted in a high frequency of embryo death at a wide range of developmental stages. When measured at midterm, a greater than threefold increase in embryo mortality was observed (24.7% vs. 6.8%). Aneuploidy of a small acrocentric chromosome was observed in three of 60 live midterm embryos recovered from NDV-injected does. No aneuploidy was observed in 52 live midterm embryos from control, saline-injected does. These observations suggest that the NDV-exposed rabbit can provide a useful model for the study of the teratological effects of virus exposure occurring at or about the time of meiosis, ovulation, and conception.
We compared the hemagglutinin-neuraminidase gene sequence among 13 strains of Newcastle disease virus (NDV) isolated over the last 50 years. Although overall homology was remarkably high, the sequence variability demonstrated the existence of at least three distinct lineages, which must have co-circulated for considerable periods. The sequence variability also appears to reflect some accumulation of mutations over time. Strictly correlating with the lineages, the translation products could be classified into three size classes. One class lacked the interchain disulfide bond, and another represented unusual precursor protein of biologically inactive form. The lineages correlated to some extent with virulence and place of isolation of the strains. However, antigenic variations, which were neither cumulative nor progressive, did not correlate with the lineages. These analyses showing multiple lineages were greatly facilitated by a precise calculation of synonymous substitutions, which had been largely free from selective pressures and had occurred frequently and evenly throughout the coding region.
The study, using sequence analysis and the phylogenetic relationship of the fusion protein gene, divided the Korean epizootic isolates of Newcastle disease virus (NDV) into several lineages to determine the molecular epidemiology of the virus. A 695 base pair fragment was amplified by polymerase chain reaction between matrix protein gene and fusion protein gene of 30 Korean NDV isolates, which were isolated from field outbreaks of Newcastle disease between 1949 and 2002. All isolates showed the amino acid sequence 112R-R-Q/R-K-R116 at the C-terminus of the F2 protein and phenylalanine (F) at the N-terminus of the F1 protein, residue 117. These amino acid sequences were identical to a known virulent motif. The region of the F gene between nucleotides 47 and 435 was compared by phylogenetic analysis. Based on nucleotide sequence, the Korean NDV isolates belonged to genotype III, V, VI and VII corresponding to isolates in 1949, 1982 to 1984, 1988 to 1997, and 1995 to 2002, respectively. These data showed that genotypes of five Korean Newcastle disease epizootics had replaced each other serially (III, V, VI and VII) in chronological order. Further, the five Korean Newcastle disease epizootics were closely related with the Newcastle disease panzootics or Newcastle disease epizootics in other countries. Present study showed that the Korean genotype V isolated before 1984 was related with European Newcastle disease epizootics in the 1970s, whereas the Korean genotypes VI and VII isolated after 1988 were more closely related with Far East Newcastle disease epizootics, especially Newcastle disease epizootics in Japan, Taiwan and China. Since 1988, the genotypes VI and VII of Far East origin were dominant in South Korea. That might be due to the increased trade of agricultural products including poultry among Far East Asian countries.
The hybrid baculovirus constructed from Autographa california nuclear polyhedrosis virus (NPV) and Bombyx mori (silkworm) NPV was used for expression of fusion glycoprotein (F) of Newcastle disease virus (NDV) strain D26. The gene encoding F protein was introduced into the improved baculovirus expression vector derived from the host-range-expanded baculovirus. In Spodoptera frugiperda (fall armyworm) cells infected with a recombinant baculovirus, HyF121, the expressed F protein was properly located onto the cell surface. After silkworm pupae were infected with HyF121, a subunit vaccine against NDV was prepared from the HyF121-infected pupae. Chickens inoculated with the subunit vaccine were protected against virulent NDV challenge.
Previously, a panel of monoclonal antibodies recognizing epitopes in four antigenic sites on the haemagglutinin-neuraminidase (HN) glycoprotein of the Australia-Victoria strain of Newcastle disease virus were used in strain comparisons. Epitopes in three sites were found to be conserved while the epitope recognized by the single antibody to site 3 was not. A new panel of antibodies is described, two of which bind to epitopes in site 3 and six of which bind to a site (site 1,4) that overlaps with sites 1 and 4 as determined by analyses of variants, temperature-sensitive mutants, and strains by assays of neutralization of infectivity and binding in a radioimmunoassay. Neutralization of heterologous strains with the panel of antibodies revealed that both new site 3 epitopes are also highly divergent, while three additional epitopes outside site 3 (those in site 1,4) are highly conserved. The new site 3 antibodies can bind to virions of several heterologous strains without neutralizing infectivity. Thus, of the 10 epitopes we have now examined, all of three in site 3 are specific with respect to neutralization of infectivity for the homologous strain, while all of seven in other sites are conserved in heterologous strains. This suggests that the strain specificity originally described for a single site 3 epitope is, instead, a property of a much more extensive, poorly conserved domain on the HN molecule.
The composition, topological distribution and biological significance of phospholipids in the membrane of Newcastle disease virus (NDV) grown in embryonated chicken eggs were investigated. Phosphatidylethanolamine and sphingomyelin were the predominant phospholipids in NDV membrane. The location of phospholipids in the lipid bilayer of the membrane was studied by assessing their reactivities with highly purified phospholipase A2 (Agkistrodon halys blomhoffi) and phospholipase D (Streptomyces chromofuscus), and the biological role of membrane phospholipids was also investigated by using pure phospholipases A2, C (Bacillus cereus) and D. Choline-containing phospholipids were found predominantly in the outer layer of the membrane. The inner layer was composed mainly of aminoglycerophospholipids, though a fair amount of them also appeared to be located in the outer half of the bilayer. When intact virion was treated with phospholipase C, marked decreases in hemolytic activity and infectivity mediated by viral fusion (F) glycoprotein were observed, but hemagglutinating and neuraminidase activities did not change significantly. Apparently complete hydrolysis of phospholipids in the outer half of the lipid bilayer with phospholipase D caused about 22% decrease in the original hemolytic activity. On the other hand, when all phosphatidylcholine and aminoglycerophospholipids in the outer half of the viral membrane were hydrolyzed with purified phospholipase A2, no significant change in viral hemolytic activity or morphology was detected. No marked change of hemagglutinating and neuraminidase activities was detected on treatment of NDV with phospholipases A2 and D. The above results suggest that the integrity of fatty acid ester of glycerophospholipids in NDV membrane is not essential for the manifestation of viral activities, though polar groups of the phospholipids in the outer half of the membrane may be involved in the function of fusion (F) glycoprotein, but not in that of hemagglutinating and neuraminidase (HN) glycoprotein of NDV.
Recombinant viruses were rescued after site-specific mutagenesis of a full-length clone of the lentogenic Newcastle disease virus (NDV) strain Clone 30. To assess the contribution of different amino acids to virulence, specific alterations were introduced into the fusion (F) protein and in the hemagglutinin-neuraminidase (HN) protein based on sequence comparison between NDV strains of different virulence. Modification of the proteolytic cleavage site in the F protein to a polybasic motif increased the intracerebral pathogenicity index (ICPI) from 0.0 to 1.28. Moreover, the additional exchange of amino acid 123 of the HN protein from tryptophan to cysteine in combination with alteration of amino acid 27 of the F protein from cysteine to arginine increased the ICPI to 1.5. The HN mutation visibly altered conformation of the protein, resulting in the formation of disulfide-linked HN dimers that may indicate that this HN conformation is beneficial for the virulent phenotype.
A reverse transcription-polymerase chain reaction (RT-PCR) was described which amplified a portion of the F and HN genes of Newcastle disease virus (NDV) isolated from recent outbreaks in Taiwan. The F protein plays an important role in determining the virulence of NDV strains. Sequencing of a region specifying the F protein cleavage site was therefore undertaken and this verified the correlation between deduced amino sequences and pathogenicity. Analysis of the deduced amino acid sequences of the F protein cleavage site showed that all recent Taiwanese isolates in 1999 were velogenic viruses. All the virulent viruses have the amino acid sequence 112RRQKR116 for the C-terminus of the F2 protein and phenylanine (F) at the N-terminus of the F1 protein, residue 117. A phylogenetic tree based on the nucleotide sequences of the F gene revealed that recent Taiwanese NDV isolates responsible for recent outbreaks were classified into two distant genotypes (VI and VII). Genotype VI virus is the first finding in Taiwan and has a highly genetic similarity to European isolates, suggesting that they might have originated from a common ancestor.
A total of 291 eight-week-old chickens were exposed to chickens infected with either of two Australian lentogenic strains (V4 and AVL NDV-1) of Newcastle disease virus (NDV). At 3 weeks after exposure, all chickens exposed to V4 infected chickens had developed haemagglutination-inhibition (HI) antibody. All chickens exposed to AVL NDV-1 virus infected chickens had developed HI antibody 5 weeks later. This sudden late appearance of HI antibody, to titres higher than those observed with V4 chickens, was explained by V4 virus being introduced to the AVL NDV-1 group of chickens. When groups of these chickens were challenged with Roakin virus (mesogenic NDV) at 3 weeks and Fontana 1083 virus (viscerotropic velogenic NDV) and Texas GB virus (neutrotropic NDV) at 3, 5, 10 and 21 weeks only three chickens developed clinical illness one of which died. These chickens were one AVL NDV-1 chicken contact challenged with Fontana 1083 virus at 3 weeks, one V4 chicken oronasally challenged with Texas GB virus at 5 weeks and one V4 chicken challenged oronasally with Fontana 1083 virus at 10 weeks. Susceptible non-vaccinated chickens died soon after challenge. Challenge by oronasal infection with 10(7.0) ELD50 of virus or contact with susceptible infected chickens enabled virulent virus to be isolated from most chickens and was accompanied by a large anamnestic increase in serum HI antibody.
Avian paramyxovirus 1 (APMV-1), also referred to as Newcastle disease virus (NDV), variants of low virulence were isolated from chickens, ducks and other unidentified species found in live-bird markets of the northeastern United States. These isolates were characterized as APMV-1 by the hemagglutination-inhibition (HI) assay utilizing NDV-specific polyclonal antisera. However, the isolates failed to react with a monoclonal antibody that has specificity for a wide variety of APMV-1 isolates. Although only highly virulent isolates require reporting to international regulatory agencies, the ability to correctly identify APMV-1 types is important for control and regulatory purposes. Protein gel patterns of the purified isolates resembled previously reported APMV-1 and anti-NDV polyclonal sera recognized the viral proteins. For three isolates oligonucleotide primers specific for the nucleoprotein, fusion protein and polymerase genes of NDV were utilized to synthesize cDNA using viral RNA as a template. Approximately 12kb of the genome was subsequently sequenced for the three isolates that included the nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase protein genes and a 5' portion of the polymerase gene. The isolates had an F protein cleavage site sequence of ERQER/LVG indicating low-virulence viruses that phylogenetically separated with other unique NDV isolates designated as a lineage 6 genotype. Additionally, a four amino acid insert was detected in the predicted phosphoprotein which complies with the "rule of six" among paramyxoviruses. These APMV-1 genotypes have not been previously reported in North America and further substantiate the heterogeneous genetic nature of these commercially important pathogens found worldwide.
Homologous and heterologous viral interference is a common occurrence that has been well studied in vitro. In the present study, homologous viral interference between the LaSota and NYP strains of Newcastle disease virus (NDV) was studied in vivo in chickens, the natural host for NDV. The LaSota strain is avirulent and widely used as a vaccine in poultry industry, while the NYP strain is highly virulent and causes acute disease and death in chickens within four to six days after infection. Chickens generally became resistant to NYP strain challenge 12 hours after intranasal or intratracheal inoculation with LaSota strain virus. The resistance was manifested by reduction in chicken morbidity and mortality, decrease in virus replication in the chicken respiratory tract (p less than 0.05), and inhibition of NYP strain induced gross and microscopic lesions. Interferon was first detected in the chicken respiratory tract and blood at 3 to 6 hours; it peaked at 12 to 24 hours and was maintained for 48 hours after viral inoculation, indicating that interferon induction might be one possible mechanism of the interference between the two strains. This study suggests a role for viral interference in vaccination against virulent viruses.