Structure of the Newcastle disease virus F protein in the post-fusion conformation

The paramyxovirus F protein is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. Previous studies of the Newcastle disease virus, human parainfluenza virus 3 and parainfluenza virus 5 F proteins revealed differences in the pre- and post-fusion structures. The NDV Queensland (Q) F structure lacked structural elements observed in the other two structures, which are key to the refolding and fusogenic activity of F. Here we present the NDV Australia-Victoria (AV) F protein post-fusion structure and provide EM evidence for its folding to a pre-fusion form. The NDV AV F structure contains heptad repeat elements missing in the previous NDV Q F structure, forming a post-fusion six-helix bundle (6HB) similar to the post-fusion hPIV3 F structure. Electrostatic and temperature factor analysis of the F structures points to regions of these proteins that may be functionally important in their membrane fusion activity.     

Identification of endoprotease activity in the trans Golgi membranes of rat liver cells that specifically processes in vitro the fusion glycoprotein precursor of virulent Newcastle disease virus

A ubiquitous host endoprotease(s) responsible for activation of the fusion glycoprotein precursor (F0) of virulent Newcastle disease virus (NDV) is an important determinant for its spreading and organ tropism in the host. To characterize the virus-activating protease (VAP), we isolated endoprotease activity from the trans Golgi membranes of rat liver cells by using F0-containing NDV particles grown in a lymphoid cell line NALM6 as substrate. The enzyme cleaved in vitro only the F0 protein of virulent NDV but not that of an avirulent strain, suggesting that it specifically recognizes pairs of basic residues at the cleavage site. Furthermore, the enzyme was found to be membrane-bound, calcium ion-dependent, and active over a broad pH range, from 6 to 8. The inhibitor spectrum of the protease together with the enzyme properties described above indicates that it is a KEX2-like enzyme. Experiments using monensin, A23187, and chloroquine indicate that the F0 cleavage of virulent NDV occurs normally in rat primary hepatocytes at or before the trans Golgi and is a calcium-dependent process. The correspondence between the characteristics of the cleavage in rat hepatocytes and those of the rat protease in vitro indicates that the endoprotease is a strong candidate for the VAP that determines the pantropic nature of virulent NDV.     

Endoproteolytic activation of newcastle disease virus fusion proteins requires an intracellular acidic environment

Effects of weak bases, chloroquine and ammonium chloride, on the intracellular cleavage of the fusion protein precursor (F0) were examined in BHK cells infected with a virulent strain of Newcastle disease virus (NDV). Most of F0 molecules synthesized during a 15-min pulse period were chased out because of cleavage into F1 and F2 within a 60-min chase period in the absence of the weak bases. In contrast, significant amounts of the precursor were found to remain uncleaved when chloroquine or ammonium chloride was present. The uncleaved fusion proteins were incorporated into progeny virions as efficiently as cleaved ones, and about the half of fusion proteins were present as F0 in the virion released by the cells treated with 0.3 mM chloroquine. Taken together with the finding that the trans cisternae of Golgi apparatus and forming secretory vesicles have an acidic pH (K. G. W. Anderson and R. K. Pathak, Cell, 40, 685-643, 1985), the present results suggest that an acidic environment in these compartments is required for intracellular proteolytic activation of NDV fusion proteins.     

A host range mutant of Newcastle disease virus with an altered cleavage site for proteolytic activation of the F protein

The primary structure of the F protein of a host range mutant of the Ulster strain of Newcastle Disease virus (NDV) has been determined by nucleotide sequence analysis and compared to that of the wild type and other NDV strains. The cleavage site of the mutant had the sequence Gly-Lys-Gln-Arg-Arg as compared to two isolated basic amino acids [Gly-Lys(Arg)-Gln-Gly-Arg] with the apathogenic strains and two pairs of basic amino acids [Arg-Arg-Gln-Lys(Arg)-Arg] with the pathogenic strains. The data indicate that the cleavability of the F protein of NDV increases with the number of arginine and lysine residues at the cleavage site and that the susceptibility of the pathogenic strains to ubiquitous host proteases depends on both pairs of basic amino acids.     

Sequence and phylogenetic analysis of the fusion protein cleavage site of Newcastle disease virus field isolates from Iran

Nine Newcastle disease virus (NDV) isolates from Newcastle disease (ND) outbreaks in different regions of Iran were characterized at molecular level. Sequence analysis revealed that the isolates shared two pairs of arginine and a phenylalanine at the N-terminus of the fusion (F) protein cleavage site similarly to other velogenic isolates of NDV characterized earlier. Eight of the nine isolates had the same amino acid sequence as VOL95, a Russian NDV isolate from 1995. However, one isolate, MK13 showed 5 amino acid substitutions, of which 3 have been reported for other velogenic NDV isolates. These results suggest that the origin of the outbreaks of ND in different parts of Iran in 1995-1998 is VOL95.     

Study on the construction of recombinant plasmid coexpressing newcastle disease virus F protein and chicken IL-2

Study on the construction of recombinant plasmid coexpressing newcastle disease virus F protein and chicken IL-2     

Phylogenetic and Pathotypic Characterization of Newcastle Disease Viruses Circulating in West Africa and Efficacy of a Current Vaccine

Newcastle disease (ND) is a deadly avian disease worldwide. In Africa, ND is enzootic and causes large economic losses, but little is known about the Newcastle disease virus (NDV) strains circulating in African countries. In this study, 27 NDV isolates collected from apparently healthy chickens in live-bird markets of the West African countries Benin and Togo in 2009 were characterized. All isolates had polybasic fusion (F)-protein cleavage sites and were shown to be highly virulent in standard pathogenicity assays. Infection of 2-week-old chickens with two of the isolates resulted in 100% mortality within 4 days. Phylogenetic analysis of the 27 isolates based on a partial F-protein gene sequence identified three clusters: one containing all the isolates from Togo and one from Benin (cluster 2), one containing most isolates from Benin (cluster 3), and an outlier isolate from Benin (cluster 1). All the three clusters are related to genotype VII strains of NDV. In addition, the cluster of viruses from Togo contained a recently identified 6-nucleotide insert between the hemagglutinin-neuraminidase (HN) and large polymerase (L) genes in a complete genome of an NDV isolate from this geographical region. Multiple strains that include this novel element suggest local emergence of a new genome length class. These results reveal genetic diversity within and among local NDV populations in Africa. Sequence analysis showed that the F and HN proteins of six West African isolates share 83.2 to 86.6% and 86.5 to 87.9% identities, respectively, with vaccine strain LaSota, indicative of considerable diversity. A vaccine efficacy study showed that the LaSota vaccine protected birds from morbidity and mortality but did not prevent shedding of West African challenge viruses.     

Carbohydrate modifications of the NDV fusion protein heptad repeat domains influence maturation and fusion activity

The amino acid sequence of the fusion protein (F) of Newcastle disease virus (NDV) has six potential N-linked glycosylation addition sites, five in the ectodomain (at amino acids 85, 191, 366, 447, and 471) and one in the cytoplasmic domain at amino acid 542. Two of these sites, at positions 191 and 471, are within heptad repeat (HR) domains implicated in fusion activity of the protein. To determine glycosylation site usage as well as the function of added carbohydrate, each site was mutated by substituting alanine for the serine or threonine in the addition signal. The sizes of the resulting mutant proteins, expressed in Cos cells, showed that sites at amino acids 85, 191, 366, and 471 are used. This conclusion was verified by comparing sizes of mutant proteins missing all four used sites with that of unglycosylated F protein. The role of each added oligosaccharide in the structure and function of the F protein was determined by characterizing stability, proteolytic cleavage, surface expression, and fusion activity of the mutant proteins. Elimination of the site in F(2) at amino acid 85 had the most detrimental effect, decreasing cleavage, stability, and surface expression as well as fusion activity. The protein missing the site at 191, at the carboxyl terminus of the HR1 domain, also showed modestly reduced surface expression and negligible fusion activity. Proteins missing sites at 366 and 471 (within HR2) were expressed at nearly wild-type levels but had decreased fusion activity. These results suggest that all carbohydrate side chains, individually, influence the folding or activity of the NDV F protein. Importantly, carbohydrate modifications of the HR domains impact fusion activity of the protein.     

Role of fusion protein cleavage site in the virulence of Newcastle disease virus

Newcastle disease virus (NDV) causes a highly contagious and economically important disease in poultry. Viral determinants of NDV virulence are not completely understood. The amino acid sequence at the protease cleavage site of the fusion (F) protein has been postulated as a major determinant of NDV virulence. In this study, we have examined the role of F protein cleavage site sequence in NDV virulence using reverse genetics technology. The sequence G-R-Q-G-R present at the cleavage site of the F protein of avirulent strain LaSota was mutated to R-R-Q-K-R, which is present in the F cleavage site of neurovirulent strain Beaudette C (BC). The resultant mutated LaSota V.F. virus did not require exogenous protease for infectivity in cell culture, indicating that the F protein was cleaved by intracellular proteases. The virulence of the mutant and parental viruses was evaluated in vivo by intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index (IVPI) tests in chickens. Our results showed that the modification of the F protein cleavage site resulted in a dramatic increase in virulence from an ICPI value of 0.00 for LaSota to a value of 1.12 for LaSota V.F. However, the ICPI value of LaSota V.F. was lower than that of BC, which had a value of 1.58. Interestingly, the IVPI tests showed values of 0.00 for both LaSota and LaSota V.F. viruses, compared to the IVPI value of 1.45 of BC. In vitro characteristics of the viruses were also studied. Our results demonstrate that the efficiency of cleavage of the F protein plays an important role if the NDV is delivered directly into the brains of chicks, but there could be other viral factors that probably affect peripheral replication, viremia, or entry into the central nervous system.     

Antipeptide antibodies for analysis of pathotype-specific variations in cleavage activation of the membrane glycoprotein precursors of Newcastle disease virus isolates in cultured cells.

Antipeptide antibodies have been produced which target regions either side of the cleavage activation sites of Newcastle disease virus (NDV) membrane glycoprotein precursors. Use of complementary pairs of antibodies in Western blot analysis of mercaptoethanol-reduced extracts of NDV-infected BHK-21 cells enabled analysis of the susceptibilities of NDV fusion protein precursors (Fo-proteins) to cleavage activation in these cells. In addition, it was possible to determine whether or not isolates produce haemagglutinin-neuraminidase (HN)-proteins in precursor forms (HNo-proteins). This assay system has been evaluated with a series of Australian isolates of NDV with well defined virulence properties in order to validate its use in pathotyping NDV isolates. Less well defined isolates also produced data consistent with their biological properties and an isolate was characterised which, hitherto, was not known to be present in Australian poultry. The applicability of this assay system in fundamental studies of the processes of cleavage activation of NDV Fo- and HNo-proteins and formatting of the antisera into ELISA systems are discussed.     

副粘病毒F蛋白HR1和HR2在特异性膜融合中的作用

目的了解副粘病毒融合蛋白（F）分子上的七肽重复序列（HR1、HR2）在特异性膜融合中的作用。方法采用基因定点突变方法，在新城疫病毒（NDV）F与人副流感病毒（hPIV）F基因上创造相同的酶切位点。酶切后采用基因重组方法将F的HR1基因片段相互交换，得到交换HR1的两个嵌合体（chimera），即NDVC-HR1和hPIVC-HR1。用同样的方法又得到交换HR2的两个嵌合体。即NDVC-HR2和hPIVC-HR2。将各种嵌合体DNA与同源及异源HNDNA共转染BHK21细胞后，在真核细胞中表达。Giemsa染色和指示基因法检测细胞融合功能，荧光强度分析（FACs）检测F蛋白的表达效率。结果交换HR1后，NDVC-HR1的融合功能只有野毒株的53．91％，hPIVC-HR1的融合功能达到野毒株的83．15％；交换HR2后，NDVC-HR2的融合功能略高于野毒株，达到野毒株的107．23％，hPIV C-HR2的融合功能却只有野毒株的12．01％。FACS分析表明，交换HR1后的NDVC-HR1和hPIVC-HR1的表达效率均下降。交换HR2后，NDVC-HR2的表达效率没有改变，而hPIVC-HR2的表达效率下降。结论NDVF的HR1对其特异性膜融合具有重要作用，而HR2则没有病毒特异性；hPIVF的HR1和HR2对hPIV的特异性膜融合都有重要作用。     

Fusion (F) protein gene of newcastle disease virus: Sequence and hydrophobicity comparative analysis between virulent and avirulent strains

The nucleotide and predicted amino acid sequences have been obtained for the fusion (F) protein gene of the avirulent strain La Sota of Newcastle disease virus (NDV). The F₁ N-terminus begins with the tripeptide Leu-Ileu-Gly instead of Phe-X-Gly as usually observed in fusion peptide. It was found that the cleavage-activation domain of the avirulent La Sota strain contained single (but no pairs of) basic residues in the sequence Gly-Arg-Gln-Gly-Arg. Hydrophobicity analysis suggested that the cleavage-activation domain became more hydrophobic and could be less accessible for host-specific protease(s); dibasic residues next to the F₁ N-terminus were shown to be important for keeping the cleavage-activation site in exposed positioning, suitable for F protein activation. Comparative sequence analysis of the NDV F proteins revealed a striking homology between lentogenic La Sota and mesogenic Beaudette C strains. Furthermore, 58 variable positions were recorded in the NDV F protein, excluding signal sequence; some of these mutations, in the cysteine-clustered region, were surmised to alter virulence.Requests for reprints should be addressed to Arsene Burny, Laboratory of Biological Chemistry, Department of Molecular Biology, Free University of Brussels, ULB, Rhode-St-Genèse, Belgium.     

Nucleotide sequence of the envelope protein genes of a highly virulent, neurotropic strain of Newcastle disease virus

The envelope glycoproteins of Newcastle disease virus (NDV), hemagglutinin-neuraminidase (HN) and fusion (F) proteins, play important roles in determining the host immune response and the virulence of that particular virus strain. The complete nucleotide sequence of the HN and F genes of a highly neurovirulent strain of NDV (Texas G. B., 1948) was determined in an effort to study the molecular basis of this strain's neurotropic properties. Comparison of the predicted amino acid sequences for the HN and F among the American NDV strains revealed that the Texas G. B. and Beaudette C envelope genes are closely related to each other and are less closely related to the avirulent B1 Hitchner strain. We have found 11 amino acid changes in the predicted HN protein between the Beaudette C and Texas G. B. strain but only 2 conservative amino acid changes (amino acids 11 and 197) in the F protein between these two strains. Although the virulence of NDV strains has been related to sequences at the cleavage site of F0, the property of neurovirulence cannot depend solely upon these sequences because there are no sequence differences between the Beaudette C and Texas G. B. strains. We suggest that the neurovirulence phenotype could be due to the molecular properties of the HN protein; however, we cannot exclude the possibility that the two conservative amino acid differences between the two F proteins could also play a role in determining the phenotypic differences between these two virus strains.     

Complete genome sequence of highly virulent neurotropic Newcastle disease virus strain Texas GB

Newcastle disease virus (NDV) strain Texas GB is a highly virulent neurotropic virus that is used as a standard vaccine challenge virus in the U.S. In this study, the complete genome sequence of strain Texas GB was determined and compared with the complete genome sequences of other NDV strains. The genome is 15,186 nucleotides (nt) long and consists of six genes in the order of 3′leader-N-P-M-F-HN-L-5′trailer. The genome contains a 55-nt leader sequence at the 3′ end and a 114-nt trailer sequence at the 5′ end. The intergenic sequences are 2, 1, 1, 31, and 47 nt between N/P, P/M, M/F, F/HN, and HN/L genes, respectively. The putative cleavage site of fusion protein showed amino acid sequence of R-R-Q-K-R↓F in position 112 to 117, which corresponds to those of virulent NDV strains. The phylogenetic analysis showed that strain Texas GB is closely related to the neurovirulent mesogenic strain Beaudette C (BC) and to NDV viruses isolated in China and Egypt than to other strains of NDV.     

Molecular epidemiological analysis of Newcastle disease virus isolated in China in 2005

Eighty-three strains of Newcastle disease virus (NDV) were obtained from outbreaks in chickens, pigeons, geese, and ducks in China in 2005 and characterized genotypically. The main functional region of the F gene (535 nucleotides) was amplified and sequenced. A phylogenetic tree based on nucleotides 47–435 of the F gene was created using sequences from 83 isolates and representative NDV sequences obtained from GenBank. Phylogenetic analysis showed that all newly characterized strains belonged to six genetic groups: I, II, III, VIb, VIIc, and VIId. All the isolates belonging to groups I and II (14 total) were lentogenic according to the amino acid sequences of the fusion protein cleavage site, and either V4 or LaSota-type, depending on the vaccines that were used. Most isolates (64 total) were classified in group VIId, a predominant genotype responsible for most Newcastle disease outbreaks since the end of the last century. One strain, NDV05-055, was in group VIIc, three pigeon strains were in group VIb, and one isolate, NDV05-041, was in group III, and characterized as a velogenic strain. This study revealed that genotype VIId was the major NDV strain responsible for the 2005 ND epizoonosis that occurred in China.     

Molecular Characterization of Virulent Newcastle Disease Virus Isolates from Chickens during the 1998 NDV Outbreak in Kazakhstan

Newcastle disease virus (NDV) infects domesticated and wild birds throughout the world and has the possibility to cause outbreaks in chicken flocks in future. To assess the evolutionary characteristics of 10 NDV strains isolated from chickens in Kazakhstan during 1998 we investigated the phylogenetic relationships among these viruses and viruses described previously. For genotyping, fusion (F) gene phylogenetic analysis (nucleotide number 47-421) was performed using sequences of Kazakhstanian isolates as compared to sequences of selected NDV strains from GenBank. Phylogenetic analysis showed that all newly characterized strains belonged to the genetic group designated as VIIb. All strains possessed a virulent fusion cleavage site (RRQRR/F) belonging to velogenic or mesogenic pathotypes with intracerebral pathogenicity indexes (ICPI) varying from 1.05 to 1.87.     

Pathotyping isolates of Newcastle disease virus using antipeptide antibodies to pathotype-specific regions of their fusion and hemagglutinin-neuraminidase proteins

Summary.  Antipeptide antibodies have been evaluated for their abilities to predict the characteristics of the cleavage motifs of the fusion protein precursors (F₀) of 25 isolates of Newcastle disease virus (NDV) with a range of virulences, grouped into 12 sets according to their monoclonal antibody reactivities. A Western blot format was used to show that antisera to synthetic peptides representing sequences at the C-termini of the F₂-polypeptides of defined pathotypes of NDV usually distinguish between pathotypes on the basis of their Fo cleavage sequences. However, exceptions were found with three groups of virulent isolates. Protein sequencing and mass spectral analysis of the F₂-polypeptide of isolate Texas GB from one of these groups, identified an anomalous cleavage/activation process which removed the amino acids required for recognition by the antisera. This probably also explained the lack of reactivity of the Roakin isolate and low reactivity of the Komarov isolate from this group. The other exceptions involved isolates in groups with cleavage region variations from the usual motif of virulent isolates or isolates with undefined cleavage motifs. Antipeptide antisera were also raised to sections of the 45 residue C-terminal extension the hemagglutinin-neuraminidase precursor (HN₀) encoded by the genes of some avirulent isolates. Western blot analysis showed that positive reactions with antibodies to peptides based on sequences between residues 577 and 613 of the HN₀ was evidence for the presence of an avirulent isolate but did not exclude the presence of other pathotypes. Antisera designed to target residues 569–577 detected HN₀ extensions of 6 residues on isolates known to encode such extensions. These antisera also enabled differentiation of isolates with HN₀ extensions of 6 residues from those with no extension, however, it was not possible to determine the virulence of isolates based on reaction with these antisera. Received May 20, 1998 Accepted August 12, 1998     

Phylogenetic Relationships among Highly Virulent Newcastle Disease Virus Isolates Obtained from Exotic Birds and Poultry from 1989 to 1996

Newcastle disease virus {NDV (avian paramyxovirus type 1 [APMV1])} isolates were recovered from imported exotic birds confiscated following importation into the United States, from waterbirds in the United States, and from poultry. The exotic birds probably originated from Central and South America, Asia, and Africa. The NDV isolates were initially characterized as highly virulent because of a short mean death time in embryonated chicken eggs. The isolates were typed as neurotropic or viscerotropic velogenic by intracloacal inoculation of adult chickens. Intracerebral pathogenicity index values for the virulent NDV isolates ranged from 1.54 to 1.90, compared to a possible maximum value of 2.0. These isolates had a dibasic amino acid motif in the fusion protein cleavage site sequence required for host systemic replication. Sequence differences were detected surrounding the fusion protein cleavage site and the matrix protein nuclear localization signal, indicating evolution of highly virulent NDV. Phylogenetically, these isolates were categorized with other highly virulent NDV strains that caused outbreaks in southern California poultry during 1972 and in cormorants in the north central United States and southern Canada during 1990 and 1992. These isolates are related to NDV that may have the APMV1 strain chicken/Australia/AV/32 or a related virus as a possible progenitor. Recent virulent NDV isolates and those recovered during disease outbreaks since the 1970s are phylogenetically distinct from current vaccine viruses and standard challenge strains.     

Genomic characterization of the first class I Newcastle disease virus isolated from the mainland of China

The complete genomic sequence of Newcastle disease virus (NDV) strain NDV08-004, isolated from domestic ducks in China, was determined in this study. The genome is 15198 nucleotides (nt) in length, follows the “rule of six” and contains a 55-nt leader sequence at the 3′ end and a 114-nt trailer sequence at the 5′ end. Compared with the full genome sequences of Class II NDV strains, the NDV08-004 isolate has a 12-nt insertion (TGGGAGACGGGG) in the phosphoprotein gene between nucleotides 2381 and 2382 of the genome (numbered according to the genomic sequence of the La Sota strain, which consists of 15186 nt). Strain NDV08-004 has the motif ¹¹²E-Q-Q-E-R-L¹¹⁷ at the cleavage site of the fusion protein, which is typical of lentogenic NDV strains, and this is in agreement with the results of pathogenic tests based on the mean death time (MDT) and the intracerebral pathogenicity index (ICPI). Phylogenetic analysis based on the full genome revealed that all the NDV strains studied could be divided into two distinct clades, namely class I and class II, and the NDV08-004 isolate characterized in this study was grouped in class I. Further phylogenetic analysis based on a 374-bp fragment of the F gene in class I strains of NDV demonstrated that NDV08-004 belongs to genotype 3, and should be therefore similar to strains obtained from live bird markets in Hong Kong in recent years.     

Molecular characterization of a Class I Newcastle disease virus strain isolated from a pigeon in China

Constant monitoring is performed to elucidate the role of natural hosts in the ecology of Newcastle disease virus (NDV). In this study, an NDV strain isolated from an asymptomatic pigeon was sequenced and analysed. Results showed that the full-length genomes of this isolate were 15,198 nucleotides with the gene order of 3′-NP-P-M-F-HN-L-5′. This NDV isolate was lentogenic, with an intracerebral pathogenicity index of 0.00 and a mean time of death more than 148?h. The isolate possessed a motif of -¹¹²E-R-Q-E-R-L¹¹⁷- at the F protein cleavage site. In addition, 7 and 13 amino acid substitutions were identified in the functional domains of fusion protein (F) and haemagglutinin-neuraminidase protein (HN) proteins, respectively. Analysis of the amino acids of neutralizing epitopes of F and HN proteins showed 3 and 10 amino acid substitutions, respectively, in the isolate. Phylogenetic analysis classified the isolate into genotype Ib in Class I. This isolate shared high homologies with the NDV strains isolated from wild birds and waterfowl in southern and eastern parts of China from 2005 to 2013. To our knowledge, this study is the first to report a NDV strain isolated from pigeon that belongs to genotype Ib in Class I, rather than to the traditional genotype VI or other sub-genotypes in Class II. This study provides information to elucidate the distribution and evolution of Class I viruses for further NDV prevention.