Use of a heteroduplex mobility assay to detect differences in the fusion protein cleavage site coding sequence among Newcastle disease virus isolates

Newcastle disease virus (NDV) is an economically important pathogen of poultry that may cause clinical disease that ranges from a mild respiratory syndrome to a virulent form with high mortality, depending on an isolate's pathotype. Infections with virulent NDV strains are required to be reported by member nations to the Office of International Epizootes (OIE). The primary determinant for virulence among NDV isolates is the presence or absence of dibasic amino acids in the fusion (F) protein cleavage activation site. Along with biological virulence determinations as the definitive tests, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criterion. Nucleotide sequence data for many NDV isolates recently isolated from infected chickens and other avian species worldwide have been deposited in GenBank. Consequently, viral genomic information surrounding the F protein cleavage site coding sequence was used to develop a heteroduplex mobility assay (HMA) to aid in further identification of molecular markers as predictors of NDV virulence. Using common vaccine strains as a reference, we were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. This technique was also used to examine NDV isolates not previously characterized. We were able to distinguish vaccine-like viruses from other isolates potentially virulent for chickens. This technique will help improve international harmonization of veterinary biologics as set forth by the OIE and the Veterinary International Cooperation on Harmonization of Technical Requirements of Veterinary Medicinal Products. Ultimately, the HMA could be used for initial screening among a large number of isolates and rapid identification of potentially virulent NDV that continue to threaten commercial poultry worldwide.     

Generation of velogenic Newcastle disease viruses from a nonpathogenic waterfowl isolate by passaging in chickens

A benign Newcastle disease virus (NDV) recently became highly virulent during replication in domestic chickens. It is still unclear whether NDVs circulating among wild waterfowl also have the potential to become highly pathogenic (velogenic) in chickens. To demonstrate experimentally the generation of velogenic NDV from a nonpathogenic waterfowl isolate, we passaged an avirulent goose isolate in chickens. After nine consecutive passages by air-sac inoculation, followed by five passages in chick brain, the virus became highly virulent in chickens, producing a 100% mortality rate, and demonstrating typical velogenic properties in pathogenicity tests. Sequence analysis at the fusion protein cleavage site showed that the original isolate contained the typical avirulent type sequence, E-R-Q-E-R/L, which progressed incrementally to a typical virulent type, K-R-Q-K-R/F, during repeated passage in chickens. These results demonstrate that avirulent viruses, maintained in wild waterfowl in nature and bearing the consensus avirulent type sequence, have the potential to become velogenic after transmission to and circulation in chicken populations. The results also suggest that chickens provide a mechanism for the selection of virulent viruses from an avirulent background.     

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.     

Newcastle disease outbreaks in Kazakhstan and Kyrgyzstan during 1998, 2000, 2001, 2003, 2004, and 2005 were caused by viruses of the genotypes VIIb and VIId

Newcastle disease virus (NDV) infects domesticated and wild birds throughout the world, and infections with virulent NDV strains continue to cause disease outbreaks in poultry and wild birds. To assess the evolutionary characteristics of 28 NDV strains isolated from chickens in Kazakhstan and Kyrgyzstan during 1998, 2000, 2001, 2003, 2004, and 2005, 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 and Kyrgyzstanian isolates as compared to sequences of selected NDV strains from GenBank. Phylogenetic analysis demonstrated that the 14 newly characterized strains from years 1998 to 2001 belong to the NDV genotype VIIb, whereas the 14 strains isolated during 2003–2005 were of genotype VIId. All strains possessed a virulent fusion protein cleavage site (R-R-Q-R/K-R-F) and had intracerebral pathogenicity indexes in day-old chickens that ranged from 1.05 to 1.87, both properties typical of NDV strains classified in the mesogenic or velogenic pathotype.     

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.     

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.     

Genetic comparisons between lentogenic Newcastle disease virus isolated from waterfowl and velogenic variants

Avirulent Newcastle disease viruses (NDV) harbored by waterfowl have the potential to become virulent after transmission to and circulation within chicken populations. In order to investigate how virulent viruses are selected from an avirulent background, we compared the complete sequences of the avirulent NDV isolate Goose/Alaska/415/91 and its virulent variant strain 9a5b, which was obtained by nine and five passages in the chick air sac and brain, respectively. Seven amino acid substitutions were detected in the M, F, and HN proteins. Two were detected between variants 9a3b and 9a5b (128P to H and 495E to K in HN protein) that were passed through the brain. Pathogenicity determined by the MDT and IVPI tests also differed between 9a3b and 9a5b. These results suggest that in addition to the F cleavage site sequence, these two amino acids in HN protein are also related to the pathogenicity of NDV in chickens.     

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.     

Pigeon paramyxovirus type 1 variants with polybasic F protein cleavage site but strikingly different pathogenicity

Newcastle disease viruses (NDV) isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) are mostly of mesogenic pathotype and characterized by a polybasic amino acid sequence motif at the fusion protein (F) cleavage site. This feature also applies to isolate R75/98 from Germany. Its genome consists of 15,192 nucleotides and it specifies an intracerebral pathogenicity index (ICPI) of 1.1, as is typical for mesogenic NDV. Recombinant R75/98 (rR75/98) derived by reverse genetics also possesses a polybasic F protein cleavage site but exhibits ICPI of 0.28, indicating a lentogenic virus. While ten virus passages of rR75/98 on embryonated chicken eggs did not result in any alteration of virus characteristics, virus which had been re-isolated from the brain of an intracerebrally inoculated chicken showed an increase in virulence, characterized by an ICPI of 0.93. Comparison of whole genome sequences of rR75/98 and re-isolated rR75/98 (RrR75/98) demonstrated only two amino acid differences, one in the F protein (N472 K) and one in the polymerase protein (K2168R). This result indicates that only very few amino acid alterations are sufficient to modulate virus virulence in the presence of a polybasic amino acid sequence at the proteolytic F protein cleavage site.     

Molecular analyses of the hemagglutinin genes of H5 influenza viruses: origin of a virulent turkey strain

Comparative sequence analysis of the hemagglutinin (HA) genes of a highly virulent H5N8 virus isolated from turkeys in Ireland in 1983 and a virus of the same subtype detected simultaneously in healthy ducks showed only four amino acid differences between these strains. Partial sequencing of six of the other genes and antigenic similarity of the neuraminidases established the overall genetic similarity of these two viruses. Comparison of the complete sequence of two H5 gene sequences and partial sequences of other virulent and avirulent H5 viruses provides evidence for at least two different lineages of H5 influenza virus in the world, one in Europe and the other in North America, with virulent and avirulent members in each group. In vivo studies in domestic ducks showed that all of the H5 viruses that are virulent in chickens and turkeys replicate in the internal organs of ducks but did not produce any disease signs. Additionally, both viruses isolated from turkeys and ducks in Ireland were detected in the blood. These studies provide the first conclusive evidence for the possibility that fully virulent influenza viruses in domestic poultry can arise directly from viruses in wild aquatic birds. Studies on the cleavability of the HA of virulent and avirulent H5 viruses showed that the principles established for H7 viruses (F. X. Bosch, M. Orlich, H. D. Klenk, and R. Rott, 1979, Virology 95, 197-207; F. X. Bosch, W. Garten, H. D. Klenk, and R. Rott, 1981, Virology 113, 725-735) also apply to the H5 subtype. These are (1) only the HAs of virulent influenza viruses were cleaved in tissue culture in the absence of trypsin and (2) virulent H5 influenza viruses contain a series of basic amino acids at the cleavage site of the HA, whereas avirulent strains contain only a single arginine with the exception of the avirulent Chicken/Pennsylvania virus. Thus, a series of basic amino acids at the cleavage site probably forms a recognition site for the enzyme(s) responsible for cleavage.     

Differentiation of virulent and non-virulent strains of Newcastle disease virus within 24 hours by polymerase chain reaction.

Fast diagnosis of Newcastle disease is a prerequisite for confining outbreaks. Diagnosis implies the differentation of virulent and non-virulent Newcastle disease viruses (NDV). However, conventional methods, i.e. isolation of the virus and determination of the intracerebal pathogenicity index, take at least 5 days. Therefore, we investigated whether diagnosis can be performed by using the reverse transcrip-tase-polymerase chain reaction (RT-PCR) on RNA isolated directly from tissue homogenate. Two oligonucleotide primers, representing the sequence at the cleavage site of the F protein of either virulent or non-virulent NDV strains, respectively, were used to differentiate NDV. Using the RT-PCR we were able to differentiate 15 NDV reference strains, 11 of which were virulent and 14 non-virulent. The RT-PCR was further validated by using homogenate of brain, trachea, lung and spleen from 12 chicken flocks and one turkey flock suspected of Newcastle disease. The RT-PCR detected virulent NDV in samples of seven flocks and non-virulent NDV in two out of three flocks in agreement with conventional methods. However the RT-PCR failed to detect virus in 1/3 flocks from which non-virulent virus was isolated. The results are discussed. We conclude that the RT-PCR described can be used to confirm diagnosis of Newcastle disease within 24 h using RNA isolated directly from tissue homogenate.     

Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus

The nucleotide sequence of the mRNA encoding the fusion (F0) protein of a virulent strain of Newcastle disease virus was determined. A single open reading frame in the sequence encodes a protein of 553 amino acids with a calculated molecular weight of 59058. The amino acid sequence predicted several structural features involving the fusion-inducing hydrophobic stretch (residues 117-142) and the cleavage-activation site (residues 112-116) to generate the disulfide-linked F1 and F2 subunits. The cleavage-activation site as well as a part of the fusion-inducing sequence were compared among a series of virulent and avirulent strains by the chain-termination method using a synthetic oligonucleotide primer. It was found that without exception, the cleavage-activation site of virulent strains consisted of two dibasic residues with an intervening glutamine, Arg-Arg-Gln-Arg-Arg, whereas the corresponding region of avirulent strains was made of a sequence with single basic residues scattered among uncharged residues, Gly-LysArg-Gln-GlySer-Arg. On the basis of these observations and the previous results showing a strict correlation between the pathogenicity and the cleavability of the fusion protein of NDV (Y. Nagai, H-D. Klenk, and R. Rott, Virology, 72, 494-508, 1976), we propose the importance of the dibasic residues for efficient proteolytic activation of the fusion protein and for the pantropic property of NDV. Some strains were found to have Leu-Ile-Gly as the N-terminus of F1, whereas others contained Phe-Ile-Gly, indicating that Phe-X-Gly is not always conserved at F1 N-terminus of paramyxovirus.     

Comparison of the positions and efficiency of cleavage activation of fusion protein precursors of virulent and avirulent strains of Newcastle disease virus: insights into the specificities of activating proteases

The F1- and F2-polypeptide components of in ovo activated fusion proteins of one virulent (AV or Australia-Victoria) strain, one low-virulence (EG or Eaves-Grimes) strain, and two avirulent (V4 or Queensland and WA2116) strains of Newcastle disease virus (NDV) were isolated and subjected to structural analysis. This included complementary application of amino acid analysis, fast atom bombardment-mass spectrometry, and N-terminal sequence analysis to fragments isolated from AspN protease digests of the F2-polypeptides using HPLC. As a result, the complete sequences of the F2-polypeptides were determined, including documentation of glycosylation of asparagine 54. The sequence of the cleavage-activation site of the WA2116 F0-protein was found to be distinctly different from this site in any other NDV F0-protein. Cleavage activation at the C termini of the F2-polypeptide regions was found to have occurred to approximately equivalent extents at arginines 82 and 85 of the AV and EG strains, but was restricted largely to arginine 85 of the V4 strain and completely to arginine 85 of the WA2116 strain. In each case cleavage activation was apparently succeeded by trimming of the basic residues from the newly formed C termini. Immunochemical analysis with antipeptide antisera showed that the extent of cleavage was influenced by amino acids adjacent to these arginines. These data provide insight into the substrate specificities of the enzymes involved in cleavage activation of the fusion protein precursors.     

A novel genotype of beak and feather disease virus in budgerigars (<Emphasis Type="Italic">Melopsittacus undulatus</Emphasis>)

House sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Five Newcastle disease virus (NDV) strains were isolated from house sparrows living around the poultry farms in southern China. These isolates were characterized by pathogenic assays and phylogenetic analysis. The results showed that all NDV isolates except one were velogenic and virulent for chickens. These four virulent strains for chickens possess the amino acid sequence ¹¹²R/K-R-Q-K/R-R-F¹¹⁷ in the F₀ cleavage site which is typical of velogenic NDV. Phylogenetic analysis indicated that these isolates belong to genotype VII and were closely related to the strains which were isolated from NDV outbreaks in chickens since 2000. One isolate of NDV from house sparrow belong to genotype II and was proved to be vaccine strain (Chicken/U.S./LaSota/46). The result of this study proved that house sparrow can carry the virulent NDV strains and the same genotype of viruses that are circulating in poultry are existing in house sparrows living around poultry farm in southern China.     

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.     

Detection of virulent Newcastle disease virus using a phage-capturing dot blot assay

Newcastle disease virus (NDV) strains can be classified as virulent or avirulent based upon the severity of the disease. Differentiation of the virus into virulent and avirulent is necessary for effective control of the disease. Biopanning experiments were performed using a disulfide constrained phage displayed heptapeptide library against three pathotypes of NDV strains: velogenic (highly virulent), mesogenic (moderately virulent) and lentogenic (avirulent). A phage clone bearing the peptide sequence SWGEYDM capable of distinguishing virulent from avirulent NDV strains was isolated. This phage clone was employed as a diagnostic reagent in a dot blot assay and it successfully detected only virulent NDV strains.     

Rapid pathotyping of Newcastle disease virus using a single-chain Fv displayed on phage against the C-terminal end of the F2 polypeptide

Filamentous bacteriophage display technology has been used to generate specific antibody fragments for differentiating virulent and avirulent Newcastle disease virus. A single-chain Fv fragment to the motif (112)RRQ(114), present at the F2 C-terminal end of many virulent Newcastle disease virus isolates, was isolated from a phage display library derived from a rabbit immunized with a peptide conjugate. An ELISA evaluation was carried out to test its ability to differentiate between 11 avirulent and 34 virulent NDV isolates. The antibody fragment reacted with 25/28 virulent viruses with the putative motif (112)RRQ(114). The three exceptions were viruses with an arginine instead of glycine, at position 110 of the fusion protein, just preceding the cleavage site. Five of six virulent isolates, whose predicted motif was different from that usually found in virulent strains, also tested negative. However, the antibody did react with one isolate with the motif (112)KRQ(114). There was no apparent reactivity with any of the avirulent isolates tested. We conclude that this antibody may, in the future, be a useful aid for the pathotyping of NDV isolates.     

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.     

The avian response to Newcastle disease virus

Newcastle disease virus (NDV) is classified as a member of the superfamily Mononegavirales in the family Paramyxoviridae. This virus family is divided into two subfamilies, the Paramyxovirinae and the Pneumovirinae. In 1993 the International Committee on the Taxonomy of Viruses rearranged the order of the Paramyxovirus genus and placed NDV within the Rubulavirus genus among the Paramyxovirinae. The enveloped virus has a negative sense single-stranded RNA genome of 15,186 kb which codes for an RNA directed RNA polymerase, hemagglutinin-neuraminidase protein, fusion protein, matrix protein, phosphoprotein and nucleoprotein in the 5' to 3' direction. The virus has a wide host range with most orders of birds reported to have been infected by NDV. Isolates are characterized by virulence in chickens and are categorized into three main pathotypes depending on severity of disease. Lentogenic isolates are of low virulence while viruses of intermediate virulence are termed mesogenic. Highly virulent viruses that cause high mortality in birds are termed neurotropic or viscerotropic velogenic. Velogenic NDV are List A pathogens that require reporting to the Office of International Epizootics and outbreaks result in strict trade embargoes. The primary molecular determinant for NDV pathogenicity is the fusion protein cleavage site amino acid sequence. Vaccination for NDV is primarily by mass application of live-virus vaccines among commercial poultry. Although protection is measured by presence of antibodies to NDV, vaccinated B-cell depleted chickens are resistant to disease. Consequently, immune protection involves responses that are presently incompletely defined.