Search

Virulent strains of Newcastle disease virus (NDV) cause Newcastle disease (ND), a devastating disease of poultry and wild birds. Phylogenetic analyses clearly distinguish historical isolates (obtained prior to 1960) from currently circulating viruses of class II genotypes V, VI, VII, and XII through XVIII. Here, partial and complete genomic sequences of recent virulent isolates of genotypes II and IX from China, Egypt, and India were found to be nearly identical to those of historical viruses isolated in the 1940s. Phylogenetic analysis, nucleotide distances, and rates of change demonstrate that these recent isolates have not evolved significantly from the most closely related ancestors from the 1940s. The low rates of change for these virulent viruses (7.05 × 10(-5) and 2.05 × 10(-5) per year, respectively) and the minimal genetic distances existing between these and historical viruses (0.3 to 1.2%) of the same genotypes indicate an unnatural origin. As with any other RNA virus, Newcastle disease virus is expected to evolve naturally; thus, these findings suggest that some recent field isolates should be excluded from evolutionary studies. Furthermore, phylogenetic analyses show that these recent virulent isolates are more closely related to virulent strains isolated during the 1940s, which have been and continue to be used in laboratory and experimental challenge studies. Since the preservation of viable viruses in the environment for over 6 decades is highly unlikely, it is possible that the source of some of the recent virulent viruses isolated from poultry and wild birds might be laboratory viruses., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

One year after a virulent Newcastle disease virus (vNDV) outbreak in Pakistan, the causative strain was present in vaccinated chickens of multiple farms despite the existence of high-average NDV-specific antibody titers (>4.75 log2). The data suggest a possible role of vaccinated birds as reservoirs of vNDV., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Despite rampant Newcastle disease virus (NDV) outbreaks in Africa for decades, the information about the genetic characteristics of the virulent strains circulating in West and Central Africa is still scarce. In this study, 96 complete NDV fusion gene sequences were obtained from poultry sampled in Cameroon, Central African Republic, Côte d'Ivoire, and Nigeria between 2006 and 2011. Based on rational criteria recently proposed for the classification of NDV strains into classes, genotypes, and subgenotypes, we revisited the classification of virulent strains, in particular those from West and Central Africa, leading to their grouping into genotype XIV and newly defined genotypes XVII and XVIII, each with two subgenotypes. Phylogenetic analyses revealed that several (sub)genotypes are found in almost every country. In Cameroon, most strains were related to vaccine strains, but a single genotype XVII strain was also found. Only three highly similar genotype XVII strains were detected in Central African Republic. Subgenotypes XVIIa, XVIIIa, and XVIIIb cocirculated in Côte d'Ivoire, while subgenotypes XIVa, XIVb, XVIIa, XVIIb, and XVIIIb were found in Nigeria. While these genotypes are so far geographically restricted, local and international trade of domestic and exotic birds may lead to their spread beyond West and Central Africa. A high genetic diversity, mutations in important neutralizing epitopes paired with suboptimal vaccination, various levels of clinical responses of poultry and wild birds to virulent strains, strains with new cleavage sites, and other genetic modifications found in these genotypes tend to undermine and complicate NDV management in Africa.

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.

Newcastle disease (ND) is a highly contagious, severe disease of poultry caused by pathogenic strains of Newcastle disease virus (NDV; or avian paramyxovirus-1). NDV is endemic in wild birds worldwide and one of the economically most important poultry pathogens. Most of the published strains are outbreak-associated strains, while the apathogenic NDV strains that occur in wild birds, posing a constant threat to poultry with their capability to convert into more virulent forms, have remained less studied. We screened for NDV RNA in cloacal and oropharyngeal samples from wild waterfowl in Finland during the years 2006 to 2010: 39 of 715 birds were positive (prevalence, 5.5%). The partial or full-length F genes of 37 strains were sequenced for phylogenetic purposes. We also characterized viruses derived from three NDV outbreaks in Finland and discuss the relationships between these outbreak-associated and the wild-bird-associated strains. We found that all waterfowl NDV isolates were lentogenic strains of class I or class II genotype I. We also isolated a genetically distinct class I strain (teal/Finland/13111/2008) grouping phylogenetically together with only strain HIECK87191, isolated in Northern Ireland in 1987. Together they seem to form a novel class I genotype genetically differing from other known NDVs by at least 12%.

Newcastle disease (ND) is one of the most important diseases of poultry, negatively affecting poultry production worldwide. The disease is caused by Newcastle disease virus (NDV) or avian paramyxovirus type 1 (APMV-1), a negative-sense single-stranded RNA virus of the genus Avulavirus, family Paramyxoviridae. Although all NDV isolates characterized to date belong to a single serotype of APMV-1, significant genetic diversity has been described between different NDV isolates. Here we present the complete genome sequence and the clinicopathological characterization of a virulent Newcastle disease virus isolate (NDV-Peru/08) obtained from poultry during an outbreak of ND in Peru in 2008. Phylogenetic reconstruction and analysis of the evolutionary distances between NDV-Peru/08 and other isolates representing established NDV genotypes revealed the existence of large genomic and amino differences that clearly distinguish this isolate from viruses of typical NDV genotypes. Although NDV-Peru/08 is a genetically distinct virus, pathogenesis studies conducted with chickens revealed that NDV-Peru/08 infection results in clinical signs characteristic of velogenic viscerotropic NDV strains. Additionally, vaccination studies have shown that an inactivated NDV-LaSota/46 vaccine conferred full protection from NDV-Peru/08-induced clinical disease and mortality. This represents the first complete characterization of a virulent NDV isolate from South America.

Eight Newcastle disease virus isolates from Pakistan were sequenced and characterized. A PCR matrix gene assay, designed to detect all avian paramyxovirus 1, did not detect four of the isolates. A new matrix gene test that detected all isolates was developed. Phylogenetic analysis and pathotyping confirmed that virulent viruses of different genotypes are circulating in Pakistan.

As part of West Nile virus surveillance programs in Rhode Island and eastern Texas between 2000 and 2007, brain tissue was collected from 5,608 dead birds representing 21 avian orders found in public places or reported by homeowners. Fifteen Newcastle disease virus isolates were recovered only from birds of the order Columbiformes and were positively identified by the USDA-validated real-time reverse transcription-PCR assay targeting the matrix gene and more specifically as pigeon paramyxovirus serotype 1 (PPMV-1) by hemagglutinin inhibition with monoclonal antibodies. Based upon partial genomic sequencing and phylogenetic analysis, the newly isolated viruses represent a distinct sublineage within class II genotype VIb. All of the viruses (15/15) were classified as virulent based upon their fusion cleavage site motif ((112)RRKKRF(117)) and intracerebral pathogenicity indices of >0.7 (ranging from 0.98 to 1.35); however, these viruses escaped detection by the fusion gene-based real-time PCR test for virulence. Modifications introduced to the probe site of the fusion gene-based assay allowed rapid virulence detection within this distinct sublineage.

Three cases of Newcastle disease virus (NDV) found in nature had the lentogenic motif (112)G-R-Q-G-R-L(117) in their fusion protein cleavage sites. However, both intracerebral pathogenicity and intravenous pathogenicity indexes showed that these NDV isolates were virulent. In comparison with the LaSota live virus vaccine, these viruses had significant genetic variations in the hemagglutinin-neuraminidase gene.

Thirty Newcastle disease virus (NDV) strains isolated from outbreaks in China during 1996 to 2005 were characterized pathotypically and genotypically. All strains except one were velogenic. An analysis of the variable region (nucleotides 47 to 420) of the F gene indicated that 6 isolates belonged to genotype II, 3 to genotype III, 1 (isolated from a pigeon) to genotype VI, and 20 to genotype VII. Isolates belonging to genotype VII were further divided into five subtypes, VIIa, VIIb, VIIc, VIId, and VIIe, and subtype VIId was made up of VIId1 to VIId5. These results showed that genotype VII isolates might have been the most prevalent in China during the past two decades. Genotype VII isolates shared high homology, but the homology was less than that between genotype VII viruses and the vaccine virus LaSota. Among these NDV isolates, 25 isolates had the velogenic motif (112)R/K-R-Q-K/R-R-F(117) that is consistent with results of the biological tests. However, four of five LaSota-type isolates that contained the lentogenic motif (112)G-R-Q-G-R-L(117) were velogenic, except SY/03, in the view of the biological test. The majority of genotype VII isolates had lost one or two N-glycosylation sites. Finally, a cross-protection experiment in which specific-pathogen-free chickens vaccinated with LaSota were challenged by six NDV isolates showed that more than three isolates were antigenic variants that could be responsible for recent outbreaks of Newcastle disease.

Newcastle disease viruses isolated from Hong Kong live bird markets (LBMs) were not detected by a USDA-validated matrix gene real-time reverse transcription-PCR (RT-PCR) assay. Based upon phylogenetic analysis of the fusion gene, these viruses were related to lentogenic class I viruses found in U.S. LBMs and wild waterfowl. An alternative real-time RT-PCR assay which complements the matrix gene assay was developed to efficiently detect class I viruses.

Newcastle disease virus (NDV) strains are classified as having high (velogenic), intermediate (mesogenic), or low (lentogenic) pathogenesis and virulence in chickens. Recent studies have established that the hemagglutinin-neuraminidase (HN) protein plays an important role in viral tropism and virulence. A monoclonal antibody (AVS-I) has previously been shown to be specific for lentogenic strains of NDV (Srinivasappa et al., Avian Dis. 30:562-567, 1986) and is routinely used to identify these strains. We have used competition antibody binding assays with a previously characterized panel of monoclonal antibodies, binding to chimeric HN proteins, and the characterization of an escape mutant to localize the binding site of AVS-I to the extreme carboxy terminus of the protein. In addition, we have shown that AVS-I does recognize at least one mesogenic strain and one velogenic strain of the virus, calling into question the potential of this antibody as a diagnostic reagent for avirulent NDV strains.

We have evaluated a diagnostic system based on the loop-mediated isothermal amplification (LAMP) assay for the rapid, simple, and sensitive detection of Newcastle disease virus (NDV) directly from culture isolates as well as clinical samples. By using one set of specific primers targeting the fusion protein gene, the LAMP assay rapidly amplified the target gene within 2 h, requiring only a regular laboratory water bath or heat block for reaction. The results obtained from testing the genomes of 38 NDV strains, other different viruses, and clinical samples of experimentally infected chickens showed that LAMP was as sensitive and specific as nested PCR. All LAMP-positive samples were positive by nested PCR. The LAMP assay is faster than nested PCR, cost-effective, and easy to perform. Our results clearly demonstrate that the LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV infection.

Isolates from the 2002-2003 virulent Newcastle disease virus (v-NDV) outbreak in southern California, Nevada, Arizona, and Texas in the United States were compared to each other along with recent v-NDV isolates from Mexico and Central America and reference avian paramyxovirus type 1 strains. Nucleotide sequencing and phylogenetic analyses were conducted on a 1,195-base genomic segment composing the 3' region of the matrix (M) protein gene and a 5' portion of the fusion (F) protein gene including the M-F intergenic region. This encompasses coding sequences for the nuclear localization signal of the M protein and the F protein cleavage activation site. A dibasic amino acid motif was present at the predicted F protein cleavage activation site in all v-NDVs, including the California 2002-2003, Arizona, Nevada, Texas, Mexico, and Central America isolates. Phylogenetic analyses demonstrated that the California 2002-2003, Arizona, Nevada, and Texas viruses were most closely related to isolates from Mexico and Central America. An isolate from Texas obtained during 2003 appeared to represent a separate introduction of v-NDV into the United States, as this virus was even more closely related to the Mexico 2000 isolates than the California, Arizona, and Nevada viruses. The close phylogenetic relationship between the recent 2002-2003 U.S. v-NDV isolates and those viruses from countries geographically close to the United States warrants continued surveillance of commercial and noncommercial poultry for early detection of highly virulent NDV.

A real-time reverse-transcription PCR (RRT-PCR) was developed to detect avian paramyxovirus 1 (APMV-1) RNA, also referred to as Newcastle disease virus (NDV), in clinical samples from birds. The assay uses a single-tube protocol with fluorogenic hydrolysis probes. Oligonucleotide primers and probes were designed to detect sequences from a conserved region of the matrix protein (M) gene that recognized a diverse set (n = 44) of APMV-1 isolates. A second primer-probe set was targeted to sequences in the fusion protein (F) gene that code for the cleavage site and detect potentially virulent NDV isolates. A third set, also directed against the M gene, was specific for the North American (N.A.) pre-1960 genotype that includes the common vaccine strains used in commercial poultry in the United States. The APMV-1 M gene, N.A. pre-1960 M gene, and F gene probe sets were capable of detecting approximately 10(3), 10(2), and 10(4) genome copies, respectively, with in vitro-transcribed RNA. Both M gene assays could detect approximately 10(1) 50% egg infective doses (EID(50)), and the F gene assay could detect approximately 10(3) EID(50). The RRT-PCR test was used to examine clinical samples from chickens experimentally infected with the NDV strain responsible for a recent epizootic in the southwestern United States. Overall, a positive correlation was obtained between the RRT-PCR results and virus isolation for NDV from clinical samples.

Sequences encompassing cleavage sites of fusion protein genes were obtained for avian paramyxovirus 1 isolates from cormorants in Canada. All isolates have the virulent cleavage site SRGRRQKR*FVG. They form a distinct cluster within isolates obtained around the world and may represent a novel genotype closely related to genotype V.

Seven Newcastle disease (ND) virus (NDV) isolates which were recovered from ND outbreaks in chicken and pigeon flocks in China and Taiwan between 1996 and 2000 were genotypically and pathotypically characterized. By phylogenetic analysis of the fusion protein genes, isolates Ch-A7/96, Ch/98-3, Ch/99, Ch/2000, and TW/2000 were placed into two novel subgenotypes, VIIc and VIId. Isolate Ch/98-1 was grouped into subgenotype VIb, while Ch-W6/96 was proven to be a mixture of isolates Ch-A7/96 and Ch/98-1. These isolates were pathotyped as viscerotropic velogenic for Ch/98-3, Ch/99, Ch/2000, and TW/2000; neurotropic velogenic for Ch-A7/96; and mesogenic for Ch/98-1. Three separate, comparative, genetic analyses of the F genes, including genetic distance measurement, phylogenetic tree analysis, and residue substitution analysis, were performed with our isolates and selected NDV strains from GenBank. Results showed that the close genetic similarity provided evidence for the epidemiological linkage between the outbreaks in China and Taiwan and that the 1990s outbreaks in Asia, the Middle East, Africa, and Europe constituted the fourth panzootic of ND. In combination with epidemiological analysis, an evolutionary model of the NDV strains, representative of the direction of transmission within the NDV strains, was proposed, and epidemiology of NDV transmission was evaluated with emphasis on molecular aspects. Finally, a cross-protective experiment indicated that at least one strain (Ch-A7/96) among our NDV isolates was an antigenic variant, responsible for recent outbreaks of ND in vaccinated chicken flocks.

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.

Degenerate oligonucleotide primers were synthesized to amplify nucleotide sequences from portions of the fusion protein and matrix protein genes of Newcastle disease virus (NDV) genomic RNA that could be used diagnostically. These primers were used in a single-tube reverse transcription PCR of NDV genomic RNA coupled to direct nucleotide sequencing of the amplified product to characterize more than 30 NDV isolates. In agreement with previous reports, differences in the fusion protein cleavage sequence that correlated genotypically with virulence among various NDV pathotypes were detected. By using sequences generated from the matrix protein gene coding for the nuclear localization signal, lentogenic viruses were again grouped phylogenetically separate from other pathotypes. These techniques were applied to compare neurotropic velogenic viruses isolated from an outbreak of Newcastle disease in cormorants and turkeys. Cormorant NDV isolates and an NDV isolate from an infected turkey flock in North Dakota had the fusion protein cleavage sequence 109SRGRRQKRFVG119. The R-for-G substitution at position 110 may be unique for the cormorant-type isolates. Although the amino acid sequences from the fusion protein cleavage site were identical, nucleotide sequence data correlate the outbreak in turkeys to a cormorant virus isolate from Minnesota and not to a cormorant virus isolate from Michigan. On the basis of sequence information, the cormorant isolates are virulent viruses related to isolates of psittacine origin, possibly genotypically distinct from other velogenic NDV isolates. These techniques can be used reliably for Newcastle disease epidemiology and for prediction of pathotypes of NDV isolates without traditional live-bird inoculations.

Using the microtiter system, titration of Newcastle disease virus infectivity and neutralizing antibodies was carried out in chicken embryo fibroblasts grown in "U" or flat-bottomed plates. Infectivity was detected by a combined hemadsorption-hemagglutination method. Inhibition of that reaction indicated the presence of neutralizing antibodies. A 24-h microneutralization test was developed and compared to the plaque neutralization and microhemagglutination inhibition test. Reproducibility of the microneutralization test was statistically analyzed.

A liquid antibody microculture plaque assay and the variables that govern its effectiveness are described. The assay is based on the principle that low concentrations of homologous antibody can inhibit secondary plaque formation without inhibiting formation of primary plaques. Thus, clear plaques that followed a linear dose response were produced. The assay was found to be more rapid, less cumbersome, and less expensive than assays using agar overlays and larger tissue culture plates. It was reproducible, quantitative, and had about the same sensitivity as the agar overlay technique in measuring infectious coxsackievirus type B-3. It was more sensitive in assaying adenovirus type 3 and Western equine encephalomyelitis, vesicular stomatitis, Semliki forest, Sendai, Sindbis, and Newcastle disease viruses than were liquid, carboxymethylcellulose, and methylcellulose microculture plaque assays. The variables influencing sensitivity and accuracy, as determined by using coxsackievirus type B-3, were: (i) the inoculum volume of virus; (ii) the incubation period of virus; and (iii) the incubation temperature.

In an effort to improve current technology for detection of Newcastle disease virus in convalescent birds, a procedure has been developed for efficient reactivation of virus that has been neutralized by antibody. The reactivation capabilities of fluorocarbon treatment, ultrasonic treatment, pH extremes, and proteolytic digestion were evaluated using the LaSota strain of virus. Reactivation was maximum after proteolytic digestion with either trypsin or papain, and reactivation effciency was up to 100%, depending on the enzyme used for digestion and the amount of antibody in the neutralization mixture. Reactivation at pH extremes was considerably less efficient than reactiviation by proteolytic digestion, and neither fluorocarbon nor ultrasonic treatments effectively recovered antibody-neutralized Newcastle disease virus.