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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.)

Newcastle disease (ND), caused by the virulent Newcastle disease virus (NDV), is one of the most important viral diseases of birds globally, but little is currently known regarding enzootic trends of NDV in northeastern China, especially for class I viruses. Thus, we performed a surveillance study for NDV in northeastern China from 2013 to 2015. A total 755 samples from wild and domestic birds in wetlands and live bird markets (LBMs) were collected, and 10 isolates of NDV were identified. Genetic and phylogenetic analyses showed that five isolates from LBMs belong to class I subgenotype 1b, two (one from wild birds and one from LBMs) belong to the vaccine-like class II genotype II, and three (all from wild birds) belong to class II subgenotype Ib. Interestingly, the five class I isolates had epidemiological connections with viruses from southern, eastern, and southeastern China. Our findings, together with recent prevalence trends of class I and virulent class II NDV in China, suggest possible virus transmission between wild and domestic birds and the potential for an NDV epidemic in the future., (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.)

In West and Central Africa, virulent Newcastle disease virus (NDV) strains of the recently identified genotypes XIV, XVII, and XVIII are enzootic in poultry, representing a considerable threat to the sector. The increasing number of reports of virulent strains in wild birds at least in other parts of the world raised the question of a potential role of wild birds in the spread of virulent NDV in sub-Saharan Africa as well. We investigated 1,723 asymptomatic birds sampled at live-bird markets and sites important for wild-bird conservation in Nigeria and 19 sick or dead wild birds in Côte d'Ivoire for NDV class I and II. Typical avirulent wild-type genotype I strains were found in wild waterfowl in wetlands in northeastern Nigeria. They were unrelated to vaccine strains, and the involvement of inter- or intracontinental migratory birds in their circulation in the region is suggested. Phylogenetic analyses also revealed that genotype VI strains found in pigeons, including some putative new subgenotype VIh and VIi strains, were introduced on multiple separate occasions in Nigeria. A single virulent genotype XVIII strain was found in a dead wild bird in Côte d'Ivoire, probably as a result of spillover from sick poultry. In conclusion, screening of wild birds and pigeons for NDV revealed the presence a variety of virulent and avirulent strains in West Africa but did not provide strong evidence that wild birds play an important role in the spread of virulent strains in the region.

Newcastle disease, one of the most important health problems that affects the poultry industry around the world, is caused by virulent strains of Newcastle disease virus. Newcastle disease virus is considered to be endemic in several countries in the Americas, including Mexico. In order to control Newcastle disease outbreaks and spread, intensive vaccination programs, which include vaccines formulated with strains isolated at least 60 years ago, have been established. These vaccines are dissimilar in genotype to the virulent Newcastle disease viruses that had been circulating in Mexico until 2008. Here, 28 isolates obtained between 2008 and 2011 from different regions of Mexico from free-living wild birds, captive wild birds, and poultry were phylogenetically and biologically characterized in order to study the recent epidemiology of Newcastle disease viruses in Mexico. Here we demonstrate that, until recently, virulent viruses from genotype V continued to circulate and evolve in the country. All of the Newcastle disease viruses of low virulence, mostly isolated from nonvaccinated free-living wild birds and captive wild birds, were highly similar to LaSota (genotype II) and PHY-LMV42 (genotype I) vaccine strains. These findings, together with the discovery of two virulent viruses at the Mexican zoo, suggest that Newcastle disease viruses may be escaping from poultry into the environment.

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 virus (NDV) is one of the most important viral diseases of birds. Wild birds constitute a natural reservoir of low-virulence viruses, while poultry are the main reservoir of virulent strains. Exchange of virus between these reservoirs represents a risk for both bird populations. Samples from wild and domestic birds collected between 2006 and 2010 in Luxembourg were analyzed for NDV. Three similar avirulent genotype I strains were found in ducks during consecutive years, suggesting that the virus may have survived and spread locally. However, separate introductions cannot be excluded, because no recent complete F gene sequences of genotype I from other European countries are available. Detection of vaccine-like strains in wild waterbirds suggested the spread of vaccine strains, despite the nonvaccination policy in Luxembourg. Among domestic birds, only one chicken was positive for a genotype II strain differing from the LaSota vaccine and exhibiting a so-far-unrecognized fusion protein cleavage site of predicted low virulence. Three genotype VI strains from pigeons were the only virulent strains found. The circulation of NDV in wild and free-ranging domestic birds warrants continuous surveillance because of increased concern that low-virulence wild-bird viruses could become more virulent in domestic populations.

We report here the complete genomic sequence of a novel Newcastle disease virus (NDV) strain, egret/China/Guangxi/2011, isolated from an egret in Guangxi Province, southern China. A phylogenetic analysis based on a fusion gene comparison with different NDV strains revealed that egret/China/Guangxi/2011 was phylogenetically close to genotype VIIa NDV, and the deduced amino acid sequence was (112)R-R-R-K-R-F(117) at the fusion protein cleavage site. The whole nucleotide sequence had the highest homology (93.3%) with the sequence of strain chicken/Sukorejo/019/10 (GenBank accession number HQ697255). This study will help us to understand the epidemiology and molecular characteristics of Newcastle disease virus in a migratory egret.

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%.

The complete genome sequence of an African Newcastle disease virus (NDV) strain isolated from a chicken in Togo in 2009 was determined. The genome is 15,198 nucleotides (nt) in length and is classified in genotype VII in the class II cluster. Compared to common vaccine strains, the African strain contains a previously described 6-nt insert in the downstream untranslated region of the N gene and a novel 6-nt insert in the HN-L intergenic region. Genome length differences are a marker of the natural history of NDV. This is the first description of a class II NDV strain with a genome of 15,198 nt and a 6-nt insert in the HN-L intergenic region. Sequence divergence relative to vaccine strains was substantial, likely contributes to outbreaks, and illustrates the continued evolution of new NDV strains in West Africa.

Newcastle disease virus (NDV) was isolated from an outbreak in layer chickens in the Dominican Republic in 2008. Infections with this isolate led to a 100% apparent case fatality rate in birds. Complete genome sequencing revealed that the isolate does not belong to any of the previously described NDV genotypes. Similarly, large differences were observed in the amino acid sequence of the fusion and hemagglutinin-neuraminidase proteins in comparison with all known NDV genotypes, suggesting the existence of an unknown reservoir for NDV. The work presented here represents the first complete genome sequence of NDV in the Dominican Republic.

Eight highly virulent Newcastle disease virus (NDV) strains were isolated from vaccinated commercial chickens in Indonesia during outbreaks in 2009 and 2010. The complete genome sequences of two NDV strains and the sequences of the surface protein genes (F and HN) of six other strains were determined. Phylogenetic analysis classified them into two new subgroups of genotype VII in the class II cluster that were genetically distinct from vaccine strains. This is the first report of complete genome sequences of NDV strains isolated from chickens in Indonesia.

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.

The persistence of 3 low-pathogenicity avian influenza viruses (LPAIV) (H4N6, H5N1, and H6N8) and one human influenza virus (H1N1) as well as Newcastle disease virus (NDV) and enteric cytopathogenic bovine orphan (ECBO) virus was investigated in lake sediment, duck feces, and duck meat at 30, 20, 10, and 0°C using a germ carrier technique. Virus-loaded germ carriers were incubated in each substrate, and residual infectivity of the eluted virus was quantified on cell culture after regular intervals for a maximum of 24 weeks. Data were analyzed by a linear regression model to calculate T(90) values (time required for 90% loss of virus infectivity) and estimated persistence of the viruses. In general, the persistence of all of the viruses was highest in lake sediment, followed by feces, and was the lowest in duck meat at all temperatures. For the avian influenza virus subtypes, T(90) values in sediment ranged from 5 to 11, 13 to 18, 43 to 54, and 66 to 394 days at 30, 20, 10, and 0°C, respectively, which were 2 to 5 times higher than the T(90) values of the viruses in the feces and meat. Although the individual viruses vary in tenacity, the survival time of influenza viruses was shorter than that of NDV and ECBO virus in all substrates. The results of this study suggest that lake sediment may act as a long-term source of influenza viruses in the aquatic habitat, while the viruses may remain infectious for extended periods of time in duck feces and meat at low temperatures, allowing persistence of the viruses in the environment over winter.

Virulent strains of Newcastle disease virus ([NDV] also known as avian paramyxovirus type 1) can be discriminated from low-virulence strains by the presence of multiple basic amino acid residues at the proteolytic cleavage site of the fusion (F) protein. However, some NDV variants isolated from pigeons (pigeon paramyxovirus type 1 [PPMV-1]) have low levels of virulence, despite the fact that their F protein cleavage sites contain a multibasic amino acid sequence and have the same functionality as that of virulent strains. To determine the molecular basis of this discrepancy, we examined the role of the internal proteins in NDV virulence. Using reverse genetics, the genes encoding the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), and large polymerase protein (L) were exchanged between the nonvirulent PPMV-1 strain AV324 and the highly virulent NDV strain Herts. Recombinant viruses were evaluated for their pathogenicities and replication levels in day-old chickens, and viral genome replication and plaque sizes were examined in cell culture monolayers. We also tested the contributions of the individual NP, P, and L proteins to the activity of the viral replication complex in an in vitro replication assay. The results showed that the replication proteins of Herts are more active than those of AV324 and that the activity of the viral replication complex is directly related to virulence. Although the M protein affected viral replication in vitro, it had only a minor effect on virulence.

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.

Low-virulence Newcastle disease viruses (loNDV) are frequently recovered from wild bird species, but little is known about their distribution, genetic diversity, or potential to cause disease in poultry. NDV isolates recovered from cloacal samples of apparently healthy waterfowl and shorebirds (WS) in the United States during 1986 to 2005 were examined for genomic diversity and their potential for virulence (n = 249). In addition 19 loNDV isolates from U.S. live bird markets (LBMs) were analyzed and found to be genetically distinct from NDV used in live vaccines but related to WS-origin NDV. Phylogenetic analysis of the fusion protein identified nine novel genotypes among the class I NDV, and new genomic subgroups were identified among genotypes I and II of the class II viruses. The WS-origin viruses exhibited broad genetic and antigenic diversity, and some WS genotypes displayed a closer phylogenetic relationship to LBM-origin NDV. All NDV were predicted to be lentogenic based upon sequencing of the fusion cleavage site, intracerebral pathogenicity index, or mean death time in embryo assays. The USDA real-time reverse transcription-PCR assay, which targets the matrix gene, identified nearly all of the class II NDV tested but failed to detect class I viruses from both LBM and WS. The close phylogenetic proximity of some WS and LBM loNDV suggests that viral transmission may occur among wild birds and poultry; however, these events may occur unnoticed due to the broad genetic diversity of loNDV, the lentogenic presentation in birds, and the limitations of current rapid diagnostic tools.

An unknown virus was isolated from a lung biopsy sample and multiple other samples from a patient who developed a lethal case of pneumonia following a peripheral blood stem cell transplant. A random PCR-based molecular screening method was used to identify the infectious agent as avian paramyxovirus 1 (APMV-1; a group encompassing Newcastle disease virus), which is a highly contagious poultry pathogen that has only rarely been found in human infections. Immunohistochemical analysis confirmed the presence of APMV-1 antigen in sloughed alveolar cells in lung tissue from autopsy. Sequence from the human isolate showed that it was most closely related to virulent pigeon strains of APMV-1. This is the most completely documented case of a systemic human infection caused by APMV-1 and is the first report of an association between this virus and a fatal disease in a human.

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.

Paramyxoviruses such as human parainfluenza viruses that bear inserts encoding protective antigens of heterologous viruses can induce an effective immunity against the heterologous viruses in experimental animals. However, vectors based on common human pathogens would be expected to be restricted in replication in the adult human population due to high seroprevalence, an effect that would reduce vector immunogenicity. To address this issue, we evaluated Newcastle disease virus (NDV), an avian paramyxovirus that is serotypically distinct from common human pathogens, as a vaccine vector. Two strains were evaluated: the attenuated vaccine strain LaSota (NDV-LS) that replicates mostly in the chicken respiratory tract and the Beaudette C (NDV-BC) strain of intermediate virulence that produces mild systemic infection in chickens. A recombinant version of each virus was modified by the insertion, between the P and M genes, of a gene cassette encoding the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN) protein, a test antigen with considerable historic data. The recombinant viruses were administered to African green monkeys (NDV-BC and NDV-LS) and rhesus monkeys (NDV-BC only) by combined intranasal and intratracheal routes at a dose of 10(6.5) PFU per site, with a second equivalent dose administered 28 days later. Little or no virus shedding was detected in nose-throat swabs or tracheal lavages following immunization with either strain. In a separate experiment, direct examination of lung tissue confirmed a highly attenuated, restricted pattern of replication by parental NDV-BC. The serum antibody response to the foreign HN protein induced by the first immunization with either NDV vector was somewhat less than that observed following a wild-type HPIV3 infection; however, the titer following the second dose exceeded that observed with HPIV3 infection, even though HPIV3 replicates much more efficiently than NDV in these animals. NDV appears to be a promising vector for the development of vaccines for humans; one application would be in controlling localized outbreaks of emerging pathogens.

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.

An earlier report (M. Sakaguchi et al., Vaccine 16:472-479, 1998) showed that recombinant Marek's disease virus type 1 (rMDV1) expressing the fusion (F) protein of Newcastle disease virus (NDV-F) under the control of the simian virus 40 late promoter [rMDV1-US10L(F)] protected specific pathogen-free chickens from NDV challenge, but not commercial chickens with maternal antibodies against NDV and MDV1. In the present study, we constructed an improved polyvalent vaccine based on MDV1 against MDV and NDV in commercial chickens with maternal antibodies. The study can be summarized as follows. (i) We constructed rMDV1 expressing NDV-F under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10P(F)]. (ii) Much less NDV-F protein was expressed in cells infected with rMDV1-US10P(F) than in those infected with rMDV1-US10L(F). (iii) The antibody response against NDV-F and MDV1 antigens of commercial chickens vaccinated with rMDV1-US10P(F) was much stronger and faster than with rMDV1-US10L(F), and a high level of antibody against NDV-F persisted for over 80 weeks postvaccination. (iv) rMDV1-US10P(F) was readily reisolated from the vaccinated chickens, and the recovered viruses were found to express NDV-F. (v) Vaccination of commercial chickens having maternal antibodies to rMDV1-US10P(F) completely protected them from NDV challenge. (vi) rMDV1-US10P(F) offered the same degree of protection against very virulent MDV1 as the parental MDV1 and commercial vaccines. These results indicate that rMDV1-US10P(F) is an effective and stable polyvalent vaccine against both Marek's and Newcastle diseases even in the presence of maternal antibodies.

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.

Temperature-sensitive (ts) mutants of Newcastle disease virus have been isolated and characterized genetically (complementation), biochemically (RNA synthesis) and biologically (fusion from within and hemadsorption). Fifteen of these mutants have been divided into five complementation groups. Groups A (five mutants) and E (one mutant) are ts for RNA synthesis (RNA-) as well as for the other functions. Group B contains four RNA+ mutants of which one is ts for fusion, one for hemadsorption and two for neither function. Group C contains one RNA+ mutant which is a poor cell fuser. Group D contains two RNA+ mutants which are ts for fusion. In addition, two noncomplementing mutants (group BC) fail to complement both group B and group C mutants while exhibiting complementation with mutants in groups A, D, and E.

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.

Evidence is presented which confirms that temperature-sensitive (ts) mutants with an RNA- phenotype are spontaneously selected in persistent infection of cell lines with Newcastle disease virus. Persistently infected BHK-21 cells, maintained since 1973, produce no interferon and are completely susceptible to vesicular stomatitis virus. Persistent infection of a canine kidney cell line (MDCK) terminated with destruction of all cells at about 100 days. Even under these conditions, a high proportion (33%) of RNA- temperature-sensitive mutants was present in the virus population 60 days after the infection was initiated.

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.

The antigenic relationship between pairs of plaque mutants of Newcastle disease virus (NDV) derived from laboratory strains and from isolates from the 1971-72 California epizootic were examined by kinetic neutralization test. Comparing four sets of mutants from laboratory strains by both kinetic neutralization and hemagglutination inhibition tests, a similarity was found in the antigenic relationship expressed as an r value with both tests. However, kinetic neutralization was the more precise as well as sensitive assay. Antigenic diversity was greatest between pairs of mutants from different strains, but distinctions could also be made between mutants from the same strains such as Herts-L and Herts-S with an r value of 36%. Examination of mutants from the California epizootic isolated from separate locations and at different times showed antigenic divergence which was greatest between two red-plaque mutants with an r value of 39%. Antigenic distinctions were found between a red- and clear-plaque mutant obtained from isolates taken from brain and tracheal swabs of one infected chicken. In addition to antigenic divergence found between pairs of some mutants, two of the clear-plaque mutants reacted more avidly with antibody than did the corresponding red-plaque partner. Thus, both differences in antigenicity and avidity can be found among these NDV mutants. The antigenic variation found among these mutants is similar to that found within a serotype. This would imply that at the present NDV is a single serological type.

Avirulent (lentogenic) strains of Newcastle disease virus form plaques on chicken embryo lung monolayers in 48 to 72 h.

The temperature-sensitive defects of virus mutants isolated from L cells persistently infected with Newcastle disease virus (NDV) were analyzed. Genetic grouping of the mutants by complementation tests was attempted by using several different methods, including yield analysis, RNA synthesis, and heterozygote formation at 42 to 43 C, the nonpermissive temperature. In each case, specific interference prevented detection of complementation. This interference was shown to occur prior to or at the level of virus RNA synthesis. Temperature-shift experiments with five different NDV(pi) clones showed that virus replication begun at 37 C could not be completed at the nonpermissive temperature. The activity of the NDV-specific RNA-dependent RNA polymerase in the cytoplasm of infected chicken embryo cells was not stable and could not be demonstrated directly. However, indirect measurement of RNA polymerase activity at the nonpermissive temperature was accomplished by studying the kinetics of virus-specific RNA synthesis in infected cells after temperature shift. Two types of response were obtained: with three NDV(pi) clones, virus-specific RNA synthesis ceased immediately upon transfer of infected cells to 42 to 43 C, whereas in cells infected with two other NDV(pi) clones, RNA synthesis continued for several hours at this temperature. These results suggested that there may be two types of ts defects in NDV(pi), both associated with virus-specific RNA polymerase activity.

Development of Newcastle disease, after experimental and natural infection with the virulent strain VLT of Newcastle disease virus, and its growth and distribution in some selected tissues as assayed by the enumeration of plaques are reported.

The synthesis of different viral ribonucleic acid (RNA) species was studied in chick embryo (CE) and mouse L-cell cultures infected with the Herts strain of Newcastle disease virus (NDV(o)) and a mutant isolated from persistently infected L cells (NDV(pi)). In CE cell cultures, both viruses synthesized significant amounts of 54, 36, and 18S RNA. However, in L cells, synthesis of 54S virion RNA was markedly reduced. From these results, it seems likely that the low yield of infective virus in L cells is due to a deficient synthesis of 54S RNA in this host. On this basis, however, it is apparent that the "covert" replication of NDV(o) in L cells is due to factors other than viral RNA synthesis. When low concentrations of interferon were used to pretreat CE cells, a differential effect on the synthesis of various RNA species was observed. The 18S RNA of NDV(o) was more sensitive to interferon action than the 36 and the 54S RNA species. In contrast, the 18S RNA of NDV(pi) was less sensitive than the 36S and the 54S RNA. The inhibition of 54S RNA synthesis correlated with the reduction of viral yield and explained the greater sensitivity of NDV(pi) to interferon.

The thermal stability of Newcastle disease virus has been characterized in terms of the rate constants for inactivation of hemagglutinating activity (HA), neuraminidase activity (NA), and infectivity. Inactivation of HA results in the concomitant loss of NA. Infectivity, however, is much more thermolabile. Disintegration of the virus particle is not responsible for the identical rate constants for inactivation of HA and NA, nor is their parallel inactivation uncoupled in envelope fragments produced by pretreating the virus with phospholipase-C. The data indicate that a common envelope factor(s) can influence the thermal stability of both activities.

RNA-dependent DNA polymerase activity was found in peparations of a mutant of Newcastle disease virus. The enzyme activity was not found in wild-type virus preparations.

Standard procedures have been described for measuring paramyxovirus-induced hemolysis. The choice of these procedures is based on the analysis of the behavior of eight different strains of Newcastle disease virus. Significant strain-specific differences in hemolytic activity have been found. The presence of at least two kinds of inhibitors of hemolysis in virus preparations necessitates the use of purified virus when comparisons of hemolytic activities are to be made. In addition, it has been stressed that both hemagglutination titers and hemolysis determinations provide only relative values. Thus, quantitative comparisons can be made only with results obtained on the same day and with the same erythrocyte preparation.

Virus mutants (NDV(pi)) recovered from L cells persistently infected with Newcastle disease virus (NDV, Herts strain) are temperature-sensitive (ts) at 43 C, although the wild-type virus (NDV(o)) which initiated the persistent infection replicates normally at that temperature. To study the relationship between the ts marker of NDV(pi) and the other properties which distinguish this virus from NDV(o), NDV(pi) ts(+) revertants were selected at the nonpermissive temperature and NDV(o) ts mutants were generated by treating NDV(o) with nitrous acid. Spontaneously-occurring ts mutants in the Herts NDV population were also isolated. The different virus populations were characterized with regard to plaque size, virulence for eggs, and thermal stability of infectivity, hemagglutinin, and neuraminidase. The NDV(pi) ts(+) revertants, although no longer temperature-sensitive, retained NDV(pi) properties, whereas both spontaneously-occurring and mutagen-induced ts mutants remained wild-type in their other properties. These findings showed that the properties which characterized NDV(pi) were independent of the ts marker. However, the ts marker and the other markers of NDV(pi) were coselected during the persistent infection, and the combination of those markers appeared to be important in the outcome of NDV infection of L cells. NDV(pi) replicated productively in L cells, whereas NDV(o), the NDV(pi) ts(+) revertants, and the spontaneously-occurring ts mutants all yielded covert infections in L cells. The role of the selection of ts mutants in persistent infection was confirmed as follows: L cells were persistently infected with NDV(pi) ts(+) revertants and NDV(o) ts mutants. Virus recovered from the persistently infected cultures after eight cell passages was always temperature-sensitive and of smaller plaque size than the parental virus in chicken embryo cell cultures. Similar results were obtained with virus recovered from L-cell cultures persistently infected with two other velogenic strains of NDV, the Texas-GB and Kansas-Man strains. These results strongly suggest that selection of ts mutants during the persistent infection was not random and played a role in establishment or maintenance of the persistent infection, or both.

Virus mutants (NDV(pi)) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus have been previously reported by this laboratory to differ from the wild-type virus (NDV(o)) in several physical and biological properties. It has now been determined that, in addition to these differences, the NDV(pi) mutants are also spontaneously selected temperature-sensitive mutants. The temperature sensitivity of 10 NDV(pi) clones was confirmed by temperature inhibition, plaquing efficiency, and single-cycle yield experiments. The cut-off temperature, at which more than 90% of virus replication is inhibited was between 41 and 42 C. All 10 NDV(pi) clones were also found to be defective in virus-specific ribonucleic acid (RNA) synthesis in infected chick embryo cells at 42 C and are tentatively classified as RNA(-). The possible relationships of the temperature sensitivity, the other NDV(pi) properties, and the maintenance of the persistently infected state are discussed.

The virions of Newcastle disease virus (NDV) contained an enzyme that catalyzed the incorporation of ribonucleotides into ribonucleic acid (RNA). Optimal conditions for this polymerase activity were identical to the conditions for the vesicular stomatitis virus (VSV) polymerase, and both enzymes were active for longer times at 32 C than at 37 C. However, the specific activity of the NDV polymerase was less than 3% that of the VSV polymerase. Product RNA species from the NDV and VSV polymerase reactions annealed specifically to the homologous virion RNA species. Transcriptive intermediates containing product RNA attached to the respective virion RNA could be identified in both systems.