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.     

Virulent Newcastle disease in Australia: molecular epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998-2000.

Gene sequence analysis of fusion (F) gene cleavage motifs and haemagglutinin-neuraminidase (HN) carboxyl-terminal extension sequences was used to analyse Newcastle disease viruses (NDV) associated with virulent outbreaks of the disease which occurred in New South Wales, Australia in 1998-2000. PCR fragments were amplified directly from diseased tissue or allantoic fluids and sequence analyses used for phylogenetic comparisons between these viruses and Australian reference NDV. F and HN gene sequence comparison showed a strong relationship to sequences derived from endemic Australian NDV rather than those of overseas viruses or wild bird isolates. Prior to notification of the 1998 outbreak, an NDV was isolated from chickens suffering respiratory disease that appeared to be the progenitor virus from which the virulent virus originated. In turn, these viruses are closely related to two previously isolated 'ancestor' viruses that have the same unique HN extension sequence.     

Molecular epidemiological investigation of velogenic Newcastle disease viruses from village chickens in Cambodia

Three isolates of Newcastle disease virus (NDV) were isolated from tracheal samples of dead village chickens in two provinces (Phnom Penh and Kampong Cham) in Cambodia during 2011–2012. All of these Cambodian NDV isolates were categorized as velogenic pathotype, based on in vivo pathogenicity tests and F cleavage site motif sequence (¹¹²RRRKRF¹¹⁷). The phylogenetic analysis and the evolutionary distances based on the sequences of the F gene revealed that all the three field isolates of NDV from Cambodia form a distinct cluster (VIIh) together with three Indonesian strains and were assigned to the genotype VII within the class II. Further phylogenetic analysis based on the hyper-variable region of the F gene revealed that some of NDV strains from Malaysia since the mid-2000s were also classified into the VIIh virus. This indicates that the VIIh NDVs are spreading through Southeast Asia. The present investigation, therefore, emphasizes the importance of further surveillance of NDV in neighboring countries as well as throughout Southeast Asia to contain further spreading of these VIIh viruses.     

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

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

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.     

Complete genome sequence of a virulent Newcastle disease virus isolated from an outbreak in chickens in Egypt

The complete genome sequence of a virulent Newcastle disease virus (NDV) isolated from chickens in Egypt was determined and compared to the sequence of NDV strains isolated from different parts of the world. The genome is 15,186 nucleotides (nt) long and consists of 6 genes in the order of 3′-N-P-M-F-HN-L-5′. The genome contains a 55-nt leader region at the 3′ end and a 114-nt trailer region at the 5′ end. Interestingly, the phylogenetic analysis showed that strain Egypt is closely related with the NDV strains isolated in China. In addition, the sequence of the fusion protein cleavage site of strain Egypt was identical to that of the NDV strain recently isolated in Mali. Determination of complete genome sequences of additional NDV strains from Africa is necessary to understand the epidemiology of currently circulating viruses in Africa.     

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

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

The use of in situ hybridization and immunohistochemistry to study the pathogenesis of various Newcastle disease virus strains and recombinants in embryonated chicken eggs

Avian paramyxovirus type 1, commonly referred to as Newcastle disease virus (NDV), is a serious pathogen of significant economic importance to the industry. To investigate the role of the fusion (F), hemagglutinin-neuraminidase (HN), and (P) phosphoprotein gene sequences in virulence, six strains of Newcastle disease virus (NDV) representing all pathotypes and seven recombinant strains created by reverse genetics were inoculated into 9-day-old chicken embryos. Tissues and chorioallantoic membranes (CAM) were harvested at 24-hour intervals post-inoculation. Riboprobe in situ hybridization and immunohistochemistry highlighted distinct tissue tropisms among the viruses. Presence of F and/or HN from virulent viruses inserted into lentogenic backbones caused dissemination of virus in a manner similar to wild type virulent viruses. Disruption of P gene decreased dissemination of velogeinic infectious clones. It is concluded that each of these genes contributes to pathogenicity.     

Pathotypical and genotypical characterization of strains of <Emphasis Type="Italic">Newcastle disease virus</Emphasis> isolated from outbreaks in chicken and goose flocks in some regions of China during 1985–2001

Summary.  Twenty-nine strains of Newcastle disease virus (NDV) isolated from outbreaks in chicken and goose flocks in several regions of China during 1985–2001 were characterized pathotypically and genotypically. All except one of these strains were velogenic. For genotyping, phylogenetic tree analysis (nt 47–420), restriction site mapping (nt 334–1682) and residue substitution analysis (residues 4–124) of the F gene were performed using sequences of our isolates and sequences of selected NDV strains from GenBank. The results revealed that most of these newly characterized strains belonged to six novel genetic groups that were designated as VIf, VIg, VIIc, VIId, VIIe and IX. The genotype IX viruses, to which the China challenge strain F48E8 used for vaccine evaluation belonged, were found only in China and still induced sporadic infections in certain areas. Isolates belonging to group VIf and VIg were distinct from previously reported members of genotype VI, with genetic distance from 2.5 to 12.1%. Subgenotype VIIc, VIId and VIIe viruses, which were distributed in clusters in the phylogenetic tree distinct from members of subgenotypes VIIa and VIIb, were responsible for disease outbreaks in chicken and goose flocks and circulated predominantly in southern China in recent years. Finally, cross-protective testing showed that specific-pathogen free (SPF) chickens vaccinated with La Sota vaccines can be fully protected against challenge by strains from genetic groups VIb, VIg, VIId and IX, indicating that the antigenic differences between strains of various genotypes are insufficient to change the cross-protection conferred by the commonly used vaccine. Received August 16, 2002; accepted January 6, 2003 Published online April 28, 2003     

Genetic and antigenic analysis of Newcastle disease viruses from recent outbreaks in Taiwan.

Portions of the haemagglutinin-neuraminidase (HN) and fusion protein (F) genes of Newcastle disease virus (NDV) isolated from recent outbreaks in Taiwan were amplified and sequenced. These isolates were velogenic, based on the amino acid sequences of the F protein cleavage site and the mean death time in chicken embryos. All the recent viruses contained the amino acid sequences 112RRQKR116 for the C-terminus of the F2 protein. The serological relatedness of recent isolates was determined using a serum neutralization (SN) test. Relatedness values, determined by a cross-SN test, revealed that all belonged to a single serotype but could be classified into distinct subtypes, suggesting that antigenic variations occurred in these isolates. Phylogenetic trees based on the nucleotide sequences of the HN and F genes revealed that recent Taiwanese isolates had evolved into two groups. Antigenic analysis also suggested that there are at least two groups of NDVs involved in recent outbreaks and that these outbreaks in Taiwan might have been caused by co-circulation of multiple velogenic NDV strains.     

Evolutionary dynamics of Newcastle disease virus

A comprehensive dataset of NDV genome sequences was evaluated using bioinformatics to characterize the evolutionary forces affecting NDV genomes. Despite evidence of recombination in most genes, only one event in the fusion gene of genotype V viruses produced evolutionarily viable progenies. The codon-associated rate of change for the six NDV proteins revealed that the highest rate of change occurred at the fusion protein. All proteins were under strong purifying (negative) selection; the fusion protein displayed the highest number of amino acids under positive selection. Regardless of the phylogenetic grouping or the level of virulence, the cleavage site motif was highly conserved implying that mutations at this site that result in changes of virulence may not be favored. The coding sequence of the fusion gene and the genomes of viruses from wild birds displayed higher yearly rates of change in virulent viruses than in viruses of low virulence, suggesting that an increase in virulence may accelerate the rate of NDV evolution.     

Nucleotide sequence analysis of the Newcastle disease virus nucleocapsid protein gene and phylogenetic relationships among the Paramyxoviridae

The nucleocapsid (N) protein genes from 24 Newcastle disease virus (NDV) isolates representing various pathotypes with different geographical and chronological origins were cloned and sequenced. The N-terminal region of the N protein to residue 401 was highly conserved among isolates with several conservative substitutions occurring that correlated with phylogenetic relationships. Variability of the N protein was detected in the C-terminal portion similar to what has been reported for other members of the Paramyxovirinae. Amino acids previously identified as invariant or highly conserved in N proteins of other paramyxoviruses were also present in the NDV protein. Phylogenetic analysis of N gene coding sequences among NDV isolates again demonstrated the existence of two major groups. One clade contained viruses that included vaccine and virulent strains isolated in the USA prior to 1970 while a second clade included vaccine and virulent viruses isolated worldwide. Comparison of N protein amino acid sequences among members of the Paramyxoviridae resulted in NDV and avian paramyxovirus 6 separating as a cluster distinct from the Rubulavirus genus. This provides further support for avian paramyxoviruses being considered for their own genus among the Paramyxovirinae.     

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

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

Phylogenetic relationships among virulent Newcastle disease virus isolates from the 2002-2003 outbreak in California and other recent outbreaks in North America

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.     

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

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

Effect of host selective pressure on Newcastle disease virus virulence

Newcastle disease virus (NDV) causes an economically important disease that can vary from clinically inapparent to highly virulent forms. Generally, NDV strains isolated from wild birds are non-pathogenic for chicken. However, there are evidences supporting the fact that avirulent viruses maintained in feral birds could have caused outbreaks of virulent NDV in poultry. The strain-specific difference in virulence is determined by structural variations on the fusion glycoprotein (F). More basic amino acids are present in the F cleavage site of virulent strains. Nevertheless, other regions have been involved in virulence determination. When we subjected an avirulent NDV isolated from a wild bird to a host change we found that the virus arose was virulent for chicken. Nucleotide changes in the F protein cleavage site amino acid sequence and in the hemagglutinin-neuraminidase protein sequence are reported.     

Fusion and Matrix Protein Gene Sequence Analysis of Paramyxoviruses of Type 1(PMV-1) Isolated from Pigeons in Slovenia

Paramyxoviruses of type 1 (PMV-l) isolated from pigeons were genetically analyzed. A part of the fusion and the matrix protein genes were amplified and sequenced, Typical amino acid sequences associated with virulence were determined at the fusion protein cleavage site in all PMV-1 isolates. All Slovene pigeon PMV-1 strains share high amino acid sequence similarity with other pigeon strains. In the phylogenetic tree, they are clustered together with pigeon PMV-1 isolates with moderate pathogenicity. Phylogenetic analysis obtained from the fusion and the matrix protein gene alignments showed the same branching order. Viruses circulating among pigeons were found to form quite unique lineage of virulent NDV strains.