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

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

Virulence of six heterogeneous-origin Newcastle disease virus isolates before and after sequential passages in domestic chickens

Four serial passages of six Newcastle disease virus (NDV) isolates were performed in two-week-old White Leghorns. The viruses were recovered from chickens (Ckn-Live Bird Market and Ckn-Australia isolates), exotic (Yellow Nape [YN] Parrot, Pheasant, and Dove isolates) and wild birds (Anhinga isolate). Infected chickens were monitored clinically and humanely killed to sample tissues for histopathology and immunohistochemistry. Pathogenicity tests, to assess the virulence of the isolates for chickens, and sequence analysis of the fusion protein cleavage site were performed before and after passages. The moderately virulent Dove isolate became highly virulent with serial passage. The originally highly virulent Pheasant isolate had an increase in the intracerebral pathogenicity index (ICPI) and the intravenous pathogenicity index (IVPI) with passages in chickens. Virulence increase was not observed with Ckn-LBM, YN Parrot, Ckn-Australia, or Anhinga isolates after four chicken passages. The results demonstrate the high risk for domestic chickens represented by some NDV-infected non-poultry species, such as doves.     

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

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

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.     

Complete genome and clinicopathological characterization of a virulent Newcastle disease virus isolate from South America

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.     

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

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

Generation of a velogenic Newcastle disease virus from cDNA and expression of the green fluorescent protein

Summary Newcastle disease virus (NDV) is a pathogen that is important in the poultry industry worldwide. Specifically, the virulent (velogenic) NDV is aparticular threat because it has now occurred frequently worldwide. The outbreaks caused by highly virulent NDV in waterfowl and especially in goose flocks, have led to greater concern in recent years as aquatic birds were previously resistant to most virulent NDV strains from chickens. The molecular determinants of host tropism, virulence and emergence of NDV isolated from diseased goose flocks are poorly understood. In the present study, we rescued a highly virulent NDV isolated from a goose using the reverse genetics approach. Infectious virus was successfully generated by cotransfection of a full-length cDNA clone of the NDV strain ZJ1 with helper plasmids. The recombinant NDV was indistinguishable from the parental wild-type virus with respect to its growth kinetics in cell culture as well as its biological properties. A recombinant NDV expressing green fluorescent protein (GFP) was generated, and GFP was subsequently detected in cells and various organs from the infected chickens.     

Separate Evolution of Virulent Newcastle Disease Viruses from Mexico and Central America

An outbreak of Newcastle disease (ND) in poultry was reported in Belize in 2008. The characteristics of three virulent Newcastle disease virus (NDV) isolates from this outbreak (NDV-Belize-3/08, NDV-Belize-4/08, and NDV-Belize-12/08) were assessed by genomic analysis and by clinicopathological characterization in specific-pathogen-free (SPF) chickens. The results showed that all three strains belong to NDV genotype V and are virulent, as assessed by the intracerebral pathogenicity index and the polybasic amino acid sequence at the fusion protein cleavage site. In 4-week-old SPF chickens, NDV-Belize-3/08 behaved as a typical velogenic viscerotropic NDV strain, causing severe necrohemorrhagic lesions in the lymphoid organs, with systemic virus distribution. Phylogenetic analysis of multiple NDV genotype V representatives revealed that genotype V can be divided into three subgenotypes, namely, Va, Vb, and Vc, and that all tested Belizean isolates belong to subgenotype Vb. Furthermore, these isolates are nearly identical to a 2007 isolate from Honduras and appear to have evolved separately from other contemporary viruses circulating in Mexico, clustering into a new clade within NDV subgenotype Vb.     

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.     

Pathogenicity and immunogenicity in chickens of Newcastle disease viruses isolated from wild ducks

Summary Three Newcastle disease viruses (NDV) isolated from wild ducks in Japan were evaluated for their biological activities, pathogenicity and immunogenicity against one-day-old chickens. One isolate was of the mesogenic type and the other two were of the lentogenic type for chicken. The lentogenic isolates could induce enough immunity in chickens to protect them from challenge with a virulent strain of NDV.     

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

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

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

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

Detection and differentiation of virulent and avirulent strains of Newcastle disease virus by real-time PCR

A rapid diagnostic method based on the melting curve SYBR Green I real-time PCR analysis was developed to detect and differentiate Newcastle disease virus (NDV) strains. Degenerated primers based on the cleavage site sequence of the F0 gene were designed to detect specific sequences characteristic of virulent and avirulent strains of NDV.Eighteen strains of NDV from four lineages were identified and grouped into virulent and avirulent strains. Peaks on the melting temperature graph with melting temperature values between 80.00 and 83.80 °C were observed for lentogenic (avirulent) strains. T_m values higher than 83.80 were observed for virulent (mesogenic and velogenic) strains. The detection limit of real-time PCR was 2 × 10² plasmid copies per reaction or 10² EID₅₀ for velogenic strains and 10³ EID₅₀ for lentogenic strains.The results obtained in this study demonstrate the possible applications for melting curve real-time PCR analysis in laboratory practice for the diagnosis and differentiation of avirulent and virulent strains of Newcastle disease virus.     

What is the potential of avirulent influenza viruses to complement a cleavable hemagglutinin and generate virulent strains?

A large pool of avirulent influenza viruses are maintained in the wild ducks and shorebirds of the world, but we know little about their potential to become virulent. It is well established that the hemagglutinin (HA) is pivitol in determining virulence and that a constellation of other genes is also necessary (R. Rott, M. Orlich, and C. Scholtissek, 1976, J. Virol. 19, 54-60). The question we are asking here is the ability of avirulent influenza viruses to provide the gene constellation that will complement the HA from a highly virulent virus and for the reassortant to be virulent. Reassortant influenza viruses were prepared between ultraviolet treated A/Chicken/Pennsylvania/1370/83 (H5N2) [Ck/Penn] and influenza viruses from natural reservoirs. These viruses included examples of the predominant subtypes in wild ducks, shorebirds, and domestic poultry. Attention was given to the influenza viruses from live poultry markets, for it is possible that these establishments may be important in mixing of influenza genes from different species and the possible transmission to domestic and mammalian species. The reassortants were genotyped by partial sequencing of each gene and were tested for virulence in chickens. Each of the reassortants contained the hemagglutinin and matrix (M) genes from Ck/Penn and a majority of genes from the viruses from natural reservoirs indicating a preferential association between the HA and M genes. The reassortants containing multiple genes from wild ducks and a cleavable HA were avirulent indicating that the gene pool in ducks may not have a high potential to provide genes that are potentially virulent. In contrast, a disproportionate number of viruses from shorebirds and all avirulent H5N2 influenza viruses from city markets provided a gene constellation that in association with cleavable H5 HA were highly virulent in chickens. An evolutionary tree based on oligonucleotide mapping established that the H5N2 influenza viruses from birds in city markets are closely related.     

Antigenic and phylogenetic studies on a variant Newcastle disease virus using anti-fusion protein monoclonal antibodies and partial sequencing of the fusion protein gene.

A virulent Newcastle disease virus (NDV) isolate, 34/90, reported to show considerable antigenic diversity from more classical strains of NDV, including vaccine strains, was evaluated phylogenetically and for the presence of neutralizing epitopes on the fusion protein. Comparison of a 309 nucleotide sequence of the fusion protein gene of 34/90 with other viruses confirmed the diversity of this virus, placing it in a discrete fifth genetic lineage with an avirulent virus isolated from waterfowl and genetically quite distant from other strains and isolates. The virus-neutralizing mAbs used in the present study were directed against at least seven distinct epitopes on the fusion protein. Of these seven, five are shared by 34/90 and the live vaccine virus Hitchner B1 and these plus an additional epitope are shared by 34/90 and strain Ulster 2C, which is used in inactivated vaccines. Two potential distinct epitopes were also shared by these three viruses. The results suggest that despite the detected antigenic and genetic variation of 34/90, it is unlikely that mutants which fail to be neutralized by antibodies induced by conventional vaccines would arise readily.     

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.     

Genetic diversity of Newcastle disease viruses isolated from domestic poultry species in Eastern China during 2005�C2008

Seventy-nine Newcastle disease viruses (NDV) isolated from clinical specimens of different poultry species including chickens, pigeons (Columba livia), geese and ostriches in Eastern China during 2005?C2008 were characterized biologically and phylogenetically. The results showed genetic diversity of these viruses: three class I viruses and one genotype I and 12 genotype II viruses of class II circulating in chickens were avirulent; four genotype VIb viruses isolated from pigeons were moderately virulent; and two genotype III viruses and 57 genotype VIId viruses were highly virulent. The three class I viruses were further classified as genotypes 2 and 3. The very high F protein sequence identity of one genotype I virus with strain Queensland V4 and 12 genotype II viruses with strain La Sota indicated that these viruses originated from the two vaccine strains. Two genotype III viruses shared greater than 99% sequence identity with the moderately virulent vaccine strain Mukteswar but exhibited significantly higher virulence, suggesting that they evolved from the vaccine virus and that the Mukteswar vaccine should be banned in China. Fifty-seven of the 63 virulent NDVs in this study belonged to genotype VIId, indicating its predominance in Eastern China. Genotype VIId viruses could be further classified into two subgroups. Four of the five NDVs isolated from pigeons belonged to genotype VIb, indicating its host-specific preference. Both the genotype VIb and VIId NDVs showed low amino acid similarity to the vaccine strains currently used in China, implying the urgent need to develop better vaccines against the most prevalent NDVs in China.     

In vivo interference by Newcastle disease virus in chickens,the natural host of the virus

Summary Homologous and heterologous viral interference is a common occurrence that has been well studied in vitro. In the present study, homologous viral interference between the LaSota and NYP strains of Newcastle disease virus (NDV) was studied in vivo in chickens, the natural host for NDV. The LaSota strain is avirulent and widely used as a vaccine in poultry industry, while the NYP strain is highly virulent and causes acute disease and death in chickens within four to six days after infection. Chickens generally became resistant to NYP strain challenge 12 hours after intranasal or intratracheal inoculation with LaSota strain virus. The resistance was manifested by reduction in chicken morbidity and mortality, decrease in virus replication in the chicken respiratory tract (p<0.05), and inhibition of NYP strain induced gross and microscopic lesions. Interferon was first detected in the chicken respiratory tract and blood at 3 to 6 hours; it peaked at 12 to 24 hours and was maintained for 48 hours after viral inoculation, indicating that interferon induction might be one possible mechanism of the interference between the two strains. This study suggests a role for viral interference in vaccination against virulent viruses.     

Molecular Epidemiology of Outbreak-Associated and Wild-Waterfowl-Derived Newcastle Disease Virus Strains in Finland,Including a Novel Class I Genotype

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%.     

Evaluation of Newcastle disease virus chimeras expressing the Hemagglutinin-Neuraminidase protein of velogenic strains in the context of a mesogenic recombinant virus backbone

A major factor in the pathogenicity of Newcastle disease virus (NDV) is the amino acid sequence of the fusion protein cleavage site, but the role of other viral genes that contribute to virulence and different clinical forms of the disease remain undefined. To assess the role of other NDV genes in virus pathogenicity, a reverse genetics system was developed using the mesogenic NDV Anhinga strain to provide a backbone for generating gene mutations or gene exchanges in attempts to enhance or attenuate the virulence of that virus. Chimeras created by exchange of the Anhinga Hemagglutinin-Neuraminidase (HN) gene with HN genes of neurotropic and viscerotropic velogenic viruses produced no significant change in virus pathogenicity as assessed by conducting the mean death time and intracerebral pathogenicity index assays and by inoculation of susceptible day-old SPF chickens. Inclusion in the recombinant construct of homotypic F genes, obtained from the parental viruses, also failed to enhance the pathotype of the recombinant viruses to a velogenic pathotype. A HN gene exchange alone within the context of the NDV Anhinga backbone failed to increase virus virulence from mesogenic to velogenic pathotype and suggests a multigenic role for NDV pathogenicity.