Quasispecies Nature of an Unusual Avian Paramyxovirus Type-1 Isolated from Pigeons

An avian paramyxovirus type-1 (APMV-1) was classified as virulent according to its Intra Cerebral Pathogenicity Index (ICPI), but as avirulent according to the motif of its F protein cleavage site. Although this atypical APMV-1 was isolated from sick, unvaccinated pigeons, it was not grouped with pigeon variants regarding its antigenic and genetic characterisation. We analysed its quasispecies nature by cloning and sequencing parts of the genome in three different genes to evaluate if heterogeneity might explain the difference observed between the ICPI and the F protein cleavage site motif. Two distinct sub-populations were detected in the phosphoprotein gene. In the fusion protein gene, two clones were found to be related to typical pigeon variants in the hypervariable domain.     

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.     

Matrix Protein Gene Sequence Analysis of Avian Paramyxovirus 1 Isolates Obtained from Pigeons

The matrix protein gene was cloned and sequenced for several recent isolates of avian paramyxovirus type 1 (APMV-1). Specifically, isolates from pigeons and doves, members of the Columbidae family were examined. APMV-1 is the causative agent of Newcastle disease and the virus is associated with disease among a diverse number of avian species. Newcastle disease virus (NDV) isolates from pigeons have also been classified as pigeon paramyxovirus type 1 (PPMV-1). Matrix protein gene sequences for PPMV-1 isolates clustered together as a group relative to isolates from other species phylogenetically. However, there were also isolates from pigeons or doves that grouped with APMV-1 isolates from other species. This indicates that PPMV-1 may be circulating among Columbidae members as a distinct lineage, but that these avian species may also harbor other NDV strains as well. Of particular interest was a dove isolate from Europe that had an aberrant fusion protein cleavage site and was an outlying member phylogenetically between the two major groups of APMV-1 isolates.     

A comparative infection study of pigeon and avian paramyxovirus type 1 viruses in pigeons: evaluation of clinical signs,virus shedding and seroconversion

The pathogenesis of pigeon paramyxovirus type 1 (PPMV-1) isolate AV324/96 and of its recombinant derivative, rgAV324, was studied in pigeons. For comparison, the virulent chicken virus FL-Herts, which is a recombinant derivative of strain Herts/33, was also included. After inoculation by the combined intraocular, intranasal and intratracheal route, clinical signs, virus shedding and serological responses were examined. Clinical signs were observed only in the FL-Herts-infected group. All virus-inoculated pigeons had positive tracheal swabs until 5 days post infection. However, only the AV324/96-infected and rgAV324-infected birds, and not the FL-Herts-infected birds, shed virus in the cloaca. The AV324/96-infected pigeons showed higher mean antibody titres than the rgAV324-infected birds, whereas the antibody titres of the FL-Herts-infected group were rather low. The results show that the pigeon strain AV324 is not virulent for pigeons, but underlines the potential risk of poultry becoming infected by PPMV-1 shed by non-symptomatic pigeons.     

Molecular Characterisation of Three Avian Paramyxovirus Type 1 Isolated from Pigeons in France

Three avian Paramyxovirus type 1 (aPMV-1) isolated from pigeons during pigeon paramyxovirosis outbreaks were molecularly characterised by sequencing parts of the six genes (NP, P, M, F, HN and L) of each strain. Virulent 99143 isolate was found to be very closely related to non-pathogenic vaccine strains of aPMV-1, even for its F protein cleavage site motif. Strains 99299 and 99106, typical pigeon paramyxovirus type 1 (pPMV-1) variants, exhibited between 10% and 20% difference with aPMV-1 at the nucleotide level. The aPMV-1 specific pattern of eight amino acids in the intracellular domain of HN protein was found different by one residue for these two isolates, and might represent a specific pattern for pPMV-1. The unique sequence of the polycistronic P gene editing site of 99299 and 99106 was characterised by four instead of three cytosine residues, and might so have an influence on the expression level of the three proteins encoded by P. This work is also the first to provide molecular data on NP, P and L genes of typical pPMV-1.     

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.     

Comparative clinico-pathological assessment of velogenic (sub-genotype VIIi) and mesogenic (sub-genotype VIm) Avian avulavirus 1 in chickens and pigeons

ABSTRACT

Newcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects a wide range of avian species worldwide. Recently, several AAvVs of diverse genotypes have emerged with varying genomic and residue substitutions, and subsequent clinical impact on susceptible avian species. We assessed the clinico-pathological influence of two different AAvV 1 pathotypes [wild bird originated-velogenic strain (sub-genotype VIIi, MF437287) and feral pigeon originated-mesogenic strain (sub-genotype VIm, KU885949)] in commercial broiler chickens and pigeons. The velogenic strain caused 100% mortality in both avian species while the mesogenic strain caused 0% and 30% mortality in chickens and pigeons, respectively. Both strains showed tissue tropism for multiple tissues including visceral organs; however, minor variances were observed according to host and pathotype. The observed gross and microscopic lesions were typical of AAvV 1 infection. Utilizing oropharyngeal and cloacal swabs, a comparable pattern of viral shedding was observed for both strains from each of the infected individuals of both avian species. The study concludes a varying susceptibility of chickens and pigeons to different wild bird-originated AAvV 1 pathotypes and, therefore, suggests continuous monitoring and surveillance of currently prevailing strains for effective control of the disease worldwide, particularly in disease-endemic countries.     

Molecular characterization of a Class I Newcastle disease virus strain isolated from a pigeon in China

Constant monitoring is performed to elucidate the role of natural hosts in the ecology of Newcastle disease virus (NDV). In this study, an NDV strain isolated from an asymptomatic pigeon was sequenced and analysed. Results showed that the full-length genomes of this isolate were 15,198 nucleotides with the gene order of 3′-NP-P-M-F-HN-L-5′. This NDV isolate was lentogenic, with an intracerebral pathogenicity index of 0.00 and a mean time of death more than 148?h. The isolate possessed a motif of -¹¹²E-R-Q-E-R-L¹¹⁷- at the F protein cleavage site. In addition, 7 and 13 amino acid substitutions were identified in the functional domains of fusion protein (F) and haemagglutinin-neuraminidase protein (HN) proteins, respectively. Analysis of the amino acids of neutralizing epitopes of F and HN proteins showed 3 and 10 amino acid substitutions, respectively, in the isolate. Phylogenetic analysis classified the isolate into genotype Ib in Class I. This isolate shared high homologies with the NDV strains isolated from wild birds and waterfowl in southern and eastern parts of China from 2005 to 2013. To our knowledge, this study is the first to report a NDV strain isolated from pigeon that belongs to genotype Ib in Class I, rather than to the traditional genotype VI or other sub-genotypes in Class II. This study provides information to elucidate the distribution and evolution of Class I viruses for further NDV prevention.     

Isolation and preliminary characterisation of a paramyxovirus from collared doves (Streptopelia decaocto)

A haemagglutinating virus was isolated from the caecal tonsils of dead collared doves (Streptopelia decaocto). The virus was identified as an avian paramyxovirus showing some antigenic relationships with PMV-7 /dove/Tennessee/4/75 and PMV-?/pigeon/Otaru/76. Of 15 collared doves and 24 feral pigeons trapped and sampled at the site at which dead doves were found, only two pigeons had significant antibody litres to the isolate. The virus was apathogenic for experimentally infected pigeons and chickens and stimulated only a weak antibody response.     

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

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

Characterization of Newcastle disease virus isolates obtained from Eurasian collared doves (Streptopelia decaocto) in Italy

Eurasian collared doves (Streptopelia decaocto) are thought to originate from India and they have colonized, throughout the centuries, the Middle East and, more recently, Mediterranean countries such as Italy and Spain. In the present paper we report of the isolation and characterization of Newcastle disease viruses (NDV) obtained from Eurasian collared doves during 2000-2001, and compare them to isolates obtained from feral pigeons (Columba livia) during the same period. All isolates could be classified as avian paramyxovirus type 1 (APMV1) and belonged to the pigeon variant group (PPMV1), as their haemagglutinating activity was inhibited by mAb 161/617 which is specific for PPMV1. The intracerebral pathogenicity indices ranged from 0.68 to 1.38 and all isolates contained multiple basic amino acids at the deduced cleavage site of the fusion protein, which is a typical feature of virulent viruses. Phylogenetic analysis of the isolates indicate that 18/20 of these form a separate cluster from the isolates obtained from pigeons in the same period. These findings suggest that different lineages are circulating in feral pigeon populations, and that a separate lineage affects Eurasian collared doves.     

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.     

Evaluation of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed “pigeon PMV-1 viruses”

Summary The amino acid sequence at the F2/F1 cleavage site was determined for 15 strains of the so-called pigeon PMV-1 (PPMV-1) variant of Newcastle disease virus (NDV) which showed close antigenic identity, determined by their reactions with a panel of 28 monoclonal antibodies, but considerable variation in their pathogenicity for chickens. Thirteen of the isolates possessed the motif¹¹²G-R-Q-K-R-F¹¹⁷. This motif was seen for one virus which had initially low pathogenicity and remained unaltered when virulence of the virus for chickens was increased by bird to bird passage. The two other viruses had the sequence¹¹²R-R-Q-K-R-F¹¹⁷ at the cleavage site which is more typical of virulent viruses, however, pathogenicity index tests indicated that these isolates were of moderate and low pathogenicity. The nucleotide sequence coding for the HN/HN0 extension region was determined for two of the PPMV-1 isolates. In both cases a stop codon was present indicating that the product for these viruses would be HN₅₇₁. We conclude that the wide variation in pathogenicity of the variant PPMV-1 for chickens is not related to variation in the amino acid motif at the F2/F1 cleavage site nor due to production of HN0 which may also influence pathogenicity. The high virulence of some of the viruses examined confirms that a double pair of basic amino acids in the region of the F2/F1 cleavage site is not necessary for the full expression of virulence.     

Newcastle disease in the European Union 2000 to 2009

Newcastle disease (ND) is a devastating disease of poultry that has to some extent been neglected by those working in the field in the past 10 to 15 years while attention has been focused on the emergence and spread of highly pathogenic avian influenza caused by a H5N1 subtype virus. During 2000 to 2009 in the European Union (EU) member states, ND viruses virulent for chickens have been detected in wild birds, domesticated pigeons and poultry. Based on these isolations it appears that the epizootic in racing pigeons caused by the variant viruses termed pigeon avian paramyxovirus type 1, which form the genetic group 4b(VIb) first seen in Europe in 1981, continued during 2000 to 2009, and the virus is probably enzootic in racing pigeons in some EU countries. This virus appears to have spread regularly to wild birds, especially those of the Columbidae family, and has been the cause of significant outbreaks in poultry. Other avian paramyxovirus type 1 viruses responsible for ND outbreaks in the EU during 2000 to 2009 have been those from genetic groups 5b(VIIb) and 5d(VIId). There is evidence that the former may well represent spread from a wild bird source and these viruses have also been isolated from wild birds, while the latter represents continuing spread from the East. Future legislation or recommendations aimed at the control and eradication of ND will need to encompass these three sources of virulent ND viruses.     

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

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

A molecular epidemiological investigation of isolates of the variant avian paramyxovirus type 1 virus (PPMV-1) responsible for the 1978 to present panzootic in pigeons.

A sequence of 375 nucleotides, which included the region encoding the cleavage activation site and signal peptide of the fusion protein gene, was determined for 178 isolates of the pigeon variant strain of Newcastle disease virus (PPMV-1). These were compared with the sequences of 47 similar isolates published by GenBank, which included 30 isolates from pigeons and 17 representatives from each sublineage of avian paramyxovirus type 1. The resulting alignment was analysed phylogenetically using maximum likelihood and the results are presented as unrooted phylogenetic trees. By phylogenetic analysis all the PPMV-1 isolates except one were placed in lineage 4b (VIb). Within this lineage there was considerable genetic heterogeneity, which appears to be predominantly influenced by the date of isolation and, to a lesser extent, geographical origins of the isolates. There were two large distinguishable groups, 4bi and 4bii. The earliest isolate available, PIQPI78442, isolated in 1978 in Iraq, was situated at the node from which the two groups diverge.     

Emergence of a virulent genotype VIIi of Newcastle disease virus in Iran

Newcastle disease (ND) is a contagious viral disease affecting numerous avian species, particularly domestic poultry, and causes devastating outbreaks. In spite of its endemicity and importance in Iran, data on the genetic characterization of ND virus (NDV) are scarce. An alarming issue that has just been raised is the occurrence of ND outbreaks with unexpected high mortality and severe clinical signs. The present study was conducted to characterize the emerging NDV genetically. An NDV strain, isolated in 2017 from commercial broilers showing severe nervous and enteric signs, was completely sequenced and found to be 15,192 nucleotides in length. The phylogenetic analysis demonstrated that the virus belonged to subgenotype VIIi, a subgenotype with potential panzootic features which has recently emerged in the Middle East and Asia. The supporting genetic pattern obtained from the complete genome, fusion and haemagglutinin gene analysis showed close relationship of the isolate with Pakistani VIIi NDVs. The analysis of the F protein showed a polybasic amino acid motif and a phenylalanine at position 117 at the cleavage site, which is a characteristic of virulent strains. The isolate showed significant differences from the previously characterized NDV strains from commercial and rural chickens in Iran. This may describe the importance of the illegal trade of pet birds from neighbouring countries leading to the emergence of new genotypes. This study introduces a newly emerging NDV VIIi subgenotype in Iran. This investigation emphasizes the necessity of effective control strategies.     

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

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

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.