Background: The remarkable diversity and mobility of Newcastle disease viruses (NDV) includes virulent viruses of genotype VI. These viruses are often referred to as pigeon paramyxoviruses 1 because they are normally isolated and cause clinical disease in birds from the Columbidae family. Genotype VI viruses occasionally infect, and may also cause clinical disease in poultry. Thus, the evolution, current spread and detection of these viruses are relevant to avian health.
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. Infections with virulent strains of Avian paramyxovirus 1 (APMV-1, synonymous with Newcastle disease virus [NDV]), a member of the family Paramyxoviridae, subfamily Paramyxovirinae, and genus Avulavirus (1), cause Newcastle disease (ND) in birds. NDV is a pathogen capable of producing a devastating disease in domestic fowl, with vast social and economic consequences (2). Chickens infected with NDV show a wide spectrum of clinical signs that vary with different virus strains (3). Hanson and Brandly (4) categorize ND viruses into three main pathological groups: lentogens are avirulent and cause mild enteric, respiratory, or subclinical disease (5); mesogens cause disease and death primarily for chickens younger than 8 weeks and produce mainly respiratory disease (6); velogens induce severe systemic infections with high mortality rates (7). According to the World Organisation for Animal Health (OIE) (8), virulent NDV strains, which include both mesogenic and velogenic strains, must meet one of the following criteria: (i) have an intracerebral pathogenicity index (ICPI) in day-old chicks (Gallus gallus) of 0.7 or greater; or (ii) have multiple basic amino acids at the C terminus of the F2 protein and phenylalanine at residue 117, which is the N terminus of the F1 protein. The term "multiple basic amino acids" refers to the presence of at least three arginine or lysine residues from positions 113 through 116. More info...
BackgroundNewcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs.MethodsAn amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow.ResultsFor all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 101 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq.ConclusionThe depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.Electronic supplementary materialThe online version of this article (10.1186/s12985-018-1077-5) contains supplementary material, which is available to authorized users.
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