Kiril M. Dimitrov

Newcastle disease vaccines-A solved problem or a continuous challenge?

Abstract Newcastle disease (ND) has been defined by the World Organisation for Animal Health as infection of poultry with virulent strains of Newcastle disease virus (NDV). Lesions affecting the neurological, gastrointestinal, respiratory, and reproductive systems are most often observed. The control of ND must include strict biosecurity that prevents virulent NDV from contacting poultry, and also proper administration of efficacious vaccines. When administered correctly to healthy birds, ND vaccines formulated with NDV of low virulence or viral-vectored vaccines that express the NDV fusion protein are able to prevent clinical disease and mortality in chickens upon infection with virulent NDV. Live and inactivated vaccines have been widely used since the 1950’s. Recombinant and antigenically matched vaccines have been adopted recently in some countries, and many other vaccine approaches have been only evaluated experimentally. Despite decades of research and development towards formulation of an optimal ND vaccine, improvements are still needed. Impediments to prevent outbreaks include uneven vaccine application when using mass administration techniques in larger commercial settings, the difficulties associated with vaccinating free-roaming, multi-age birds of village flocks, and difficulties maintaining the cold chain to preserve the thermo-labile antigens in the vaccines. Incomplete or improper immunization often results in the disease and death of poultry after infection with virulent NDV. Another cause of decreased vaccine efficacy is the existence of antibodies (including maternal) in birds, which can neutralize the vaccine and thereby reduce the effectiveness of ND vaccines. In this review, a historical perspective, summary of the current situation for ND and NDV strains, and a review of traditional and experimental ND vaccines are presented.

Newcastle Disease Viruses Causing Recent Outbreaks Worldwide Show Unexpectedly High Genetic Similarity to Historical Virulent Isolates from the 1940s

ABSTRACT 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.

Genome-wide analysis reveals class and gene specific codon usage adaptation in avian paramyxoviruses 1

In order to characterize the evolutionary adaptations of avian paramyxovirus 1 (APMV-1) genomes, we have compared codon usage and codon adaptation indexes among groups of Newcastle disease viruses that differ in biological, ecological, and genetic characteristics. We have used available GenBank complete genome sequences, and compared codon usage of class I (CI-29 sequences containing 132,675 codons) and class II (CII-259 sequences containing 1,184,925 codons) APMV-1 genomes. We also compared available complete fusion protein gene sequences (CI-175 sequences containing 96,775 codons; CII-1166 sequences containing 644,798 codons). Adaptation to Gallus gallus was compared among the different classes of viruses, among different genomic regions based on transcriptional levels, or among the fusion gene. Interestingly, distinctive codon usage determined by differences in relative synonymous codon usage and by codon adaptation indexes was observed for the two APMV-1 classes and for different transcriptional regions within classes. Furthermore, differential use of the third codon position and preferential use of codon pairs were seen for the two different classes and for selected genotypes of class II despite the fact that there were no large differences in nucleotide composition. The data suggest that codon usage has changed significantly since the two APMV-1 classes diverged, however, these changes are not significantly pronounced among viruses of the same genotype, suggesting that codon adaptation in APMV-1 occurs through a slow evolutionary process.

Identification and Complete Genome Sequence Analysis of a Genotype XIV Newcastle Disease Virus from Nigeria

ABSTRACT The first complete genome sequence of a strain of Newcastle disease virus (NDV) from genotype XIV is reported here. Strain duck/Nigeria/NG-695/KG.LOM.11-16/2009 was isolated from an apparently healthy domestic duck from a live bird market in Kogi State, Nigeria, in 2009. This strain is classified as a member of subgenotype XIVb of class II.

Repeated isolation of virulent Newcastle disease viruses of sub-genotype VIId from backyard chickens in Bulgaria and Ukraine between 2002 and 2013

Here, we report the circulation of highly related virulent Newcastle disease viruses (NDV) in Bulgaria and Ukraine from 2002 until 2013. All of these NDV isolates have the same virulence-associated cleavage site (“113RQKR↓F117”), and selected ones have intracerebral pathogenicity index values ranging from 1.61 to 1.96. These isolates are most closely related to viruses circulating in Eastern Europe, followed by viruses isolated in Asia during the same period of time. Interestingly, the majority of the viruses were isolated from backyard poultry, suggesting the possibility of a “domestic” or “urban” cycle of maintenance. The molecular characterization of the nucleotide sequence of the complete fusion protein gene of the studied viruses suggests continued circulation of virulent NDV of sub-genotype VIId in Eastern Europe, with occasional introductions from Asia. Furthermore, the high level of genetic similarity among those isolates suggests that the NDV isolates of sub-genotype VIId from Bulgaria and Ukraine may have been part of a broader epizootic process in Eastern Europe rather than separate introductions from Asia or Africa. The continuous monitoring of backyard poultry flocks for the presence of circulating virulent NDV strains will allow early identification of Newcastle disease outbreaks.

Whole-genome sequencing of genotype VI Newcastle disease viruses from formalin-fixed paraffin-embedded tissues from wild pigeons reveals continuous evolution and previously unrecognized genetic diversity in the U.S.

BackgroundNewcastle disease viruses (NDV) are highly contagious and cause disease in both wild birds and poultry. A pigeon-adapted variant of genotype VI NDV, often termed pigeon paramyxovirus 1, is commonly isolated from columbids in the United States and worldwide. Complete genomic characterization of these genotype VI viruses circulating in wild columbids in the United States is limited, and due to the genetic variability of the virus, failure of rapid diagnostic detection has been reported. Therefore, in this study, formalin-fixed paraffin-embedded (FFPE) samples were subjected to next-generation sequencing (NGS) to identify and characterize these circulating viruses, providing valuable genetic information. NGS enables multiple samples to be deep-sequenced in parallel. When used on FFPE samples, this methodology allows for retrospective studies of infectious organisms.MethodsFFPE wild pigeon tissue samples (kidney, liver and spleen) from 10 mortality events in the U.S. between 2010 and 2016 were analyzed using NGS to detect and sequence NDV genomes from randomly amplified total RNA. Results were compared to the previously published immunohistochemistry (IHC) results conducted on the same samples. Additionally, phylogenetic analyses were conducted on the complete and partial fusion gene and complete genome coding sequences.ResultsTwenty-three out of 29 IHC-positive FFPE pigeon samples were identified as positive for NDV by NGS. Positive samples produced an average genome coverage of 99.6% and an average median depth of 199. A previously described sub-genotype (VIa) and a novel sub-genotype (VIn) of NDV were identified as the causative agent of 10 pigeon mortality events in the U.S. from 2010 to 2016. The distribution of these viruses from the North American lineages match the distribution of the Eurasian collared-doves and rock pigeons in the U.S.ConclusionsThis work reports the first successful evolutionary study using deep sequencing of complete NDV genomes from FFPE samples of wild bird origin. There are at least two distinct U.S. lineages of genotype VI NDV maintained in wild pigeons that are continuously evolving independently from each other and have no evident epidemiological connections to viruses circulating abroad. These findings support the hypothesis that columbids are serving as reservoirs of virulent NDV in the U.S.

Reply to “May Newly Defined Subgenotypes Va and Vb of Newcastle Disease Virus in Poultry Be Considered Two Different Genotypes?”

We appreciate Dr. Desingu’s comments (1) on our paper entitled “Separate Evolution of Virulent Newcastle Disease Viruses from Mexico and Central America” (2). Dr. Desingu points out that the mean evolutionary distance between viruses in subgenotypes Va and Vb is higher than the threshold (10%) set for subgenotype assignment (3) and, therefore, that subgenotypes Va and Vb should be considered two distinct genotypes. In addition, he suggests that subgenotype Vc may be considered ancestral to both genotypes Va and Vb. In response to the first comment made by Dr. Desingu, we believe that subgenotypes Va, Vb, and Vc should remain subgenotypes within genotype V for the following reasons. (i) Although we reported an evolutionary distance between subgenotypes Va and Vb (10.9%) that is slightly higher than the threshold (2, 3), the supporting data for a new genotype are not sufficiently strong. In fact, the nucleotide distance criterion ( 10%) for designating a new genotype should be met for any group of viruses that is compared to all other groups. If subgenotype Va is to be considered a putative new genotype, it should have an evolutionary distance of 10% compared to all other genotypes. Using the same data set as in our previous paper (2), analysis comparing Va (putative new genotype) to merged Vb and Vc (as these will remain and constitute genotype V) shows that the distance between them is 9.8% (Table 1) and thus does not meet the 10% rule. Similarly, using a new data set with all currently available sequences, the evolutionary distances between these three subgenotypes within genotype V fall under 10% (Table 2), confirming our previous results. These observations suggest that above-the-threshold evolutionary distances between subgenotypes do not justify automatic assignment of a new genotype. Calculation of evolutionary distances between subgenotypes is influenced by how homogeneously such distances are distributed within the genotype and by the number of taxa available for analysis (groups with more strains weigh more heavily in the analysis). Adding or removing few sequences in an analyzed set may affect distances and topology. This is among the reasons explaining why our group has followed a very conservative approach in the creation of new genotypes in the past. (ii) There are currently no defined criteria for naming genotypes that originate by continued evolution of existing subgenotypes. Under the current classification, a new genotype should receive a different Roman numeral (e.g., genotype XIX) and this will inevitably result in additional confusion among researchers due to lack of consistency with existing literature. Regarding the second comment of Dr. Desingu stating that subgenotype Vc should be considered ancestral to both subgenotype Va and subgenotype Vb, we believe that there is no evidence supporting such a statement. As presented in Fig. 1 in our original paper (2), the phylogenetic tree demonstrates that subgenotypes Vb and Vc diverged from a common ancestor (bootstrap value, 65%) genetically closest to a Newcastle disease virus (NDV) strain

Rapid and sensitive virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing

Newcastle 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. A highly sensitive amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described using both egg-propagated virus and clinical samples. 1D MinION sequencing of barcoded NDV amplicons was performed on 33 egg-grown isolates, (23 unique lineages, including 15 different NDV genotypes), and from 15 clinical swab samples from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. For all egg-grown samples, NDV was detected and virulence and genotype were predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes, of which the MinION method detected both genotypes in four of those samples. Additionally, testing a dilution series of one NDV sample resulted in detection of NDV with a 50% egg infectious dose (EID50) as low as 101 EID50/ml. This was accomplished in as little as 7 minutes of sequencing time, with a 98.37% sequence identity compared to the expected consensus. The high sensitivity, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for identification of NDV of different genotypes circulating worldwide. This general method will likely be applicable to other infections agents.

Newcastle Disease Virus Infection in Quail

Newcastle disease (ND), caused by virulent strains of Newcastle disease virus (NDV), is a devastating disease of poultry worldwide. The pathogenesis of ND in quail is poorly documented. To characterize the ability of virulent NDV strains to replicate and cause disease in quail, groups of 14 two-week-old Japanese quail (Coturnix japonica) were experimentally inoculated with 108 EID50 (embryo infectious dose 50%) units of 1 of 4 virulent NDV strains: 2 isolated from quail (N2, N23) and 2 from chickens (Israel, Pakistan). At day 2 postinfection, noninfected quail (contact group) were added to each infection group to assess the efficacy of virus transmission. Tested NDV strains showed moderate pathogenicity, with highest mortality being 28% for the N2 strain and below 10% for the others. Two N2-inoculated birds showed neurological signs, such as head tremor and ataxia. Microscopic lesions were present in N2-, Israel-, and Pakistan-inoculated birds and consisted of nonsuppurative encephalitis. Contact birds showed no clinical signs or lesions. In both inoculated and contact birds, virus replication was moderate to minimal, respectively, as observed by immunohistochemistry in tissues and virus isolation from oropharyngeal and cloacal swabs. Strains originally isolated from quail resulted in higher numbers of birds shedding in the inoculation group; however, transmission appeared slightly more efficient with chicken-derived isolates. This study shows that virulent NDV strains have limited replicative potential and mild to moderate disease-inducing ability in Japanese quail.