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.

Newcastle disease (ND), caused by virulent strains of avian avulavirus 1 (AAvV-1, commonly termed Newcastle disease virus, used hereafter), is a worldwide disease that causes significant economic losses to poultry producers and is exotic to the United States poultry industry [1, 2]. However, genetic variants of Newcastle disease virus (NDV) that belong to genotype VI viruses, termed pigeon paramyxovirus 1 (PPMV-1), are endemic to the U.S. and isolated from wild columbids, including doves and pigeons [3]. Pigeon-origin NDVs are highly contagious and can result in enteric, visceral, and neurological disease in infected birds and has the potential to cause outbreaks in poultry [1].

As with other NDVs, pigeon-origin NDVs are genetically diverse, and their virulence is variable [4]. Although the pigeon-adapted variants are the cause of the third NDV panzootic [4, 5], affecting primarily pigeons and occasionally poultry [1], thorough characterization of the complete genomes of these NDV is limited [6]. Due to the rapid spread of the virus, mass mortalities of specifically Eurasian collared-doves (Streptopelia decaocto, ECDO) and rock pigeons (Columba livia, ROPI) have occurred within the U.S. [7, 8]. Both species have the ability to interact with naïve species due to the lack of geographical separation, which justifies increased biosecurity within poultry production regions where these columbids abound. For these reasons, it is important to develop a robust and reliable diagnostic assay to provide detailed genomic characterization of NDV circulating in wild columbids in the United States.

Between 2009 and 2016, there were 25 NDV-associated mortality events of pigeons in ten different states of the U.S. [9]. Previously, Isidoro-Ayza, et al. collected 70 birds from 25 events. Using viral isolation, RT-PCR, and immunohistochemistry (IHC), the presence of NDV was confirmed in 14 ECDO and 3 ROPI from 10 of those 25 mortality events [9].

IHC is a common tool in pathogen detection [10–12], it is target-based and can be subjective in some cases. The IHC, using a polyclonal antibody to the nucleoprotein, raised against a synthetic peptide [13], demonstrated widespread NDV antigen in most tissues from most birds. This NDV immunoreactivity, along with the histopathologic changes within these tissues, suggested the lethal pathogenicity of these viruses in these hosts [9]. While IHC can identify only the absence or presence of NDV, molecular methods provide genetic information allowing epidemiological and evolutionary studies to be conducted.

One of the most widely used methods to preserve and archive clinical specimens is formalin fixation and paraffin embedding (FFPE) [14]. Due to the wide use of this technique in pathology studies, significant amount of FFPE samples are stored and available for subsequent analysis. Unlike flash-frozen tissue, nucleic acids are degraded and fragmented through FFPE [15]. Next-generation sequencing (NGS) is a powerful tool used to detect and genotype infectious agents, and a random-priming approach allows unbiased detection of genetic variation [16, 17]. The use of NGS on FFPE samples allows for retrospective studies and for molecular investigations of infectious organisms [18]. While other studies have used NGS on FFPE tissues [19], the use of this approach is limited in studying animal pathogens.

The current aim was to characterize the NDV from the FFPE tissue samples described above [9], and to study the circulation and evolution of endemic pigeon viruses in different regions of the U.S. Here, to improve upon standard NDV genotyping methods, random-priming next-generation sequencing was used for whole-genome (99% coverage) characterization of NDV. The feasibility of NGS to detect, sequence, and identify pathogens in FFPE tissues from wild bird mortality events which have varying degrees of autolysis was demonstrated. This study identified the existence of undescribed genetic diversity of genotype VI NDV in the U.S.

The constant evolution of genotype VI NDV has caused failures to detect some viruses in the past [25, 27, 28]. Occasionally, these viruses have been reported to cause outbreaks in poultry, which highlights the necessity to monitor their distribution, circulation and evolution [3, 29–31]. Analysis of the genomic region of the U.S. pigeon NDV used for detection of virulent NDV showed existence of primers/probes mismatches, suggesting that some of these viruses could escape detection by real-time RT-PCR. Due to the need of an unbiased diagnostic approach, we used a previously described target-independent NGS technique [17] that is capable of detecting unknown avian pathogens and applied it on FFPE tissue samples. This is the first report of utilizing random-priming NGS on formalin-fixed, paraffin-embedded tissues from wild birds for evolutionary characterization of Newcastle disease viruses. Newcastle disease viruses of genotype VI, commonly isolated from columbids in the U.S., were identified, and their complete genome sequences were obtained. The performed phylogenetic analysis suggested the existence of a new sub-genotype within genotype VI NDV, thus revealing unknown genetic diversity.

Here, we provide evidence that there are two, independently circulating lineages of genotype VI NDV in the U.S. The phylogenetic analysis showed clear separation of the obtained 23 sequences as they clustered into two independent branches of the phylogenetic tree (Fig. 1). One group had sequences from tissues collected from MT, MA, and PA that clustered with a portion of isolates collected from northeastern states, and the other group had sequences from samples collected from mid-southern states of TX, UT, and KS that clustered with samples from Texas and Louisiana (Fig. 1). The genetic distance between these two groups (4.2% nucleotide distance) suggests a very distant epidemiological link between them. Indeed, such high genetic distance is indicative of at least four decades [32] of independent evolution of these two groups of viruses. Interestingly, the majority of sequences from the mid-south were from ECDO, while the sequences from the northeast were from mixed species including ROPI, unidentified pigeons, doves, chukars, and poultry without prevalence of one species or another, a pattern that could also be noticed in previous studies [7, 33]. The host association of the genotype VI NDV in the southern states with ECDO is puzzling and might reflect the distribution of this species which, although present in many states, predominantly inhabits the southern and southwestern parts of the U.S. with much smaller populations reported in the northeastern states [34, 35]. Because ECDO are an invasive species that first spread throughout Florida in the early 1980s [36, 37], it is possible that this species may have introduced the southern lineage of genotype VI NDV. This is supported by the estimated four decades of evolution described above (4.2% nucleotide distance). However, there is no NDV sequence data from the rest of the world that supports this hypothesis.

The lack of evidence of a direct epidemiological link between U.S. and other genotype VI NDV from other countries and continents suggest the independent maintenance of the U.S. viruses. Utilization of a large number of sequences of the 374 nt region of the fusion gene identified multiple isolations over time of viruses from sub-genotype VIa and sub-genotype VIn across the U.S. (Fig. 1, Additional file 4: Figure S1, group 2, VIn) and only incidental introductions of viruses from abroad. These findings support the hypothesis that columbids are more than likely serving as a reservoir of genotype VI NDV and harboring virulent viruses in the U.S. [1, 3, 38].

The evolutionary distance between the two groups of viruses discussed above, the observed phylogenetic topology, and bootstrap support, indicate the presence of hitherto unidentified genetic diversity among U.S. genotype VI NDV. These two groups fulfill the criteria to be classified as sub-genotypes VIa and VIn [24]. The currently established method to classify NDV based on the full fusion gene analysis provides comprehensive phylogeny (Fig. 1). The complete genome CDS tree supported the results from the full fusion gene analysis and showed bootstrap values of 100 in nearly all nodes, demonstrating the robustness of this analysis (Fig. 2). Furthermore, pigeon viruses from genotype VI have been identified in poultry within the U.S., both in the north and the south, again indicating the potential of these viruses to infect chickens and turkeys (Figs. 1 and 2) and highlighting the importance of keeping the knowledge of their genetic makeup and epidemiology up to date.

The use of FFPE tissues provided several advantages in the current study. This approach allowed complete genome characterization of archival specimens, which were sampled 2 to 7 years ago. The availability to quickly obtain deep, high quality, and detailed molecular characterization of archival samples is of significant value. Previously, the genome of the 1918 pandemic influenza A virus was determined after a 9-year effort by overlapping RT-PCR from postmortem samples; while using NGS, the complete genome was obtained in a single run [39]. Significant numbers of FFPE archival tissues samples dating as far back as the 19th century are stored in tissue banks, research institute repositories and laboratories [39–41]. Sequencing of these samples using our developed approach has the potential to provide a wealth of missing information that will provide insights in genetics, metagenomics and epidemiological knowledge.

Here, the obtained NGS results were compared to the previously performed IHC (see Additional file 2, Table S4). It was confirmed that random-priming NGS of FFPE tissue samples from wild birds can be used as a tool to produce more specific diagnostic information than IHC. While 23 samples were identified as positive for NDV by both NGS and IHC, NGS provided specific genotype and virulence information (see Additional file 2: Table S4). IHC, however, identified only the absence or presence of NDV. Additionally, three birds total, two ECDO and one ROPI, were determined to be negative by NGS (18%) where only one was negative by IHC. Interdependence among the positive detection between the two methods was observed in a high percentage of the samples but not in all of them; nevertheless, NGS provided additional data in all samples. Thus, NGS could be successfully used as a complementary test for precision diagnostics of infectious diseases in FFPE samples.

This research shows the efficacy of the use of NGS on FFPE samples, as well as its complementary value to IHC, for investigating the circulation and evolution of pigeon-derived genotype VI NDV within the U.S. It demonstrates that there are at least two distinct U.S. genotype VI sub-genotypes maintained in wild pigeons that are continuously evolving independently from each other and independently from the rest of the world.