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01.12.2018 | Research | Ausgabe 1/2018 Open Access

Virology Journal 1/2018

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.

Virology Journal > Ausgabe 1/2018
Ying He, Tonya L. Taylor, Kiril M. Dimitrov, Salman L. Butt, James B. Stanton, Iryna V. Goraichuk, Heather Fenton, Rebecca Poulson, Jian Zhang, Corrie C. Brown, Hon S. Ip, Marcos Isidoro-Ayza, Claudio L. Afonso
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Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12985-017-0914-2) contains supplementary material, which is available to authorized users.



Newcastle 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.


FFPE 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.


Twenty-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.


This 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.
Additional file 1: Table S1. Dataset used in the complete fusion gene coding sequences phylogenetic analysis. Table S2. Dataset used in the complete genome concatenated coding sequences phylogenetic analysis. Table S3. Dataset used in the partial fusion gene (374 nucleotides) phylogenetic analysis. (PDF 366 kb)
Additional file 2: Table S4. Comparison of next-generation sequencing results to immunohistochemistry results from 48 Eurasian collared-doves and rock pigeons formalin-fixed paraffin-embedded samples collected in the U.S. between 2010 and 2016. (PDF 208 kb)
Additional file 4: Figure S1. Phylogenetic analysis using the 374-nucleotide partial fusion gene sequences of genotype VI Newcastle disease viruses. The evolutionary history was inferred using the Maximum-likelihood method based on the Kimura-2 parameter model with 1000 bootstrap replicates [ 42]. The analysis involved 931 genotype VI partial fusion gene sequences (374 nucleotides). Roman numerals are used for the sub-genotype VI designation, and for a clearer view, the taxa names and bootstrap values are not shown in the radiation tree. The VIa and VIn groups are labeled with the country and years of isolate collection. Evolutionary analyses were conducted in MEGA6 [ 21]. (PDF 283 kb)
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