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Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples

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Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

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The authors would like to thank Poonam Sharma, Jeremy D. Volkening, and Dawn Williams-Coplin for their technical support during the study. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer.


This work was supported by the U.S. Department of Agriculture ARS CRIS 6040-32000-072 and the U.S. Department of State BEP/CRDF NDV 31063. Dr. Salman Latif Butt is conducting his PhD research program sponsored by the Fulbright U.S. Student Program.

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Authors and Affiliations



SLB prepared NGS libraries, performed NGS data analysis, and drafted the manuscript; KMD contributed in NGS data analysis and helped in phylogenetic analysis; JZ conducted IHC; AW, TS, and AB collected clinical tissue samples during disease outbreak and processed for transportation; CCB aided in experimental design and contributed to the drafting of the manuscript; SFR aided in sample collection during disease outbreaks; JBS and CLA designed and oversaw the tissue-based experiments, and aided with analysis of results and with writing of the manuscript.

Corresponding authors

Correspondence to James B. Stanton or Claudio L. Afonso.

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The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

No human subjects were used in this study. This article does not contain any studies with animals performed by any of the authors. Tissues were collected from dead birds being used for diagnostic purposes during disease outbreaks. Sampling was carried out by veterinarian, who took different samples as part of his routine work and under the permission of the farm owner. Since these are diagnostic specimens, sampling did not require the approval of the Ethics Committee.

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Supplementary material 1 Fig. S1 A–D: Photomicrographs demonstrating NDV nucleoprotein immunostaining within formalin-fixed paraffin-embedded tissue sections from chicken. Black arrows showing granular staining of viral nucleoprotein in the cytoplasm of cells. A) Brain. B) Spleen. C) Small intestine. D) Lung. Bar = 50 µm. (TIFF 7134 kb)

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Butt, S.L., Dimitrov, K.M., Zhang, J. et al. Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples. Virus Genes 55, 502–512 (2019).

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