Journal Article

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

Dimitrov, Kiril M., Volkening, Jeremy D., Afonso, Claudio L., Taylor, Tonya L., Miller, Patti J., Suarez, David L., Butt, Salman L., Williams-Coplin, Dawn, Stanton, James B., Lahmers, Kevin K., Rana, Asif M.

2018

Overview

Background 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. Methods An 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. Results For 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 10 1 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. Conclusion The 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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Animals / Avian orthoavulavirus 1 / Bioinformatics / Biomedical and Life Sciences / Biomedicine / Conserved sequence / Data Accuracy / Data processing / Deoxyribonucleic acid / Disease / DNA / DNA Barcoding, Taxonomic / DNA sequencing / eggs / etiological agents / Evolution / Genes / Genetic diversity / Genetic Variation / Genome, Viral / Genomes / Genotype / Genotype & phenotype / Genotypes / High-Throughput Nucleotide Sequencing - methods / Identification / Methods / MinION / Mitochondrial DNA / Nanopore sequencing / Nanopores / Negative-strand RNA viruses / Newcastle disease / Newcastle Disease - diagnosis / Newcastle Disease - virology / Newcastle disease virus / Newcastle disease virus - genetics / Newcastle disease virus - isolation & purification / Outbreaks / Phylogeny / Poultry / Poultry - virology / Poultry Diseases - diagnosis / Poultry Diseases - virology / poultry industry / prediction / protocols / Rapid sequencing / RNA / RNA, Viral - genetics / Sensitivity and Specificity / sequence analysis / Virology / Virulence / Viruses