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Research Project: Intervention Strategies to Predict, Prevent and Control Disease Outbreaks Caused by Emerging Strains of Virulent Newcastle Disease Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

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

item BUTT, SALMAN - University Of Georgia
item Taylor, Tonya
item VOLKENING, JEREMY - Base2bio
item DIMITROV, KIRIL - Consultant
item Williams Coplin, Tina
item LAHMERS, KEVIN - Virginia Tech
item Suarez, David
item Afonso, Claudio
item STANTON, JAMES - University Of Georgia

Submitted to: Virology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/10/2018
Publication Date: 11/22/2018
Publication URL:
Citation: Butt, S.L., Taylor, T.L., Volkening, J.D., Dimitrov, K., Williams Coplin, T.D., Lahmers, K.K., Suarez, D.L., Afonso, C.L., Stanton, J. 2018. Rapid virulence prediction and identification of Newcastle disease virus genotypes using third generation sequencing. Virology Journal. 15:179.

Interpretive Summary: Newcastle disease outbreaks are a global challenge to the poultry industry, as effective management of this disease is hindered by the genetic diversity and rapid evolution of Newcastle disease virus (NDV). Newcastle disease is caused by virulent strains of avian paramyxovirus 1. Rapid, sensitive and specific identification of the virus well as understanding where it came from are fundamental parts of any disease control strategy. Sequencing of viruses is becoming faster and cheaper, and advances in next-generation sequencing techniques such as long read third-generation sequencing technology allow sequencing and analysis with a short turnaround time, field portability,and low startup cost compared to other methods for surveillance and pathogen characterization. In this study, we developed a specific, sensitive, rapid sequencing and data analysis protocol using a pocket-sized sequencer powered by a computer to detect genetically type isolates from all current genotypes of NDV. This protocol was also tested on clinical swab samples collected from chickens during disease outbreaks.

Technical Abstract: Newcastle disease (ND) outbreaks are global challenges to the poultry industry, and rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV) during disease outbreaks is crucial for control efforts. However, effective diagnostics is hindered by the genetic diversity and rapid evolution of NDV. Here, a third-generation, DNA sequencing technology based on the MinION Oxford Nanopore device which provides highly sensitive detection and specific genetic identification of NDV genotypes is described. Furthermore, it is demonstrated that clinical samples are a feasible input in this assay. Total RNAs from 33 egg grown isolates representing 15 different genotypes and sub-genotypes of NDV/SP and 15 clinical swab samples from field outbreaks were reverse transcribed and amplified with tailed NDVspecific primers. DNA libraries were barcoded, pooled, and sequenced on a MinION device. Data analysis was performed in a newly developed galaxy based workflow. NDV was detected in six 10-fold serially diluted samples with 50% egg infectious dose as low as 10^1 by processing data collected from 19 minutes of sequencing with a 99.45-100% sequence identity to the expected consensus obtained in sequencing with alternative methods. Furthermore, cost efficiency was achieved by multiplexing samples (n = 33) using barcodes. The high sensitivity of PCR, combined with the fast sequencing capabilities of the MinION platform, the consensus accuracy of long reads processed with a newly developed, customized workflow and the low cost of multiplexing should allow identification of NDV genotypes worldwide and particularly in endemic countries where lack of resources creates challenges for monitoring and controlling ND.