Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools.

Shaoyuan Tan,Cheryl M.T Dvorak,Michael P Murtaugh

doi:10.3390/v11121132

Shaoyuan Tan, Cheryl M.T Dvorak + Show 1 more

Open Access

https://doi.org/10.3390/v11121132

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Journal: Viruses	Publication Date: Dec 7, 2019
Citations: 73	License type: CC BY 4.0

Affiliation: University of Minnesota

Abstract

Prompt detection and effective control of porcine reproductive and respiratory syndrome virus (PRRSV) during outbreaks is important given its immense adverse impact on the swine industry. However, the diagnostic process can be challenging due to the high genetic diversity and high mutation rate of PRRSV. A diagnostic method that can provide more detailed genetic information about pathogens is urgently needed. In this study, we evaluated the ability of Oxford Nanopore MinION direct RNA sequencing to generate a PRRSV whole genome sequence and detect and discriminate virus at the strain-level. A nearly full length PRRSV genome was successfully generated from raw sequence reads, achieving an accuracy of 96% after consensus genome generation. Direct RNA sequencing reliably detected the PRRSV strain present with an accuracy of 99.9% using as few as 5 raw sequencing reads and successfully differentiated multiple co-infecting strains present in a sample. In addition, PRRSV strain information was obtained from clinical samples containing 104 to 106 viral copies or more within 6 hours of sequencing. Overall, direct viral RNA sequencing followed by bioinformatic analysis proves to be a promising approach for identification of the viral strain or strains involved in clinical infections, allowing for more precise prevention and control strategies during PRRSV outbreaks.

Highlights

The swine industry plays an important role in feeding the world, as pork is one of the highest consumed animal proteins in the world [1,2]
A high concentration cell culture grown porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) VR2332 stock was used for RNA isolation and evaluation of MinION direct RNA whole genome sequencing
PRRSV has been a severe threat to the swine industry worldwide ever since it was first described in the late 1980s [63]

Summary

Introduction

The swine industry plays an important role in feeding the world, as pork is one of the highest consumed animal proteins in the world [1,2]. Emerging and re-emerging viral infectious diseases have been posing great challenges to the swine industry, among which porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating diseases [3,4]. PRRS remains a threat to the swine industry despite many years of combined efforts to combat and control infection and disease [10]. One of the challenges for PRRSV control is the frequent recurrence of PRRS outbreaks in swine farms [11], with a prediction that of farms reporting an outbreak today, 71% will have a recurrence of PRRSV infection within the following two years [12]. The PRRS recurrence is either caused by introduction of a new strain or the resident virus strain.

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