A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2

TY - JOUR

T1 - A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2

AU - Kvisgaard, Lise K

AU - Hjulsager, Charlotte K

AU - Fahnøe, Ulrik

AU - Breum, Solvej Ø

AU - Ait-Ali, Tahar

AU - Larsen, Lars E

N1 - Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

PY - 2013/11

Y1 - 2013/11

N2 - PRRSV is a positive-sense RNA virus with a high degree of genetic variability among isolates. For diagnostic sensitivity and vaccine design it is essential to monitor PRRSV genetic diversity. However, to date only a few full genome sequences of PRRSV isolates have been made publicly available. In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods generated robust and reliable sequences both on primary material and cell culture adapted viruses and the protocols performed well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq2000, and Ion Torrent PGM™ Sequencer). These methods will greatly facilitate the generation of more full genome PRRSV sequences globally.

AB - PRRSV is a positive-sense RNA virus with a high degree of genetic variability among isolates. For diagnostic sensitivity and vaccine design it is essential to monitor PRRSV genetic diversity. However, to date only a few full genome sequences of PRRSV isolates have been made publicly available. In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods generated robust and reliable sequences both on primary material and cell culture adapted viruses and the protocols performed well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq2000, and Ion Torrent PGM™ Sequencer). These methods will greatly facilitate the generation of more full genome PRRSV sequences globally.

KW - Animals

KW - Genome, Viral

KW - High-Throughput Nucleotide Sequencing/methods

KW - Porcine Reproductive and Respiratory Syndrome/virology

KW - Porcine respiratory and reproductive syndrome virus/genetics

KW - RNA, Viral/genetics

KW - Reverse Transcriptase Polymerase Chain Reaction/methods

KW - Swine

KW - Virology/methods

U2 - 10.1016/j.jviromet.2013.07.019

DO - 10.1016/j.jviromet.2013.07.019

M3 - Journal article

C2 - 23891870

SN - 0166-0934

VL - 193

SP - 697

EP - 705

JO - Journal of Virological Methods

JF - Journal of Virological Methods

IS - 2

ER -