High-throughput whole genome sequencing of Porcine reproductive and respiratory syndrome virus from cell culture materials and clinical specimens using next-generation sequencing technology:

Next-generation sequencing (NGS) technologies have increasingly played crucial roles in biological and medical research, but are not yet in routine use in veterinary diagnostic laboratories. We developed and applied a procedure for high-throughput RNA sequencing of Porcine reproductive and respiratory syndrome virus (PRRSV) from cell culture–derived isolates and clinical specimens. Ten PRRSV isolates with known sequence information, 2 mixtures each with 2 different PRRSV isolates, and 51 clinical specimens (19 sera, 16 lungs, and 16 oral fluids) with various PCR threshold cycle (Ct) values were subjected to nucleic acid extraction, cDNA library preparation (24-plexed), and sequencing. Whole genome sequences were obtained from 10 reference isolates with expected sequences and from sera with a PRRSV real-time reverse transcription PCR Ct ≤ 23.6, lung tissues with Ct ≤ 21, and oral fluids with Ct ≤ 20.6. For mixtures with PRRSV-1 and -2 isolates (57.8% nucleotide identity), NGS was able to distinguish them a...