Rapid genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) using MinION nanopore sequencing

The global distribution and constant evolution are challenges for the control of the porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting the swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION nanopore platform, respectively. Procedures were optimized on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The target amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and part of ORF4 and ORF6 of both type 1 and type 2 PRRSV species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S.A and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS protocol. Fifty out of sixty sera (83.3%) and eighteen out of 20 lungs (90%) had at least 80% of genome covered at a minimum of 20X sequence depth. The rapid turnaround time, portability and ease of use of this technology offer significant advantages over traditional diagnostic approaches or other sequencing platforms. The protocols presented here are valuable tools with potential for field application during PRRSV elimination programs