Rapid detection of porcine reproductive and respiratory syndrome virus using real-time MinION nanopore sequencing

PRRSV is one of the most important pathogens affecting swine worldwide. The main characteristic that makes PRRSV a difficult pathogen to control is the genetic diversity of the virus and the frequent emergence of variant strains. Therefore, sequencing of PRRSV strains has been widely used by the swine industry to monitor PRRSV strains and to gain information on the genetic differences of the viruses circulating in swine farms across the United States. In the present project we developed and validates two approaches to detect and obtain partial (ORF5) or complete PRRSV genome sequences directly from clinical samples (serum, processing fluid and oral fluid). These approached proved highly effective in determining the genetic makeup of PRRSV from those clinical samples. Importantly, the sequencing platform used in our project will allow broad application and use of the procedures developed in our project across different diagnostic laboratories across the country. These assays cost effective, as multiple samples can be batched in the same sequencing run and will provide a more economical alternative to current whole genome or ORF5 sequencing being performed for PRRSV. The assays developed in this project are now available for producers at the Animal Health Diagnostic Center (AHDC) at Cornell University.

Key Findings:
• Two sequencing approaches for PRRSV genotyping directly from clinical samples (serum, oral fluid and processing fluid) were developed in this project:
o Targeted amplicon sequencing (AmpliSeq) of PRRSV ORF5
o Whole genome sequencing
• The approach developed works for both Type I and type II PRRSV strains
• The sensitivity of the Ampliseq approach is comparable to real-time PCR, and it provides a means of rapidly detecting and identifying field PRRSV strains at the nucleotide level.
• The WGS strategy is effective at generating full genome sequences from samples with Ct up to… with a turnaround time of 11 hours from RNA to final DNA library.
• These protocols offer cost-effective tools that can be used in diagnostic laboratories in the field, providing invaluable information for veterinarians working on the control of PRRSV.