Metagenomic Detection and Genome Assembly of Novel PRRSV-2 Strain Using Oxford Nanopore Flongle Flow Cell

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a syndrome characterized by reproductive failure and respiratory complications (PRRS). Early detection and classification of PRRSV strains are vital for appropriate management strategies to minimize loss following outbreaks. The most widely used classification method for PRRSV is based on open reading frame 5 (ORF5) sequences. However, the effectiveness of the ORF5-based classification system in accurately representing genetic variation is under scrutiny because ORF5 constitutes less than 5% of the 15kb-long genome. In this study, a single Oxford Nanopore Flongle flow cell was used to identify and assemble the genome of a strain sampled in May of 2022 from a Midwest research farm. Based on comparisons with available PRRSV genomes, the assembled genome was determined to be a novel PRRSV-2 strain belonging to the 1-4-4 L1C.5 ORF5-based lineage. Phylogenetic analyses of ORF5 and whole-genome sequences demonstrated differences in clustering between PRRSV strains, supporting the inability of ORF5 to capture genome-wide variation. For example, high levels of variation were observed within ORF1a, which encodes the hypervariable nsp2 protein. Comparison of the newly assembled genome with the genome of a highly characterized strain (VR2332 PRRSV-2) identified a 100 amino acid deletion within nsp2 characteristic of NADC34-like PRRSV. Oxford Nanopore Technologies’ Flongle flow cell has been proven in this study to provide a rapid, cost-effective and accessible approach for whole-genome sequencing of PRRSV strains present within clinical samples necessary for strain-specific genome-wide characterization.