Enriched Long-Read Sequencing of Co-circulating Viruses in Complex Samples for Advanced Diagnostics and Surveillance

Viral co-infections on swine farms are common and frequent, contributing to aggravated disease outcomes and impairing economic profit and animal well-being. In addition, due to high mutation rates, the co-circulation of viruses within swine herds facilitates the emergence and re-emergence of new variants, impairing disease investigation, control, and prevention. Despite their significant cost and health relevance, viral co-infections are understudied in part due to limitations in sequencing techniques able to generate full-length sequences of multiple viruses from field samples. Furthermore, the detection and genomic characterization of novel variants are still a challenge, and many questions remain about the dynamics and interactions of co-circulating viruses in swine herds. To advance our understanding of viral co-infections, we have developed a workflow called TELSVirus, or Target-Enriched Long-read Sequencing of Virus that enables the real-time detection and genomic characterization of multiple viral pathogens from a single sample in a relatively short turnaround time (24 h). The main objective of this study was to apply the TELSVirus workflow to porcine oral fluid samples to detect and characterize genomes of target viral pathogens. Overall, this method allowed us to detect a high prevalence of co-circulating yet understudied viruses. We also demonstrated that TELSVirus limit of detection for PRRSV and IAV is comparable to that of qPCR, while providing increased genomic information. Based on these results, TELSVirus has the potential to support real-time surveillance of endemic and emergent viruses, while also improving our understanding of co-circulating viruses; their genetic diversity; and ultimately how they impact swine health and production.