SHIC-Funded Study Results in Validation of Whole Genome Sequencing Breakthrough

A Swine Health Information Center-funded study aimed to develop and validate capture probe-based enrichment for whole genome sequencing (WGS) of PRRSV. Led by Dr. Leyi Wang at the University of Illinois Urbana-Champaign, the project sought to improve the diagnostic performance of PRRSV WGS through the evaluation of a capture method that acts as a genetic magnet to pull viral material out of complex samples. Study results demonstrate that the enrichment method increased PRRSV genome coverage in oral fluids, processing fluids, lung tissue, and serum, consistently capturing >90% of the whole genome. Higher genome recovery using convenient, non-invasive sample types provides more accurate diagnostic results. The validated capture enrichment-based method for WGS enables faster identification of new variants, better differentiation between vaccine and wild-type strains, and more precise biosecurity decisions to protect herd health.

Read the industry summary for study #24-030 here.

WGS of PRRSV is a commonly used diagnostic tool for genotyping, providing significantly higher discriminatory power than ORF5 sequencing and improving viral genomic surveillance. PRRSV WGS has been growing as the preferred approach over traditional ORF5 sequencing because it captures the entire genome, whereas ORF5 sequencing captures only about 4% of the genome. Although WGS provides a complete genetic fingerprint of PRRSV, its sensitivity is often insufficient in commonly collected farm samples, such as oral fluids and processing fluids, which may have low viral loads. To address this limitation, the overarching goal of this project was to develop and validate capture probe-based enrichment for WGS of PRRSV in different sample types, including serum, lung, oral fluids, processing fluids, and tongue tip fluids.

Objectives of the study described herein include: 1) capture probe design and optimization by using over 28,000 PRRSV complete and partial genomic sequences available at the GenBank database; 2) measure sensitivity of the capture probe method through serial dilutions of available PRRSV-1 and PRRSV-2 isolates; 3) investigate clinical applications and validation through processing PRRSV-positive clinical samples and using for sequencing with capture probe enrichments. While amplicon-based methods are common for other viruses, they are poorly suited for PRRSV due to its high mutation rate and frequent genomic rearrangements. Probe-based methods offer a more robust alternative due to the increased tolerance of genetic diversity and lack of requirement for precise sequence matches to detect new variants.

A total number of 28,721 PRRSV complete and partial genomes were downloaded from GenBank, containing 1,604 complete or near complete-genomic sequences and 27,117 partial genomic sequences, that were utilized for probe selection and development. Sensitivity measurements for capture probe enrichment of PRRSV WGS utilized PRRSV laboratory strains (three North American strains 11604, PA8, VR2332, one EU strain Lelystad) which had been previously sequenced and serially diluted for evaluation. Clinical application and validation of the new technique was evaluated using 110 swine samples (30 serum samples, 33 processing fluids, 24 oral fluids, 23 lung tissues) for both probe-capture based enrichment and non-enrichment-based sequencing.

For laboratory isolates, the probe capture enrichment method effectively enhanced genome coverage, ranging from 2.6% to 95%, and achieved >90% genome coverage for RNA with Ct values up to 29.8 for three PRRSV-2 strains (11604, PA8, and VR2332). For the PRRSV-1 strain (Lelystad), the genome coverage ranged from 2.2% to 32.5%, with >90% genome coverage for samples with Ct values up to 30.2.

Results demonstrated a significant breakthrough on clinical samples: the enrichment method increased PRRSV genome coverage by nearly 46% in oral fluids, 32% in processing fluids, 25% in lung tissue, and 24% in serum. It consistently captured >90% of the whole genome even at lower viral loads (Ct 26–27) for serum, processing fluids, and lung tissue. Even in samples with Ct values as high as 33.8, enrichment significantly boosted data recovery. When probe enrichment was combined with prior ribosomal RNA (rRNA) removal in tissue samples, further enhancement of PRRSV genome coverage occurred. The data demonstrates that probe capture-based enrichment significantly increases PRRSV WGS and genome surveillance.

Improvement in PRRSV WGS can result in higher genome recovery when using convenient, non-invasive population-based sample types. The evaluation of capture probe-based enrichment demonstrates a significant advancement in the industry’s ability to monitor PRRSV and other viral swine diseases, particularly in challenging sample types common in modern production systems. Enhancing diagnostic capabilities to improve accurate detection of pathogens in swine samples aligns with SHIC’s efforts for surveillance and discovery of emerging diseases.

The Swine Health Information Center, launched in 2015 with Pork Checkoff funding, protects and enhances the health of the US swine herd by minimizing the impact of emerging disease threats through preparedness, coordinated communications, global disease monitoring, analysis of swine health data, and targeted research investments. As a conduit of information and research, SHIC encourages sharing of its publications and research. Forward, reprint, and quote SHIC material freely. For more information, visit http://www.swinehealth.org or contact Dr. Megan Niederwerder at [email protected] or Dr. Lisa Becton at [email protected].