Porcine reproductive and respiratory syndrome virus whole-genome sequencing efficacy with field clinical samples using a poly(A)-tail viral genome purification method

Abstract

The genomic surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) is based on sequencing of the ORF5 gene of the virus, which covers only 4% of the entire viral genome. It is expected that PRRSV whole-genome sequencing (WGS) will improve PRRSV genomic data and allow better understanding of clinical discrepancies observed in the field when using ORF5 sequencing. Our main objective was to implement an efficient method for WGS of PRRSV from clinical samples. The viral genome was purified using a poly(A)-tail viral genome purification method and sequenced using Illumina technology. We tested 149 PRRSV-positive samples: 80 sera, 33 lungs, 33 pools of tissues, 2 oral fluids, and 1 processing fluid (i.e., castration liquid). Overall, WGS of 67.1% of PRRSV-positive cases was successful. The viral load, in particular for tissues, had a major impact on the PRRSV WGS success rate. Serum was the most efficient type of sample to conduct PRRSV WGS poly(A)-tail assays, with a success rate of 76.3%, and this result can be explained by improved sequencing reads dispersion matching throughout the entire viral genome. WGS was unsuccessful for all pools of tissue and lung samples with Cq values > 26.5, whereas it could still be successful with sera at Cq ≤ 34.1. Evaluation of results of highly qualified personnel confirmed that laboratory skills could affect PRRSV WGS efficiency. Oral fluid samples seem very promising and merit further investigation because, with only 2 samples of low viral load (Cq = 28.8, 32.8), PRRSV WGS was successful.

Implementing a user‐friendly format to analyze PRRSV next‐generation sequencing results and associating breeding herd production performance with number of PRRSV strains and recombination events

“…Recently, the concept of applying NGS directly to field samples has been explored (Lalonde et al., 2020 ). There have been encouraging reports describing the potential recovery of a near complete‐PRRSV genome from field clinical serum samples with Ct up to 34.1, oral fluid with Ct up to 32.8 and tissue and lung samples with Ct up to 26.5 (Gagnon et al., 2021 ). RNA viruses, such as PRRSV, are challenging to sequence and characterize using high‐throughput sequencing technologies due to their genetic diversity, relatively short genome lengths and lack of conserved genome regions (Fitzpatrick et al., 2021 ).…”

Measuring How Recombination Re-shapes the Evolutionary History of PRRSV-2: A Genome-Based Phylodynamic Analysis of the Emergence of a Novel PRRSV-2 Variant

“…Sample selection criteria for WGS included having ORF5 sequences within the emerging variant's phylogenetic clade (<2% genetic distance to at least one other sequence classified as L1C-1-4-4) and cycle threshold (Ct) value ≤25 for reverse transcription polymerase chain reaction (RT-PCR) using VetMAX TM NA and EU PRRSV Reagents (Thermo Fisher Scientific, MA, USA). Oral fluids and processing fluids samples were excluded due to the low success rate for whole genome sequencing (17,18). At least one ORF5 sequence from each participating system was whole-genome sequenced.…”

Comparison of Primary Virus Isolation in Pulmonary Alveolar Macrophages and Four Different Continuous Cell Lines for Type 1 and Type 2 Porcine Reproductive and Respiratory Syndrome Virus

“…De novo PRRSV1 genomes were generated using Canu (v2.0) [35], graphmap (v0.5.2) [36], and medaka polishing (v1.0.0; ONT). Sequencing of the wild-type PRRSV2 strains was done using the protocol reported earlier [37,38]. In brief, viral RNA was isolated from PRRSV qPCR positive clinical samples with a poly(A)-tail mRNA magnetic isolation module (New England BioLabs), followed by in vitro synthesis of dsDNA (New England BioLabs), sequencing libraries preparation, purification, and normalization (Illumina Nextera XT DNA library preparation kit).…”