Monitoring of European PRRSV strains using sequencing technologies

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most economically important infectious diseases of swine. PRRSV is divided into European (Type I) and American (Type II) genotypes. PRRS is caused by a single stranded positive-sense RNA enveloped virus with a high mutation rate leading to greater heterogeneity of the nucleotide sequence between the individual strains. The high genetic virus diversity increases the risk of reduced sensitivity for real-time RT PCR diagnostic tool. The aim of the present study was to monitor circulating PRRSV strain throughout Europe using capillary ORF7 sequencing and NGS technology.

Thermo Fisher Scientific established several collaborations with laboratories and research institutes to collect field samples. More than 120 PRRSV positive samples were collected in different countries (Spain, Belgium, Netherlands, Czech Republic, Slovenia and Russia) and were sequenced in our lab. According to PRRS viral load, two strategies were performed. Samples with high PRRS viral load were sequenced using NGS workflow on Ion Torrent™ PGM™ instrument, to obtain whole genome sequences. Bioinformatics analysis is performed using an in-house developed tool, Viral Genome Assembly Pipeline (VGAP) for genome assembly and identification of a consensus sequence. For samples with low PRRS viral load, ORF7 sequencing was performed using capillary electrophoresis. Therefore, 16 samples were sequenced using NGS workflow and 33 samples were sequenced in ORF7 sequencing.

33 ORF7 sequences obtained were aligned with PRRS-EU1 (28/33) and PRRS-EU2 (5/33) reference sequences and were confirmed by BLAST analysis. Whole genome sequencing data on 16 samples, were of high quality with a mean coverage of ~4000X. A mean nucleotide read length is around 110pb. Mapping reads against PRRSV genomes available in public database highlighted different European reference strains: subtype 1 (KF203132, GQ461593, KT159248) and subtype 2 strains (KP889243). Comparison between sequences obtained showed an important variability of PRRSV strains on ORF7 fragment as well as on PRRSV complete genome. Mutations on primers/probes design can lead to important impact on detection and decrease kit’s performances: low fluorescence level, late or absence of detection.

The monitoring of circulating strains, associated with participation to Proficiency Testing Scheme, are necessary to identify and sequence new variants. New NGS technology enables to sequence the complete genome of PRRSV and is really essential to develop accurate diagnostic tools. Thermo Fisher Scientific offers different workflows for NGS and targeted sequencing to enable this monitoring.

Disclosure of Interest: None Declared.