Evaluating NGS-generated contig accuracy for detection and assignment of PRRSV against a reference genome in experimentally mixed PRRSV infection samples.

Mixed infections with multiple Porcine reproductive and respiratory syndrome virus (PRRSV) are common in swine production and complicate diagnostic interpretation, particularly when whole genomes cannot be recovered through next-generation sequencing (NGS). This study evaluated the ability of NGS to detect and characterize multiple PRRSV-2 strains present in controlled viral mixtures that varied in strain number, viral load, and proportional abundance, ultimately providing guidance for interpreting NGS outputs. Nine PRRSV-2 viruses, including three wild-type viruses and six commercial vaccine viruses, were spiked into processing fluid at Ct values of 21 and 27 to generate standardized combinations that reflect field conditions. Across all mixtures, NGS resulted in contigs that identified the correct strain composition in 89.7% of samples. NGS detected virus present at 10% concentration in 95.8% (23 of 24) of samples containing two viruses diluted at a 1:9 mixture. Contig nucleotide identity to reference strains decreased as viral load declined, yet accurate strain assignment was still achieved in most cases using a threshold of at least 98% nucleotide identity for high-concentration samples and at least 96% for lower-concentration samples. Detection failures occurred in 10.3% (8 of 78) of mixtures, 75% (6 of 8) of which contained four or more strains, indicating reduced NGS sensitivity under highly complex conditions with the same viral species. In different instances, NGS failed to detect MLV vaccines when 2 or more wild-type viruses were present. In 0.7% (8 of 1,147) of contigs, assembly artifacts were identified that proved to be artificial recombinant contigs, and 0.61% (7 of 1,147) were classified as inconclusive because they matched two highly similar vaccine strains at greater than 98% nucleotide identity. These results demonstrate that meaningful interpretation of PRRSV variant composition is possible from partial genome recovery, provided appropriate identity thresholds are applied and reference genetic sequences are available for comparison. The findings provide practical guidance for diagnostic laboratories and veterinarians who rely on NGS-derived contigs for PRRSV classification in field samples.