Deep Sequencing of Porcine Reproductive and Respiratory Syndrome Virus ORF7: A Promising Tool for Diagnostics and Epidemiologic Surveillance

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major concern worldwide. Control of PRRSV is a challenging task due to various factors, including the viral diversity and variability. In this study, we evaluated an amplicon library preparation protocol targeting the ORF7 region of both PRRSV species, Betaarterivirus suid 1 and Betaarterivirus suid 2. We designed tailed primers for a two-step PCR procedure that generates ORF7-specific amplicon libraries suitable for use on Illumina sequencers. We tested the method with serum samples containing common laboratory strains and with pooled serum samples (n = 15) collected from different pig farms during 2019-2021 in Hungary. Testing spiked serum samples showed that the newly designed method is highly sensitive and detects the viral RNA even at low copy numbers (corresponding to approx. Ct 35). The ORF7 sequences were easily assembled even from clinical samples. Two different sequence variants were identified in five samples, and the Porcilis MLV vaccine strain was identified as the minor variant in four samples. An in-depth analysis of the deep sequencing results revealed numerous polymorphic sites along the ORF7 gene in a total of eight samples, and some sites (positions 12, 165, 219, 225, 315, 345, and 351) were found to be common in several clinical specimens. We conclude that amplicon deep sequencing of a highly conserved region of the PRRSV genome could support both laboratory diagnosis and epidemiologic surveillance of the disease.