Jakab Szilvia, Bali Krisztina, Freytag Csongor, Pataki Anna, Fehér Enikő, Halas Máté, Jerzsele Ákos, Szabó István, Szarka Krisztina, Bálint Ádám, Bányai Krisztián
Veterinary Medical Research Institute, Hungária krt. 21., H-1143 Budapest, Hungary.
National Laboratory for Infectious Animal Diseases, Antimicrobial Resistance, Veterinary Public Health and Food Chain Safety, Hungária krt. 21., H-1143 Budapest, Hungary.
Animals (Basel). 2023 Oct 15;13(20):3223. doi: 10.3390/ani13203223.
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, and . 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 ( = 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.
猪繁殖与呼吸综合征病毒（PRRSV）是全球关注的主要问题。由于包括病毒多样性和变异性在内的各种因素，控制PRRSV是一项具有挑战性的任务。在本研究中，我们评估了一种针对PRRSV两种毒株ORF7区域的扩增子文库制备方案。我们设计了用于两步PCR程序的带尾引物，该程序可生成适用于Illumina测序仪的ORF7特异性扩增子文库。我们用含有常见实验室毒株的血清样本以及2019 - 2021年期间从匈牙利不同养猪场收集的混合血清样本（n = 15）对该方法进行了测试。对加标血清样本的测试表明，新设计的方法高度灵敏，即使在低拷贝数（对应约Ct 35）时也能检测到病毒RNA。即使从临床样本中也能轻松组装出ORF7序列。在五个样本中鉴定出两种不同的序列变体，并且在四个样本中鉴定出Porcilis MLV疫苗株为次要变体。对深度测序结果的深入分析揭示了总共八个样本中沿ORF7基因存在大量多态性位点，并且发现一些位点（第12、165、219、225、315、345和351位）在几个临床标本中很常见。我们得出结论，PRRSV基因组高度保守区域的扩增子深度测序可为该疾病的实验室诊断和流行病学监测提供支持。
