Liu Rongxiao, Fu Shi, Chai Yukun, Liu Nian, Liu Benjin, Luo Lingzhi, Yang Wei, Dong Xiumei, Cui Jin
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China.
Front Cell Infect Microbiol. 2025 Aug 26;15:1653170. doi: 10.3389/fcimb.2025.1653170. eCollection 2025.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant pathogen posed a serious threat to the global swine industry. In this study, a BAC-based reverse genetics platform was established using a highly pathogenic PRRSV (HP-PRRSV) strain. Three recombinant reporter viruses were constructed by inserting the enhanced green fluorescent protein (EGFP) gene into three different intergenic regions of the complete PRRSV-L251 genome. Immunofluorescence assays combined with viral growth kinetics and reporter gene stability assessments indicated that rL251-ORF4-5a-EGFP maintained relatively stable expression during serial passage, and viral titers at 72 hours post-infection (hpi) were comparable to the parental virus. Subsequently, we identified four candidate compounds with potential anti-PRRSV activity using rPRRSV-L251-ORF4-5a-EGFP, indicating that this platform can be used as a visual assessment tool for antiviral drug screening. This study demonstrated that the ORF4-5a interval region is a feasible and promising site for exogenous gene insertion, and provided a robust technical platform for PRRSV vaccine development and pathogenesis studies.
猪繁殖与呼吸综合征病毒（PRRSV）是对全球养猪业构成严重威胁的重要病原体。在本研究中，利用高致病性PRRSV（HP-PRRSV）毒株建立了基于细菌人工染色体（BAC）的反向遗传学平台。通过将增强型绿色荧光蛋白（EGFP）基因插入完整PRRSV-L251基因组的三个不同基因间隔区，构建了三种重组报告病毒。免疫荧光分析结合病毒生长动力学和报告基因稳定性评估表明，rL251-ORF4-5a-EGFP在连续传代过程中保持相对稳定的表达，感染后72小时（hpi）的病毒滴度与亲本病毒相当。随后，我们使用rPRRSV-L251-ORF4-5a-EGFP鉴定了四种具有潜在抗PRRSV活性的候选化合物，表明该平台可作为抗病毒药物筛选的可视化评估工具。本研究表明，ORF4-5a间隔区是外源基因插入的一个可行且有前景的位点，并为PRRSV疫苗开发和发病机制研究提供了一个强大的技术平台。
