Li Yang, Wang Yumiao, Pei Xiuxiu, Wu Yongshuai, Chen Shao, Weng Han, Jing Yang, Ma Zhiqian, Li Zhiwei, Zheng Zifang, Feng Yingtong, Xu Lele, Guo Xuyang, Liu Xiao, Zhang Jianwu, Zheng Haixue, Xiao Shuqi
State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730000, China.
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, China.
Appl Microbiol Biotechnol. 2025 May 2;109(1):109. doi: 10.1007/s00253-025-13502-5.
PRRSV is an immunosuppressive virus that is prone to secondary infection by other viral and microbial pathogens, exacerbating its harm to pigs. The development of multivalent vaccines that can prevent and control multiple pathogens is of great significance for the pig industry. In order to evaluate the potential of commercial PRRSV vaccines as the viral vectors for the trivalent vaccine, this study first embedded mCherry or EGFP genes for fluorescent proteins into the infectious clone of the HP-PRRSV MLV vaccine strain GD to construct a recombinant strain. Furthermore, we constructed a recombinant PRRSV strain, rPRRSV-mCherry-EGFP, that expresses both EGFP and mCherry proteins simultaneously and evaluated the stability and immunogenicity of the exogenous proteins. The results showed that MARC-145 cells infected with rPRRSV-mCherry-EGFP could simultaneously express both EGFP and mCherry exogenous proteins, and the number of EGFP and mCherry positive cells increased with virus infection, providing more fluorescent tool options for virus visualization and high-throughput screening of anti-PRRSV drugs. More importantly, the recombinant rPRRSV-mCherry-EGFP can be passaged in MARC-145 cells for at least 10 generations. It can induce piglets to produce antibodies against PRRSV, EGFP, and mCherry, indicating that the embedded two exogenous genes can also induce good immune responses. Our research suggests that using the infectious clone of a commercial HP-PRRSV-attenuated vaccine as a vector and embedding immune genes of two other pathogens to construct a recombinant strain may be an effective attempt to achieve cross-protection against three pathogens, providing an important research basis for the design of trivalent vaccines. KEY POINTS: • A recombinant PRRSV that expressed two foreign proteins was constructed. • The recombinant PRRSV could induce antibodies against PRRSV, EGFP, and mCherry. • PRRSV MLV strain had the potential to be used as a viral vector for triple vaccines.
猪繁殖与呼吸综合征病毒（PRRSV）是一种免疫抑制性病毒，容易继发其他病毒和微生物病原体感染，加剧其对猪的危害。开发能够预防和控制多种病原体的多价疫苗对养猪业具有重要意义。为了评估商业PRRSV疫苗作为三价疫苗病毒载体的潜力，本研究首先将用于荧光蛋白的mCherry或EGFP基因嵌入HP-PRRSV MLV疫苗株GD的感染性克隆中，构建重组株。此外，我们构建了同时表达EGFP和mCherry蛋白的重组PRRSV株rPRRSV-mCherry-EGFP，并评估了外源蛋白的稳定性和免疫原性。结果表明，感染rPRRSV-mCherry-EGFP的MARC-145细胞可同时表达EGFP和mCherry外源蛋白，且EGFP和mCherry阳性细胞数量随病毒感染而增加，为病毒可视化和抗PRRSV药物的高通量筛选提供了更多荧光工具选择。更重要的是，重组rPRRSV-mCherry-EGFP可在MARC-145细胞中传代至少10代。它能诱导仔猪产生针对PRRSV、EGFP和mCherry的抗体，表明嵌入的两个外源基因也能诱导良好的免疫反应。我们的研究表明，利用商业HP-PRRSV减毒疫苗的感染性克隆作为载体，嵌入另外两种病原体的免疫基因构建重组株，可能是实现对三种病原体交叉保护的有效尝试，为三价疫苗的设计提供重要研究依据。要点：• 构建了表达两种外源蛋白的重组PRRSV。• 重组PRRSV可诱导产生针对PRRSV、EGFP和mCherry的抗体。• PRRSV MLV株有潜力用作三联疫苗的病毒载体。
