Zhang Baoge, Cai Jinshuang, Zhu Chenguang, Zhang Yuyu, Wu Jiaqiang, Li Yufeng
Key Laboratory of Bacteriology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan, China.
J Virol. 2025 Jun 17;99(6):e0034125. doi: 10.1128/jvi.00341-25. Epub 2025 May 12.
The pathogenesis of naïve porcine circovirus type 3 (PCV3) in pigs is not accurately elucidated due to virus isolation failure . In this study, naïve PCV3 was successfully rescued and used to infect cesarean-derived (CD) pigs. The PCV3 genome was synthesized, cloned, and inserted into the pBluescript SK vector. PK-15 cells transfected with the recombinant plasmid pSK-PCV3 were passaged, and Cap protein expression was confirmed by immunofluorescence assay (IFA) and western blotting. Virus particles (~20 nm) were observed via transmission electron microscopy (TEM). The viral growth curve was plotted to determine the replication of the virus within the cells. Nocodazole treatment demonstrated that PCV3 replication is dependent on microtubule polymerization in the cell. Cells infected with PCV3 or PCV3-positive clinical samples (wPCV3) showed only cytoplasmic fluorescence. PCV3 can successfully infect CD pigs, resulting in persistent viremia, and increased viral loads in tissues and an antibody delay were observed. Inflammatory lesions were also observed in the lungs, lymph nodes, livers, and intestines of infected pigs. Stimulation of peripheral blood mononuclear cells (PBMCs) with virus-like particles (VLPs) containing the Cap protein significantly inhibited cell proliferation. scRNA-seq revealed a significant reduction in T helper 2 (Th2) cells and the migration of T helper 1 (Th1) cells toward the late differentiation stage following infection. In particular, the decrease in Th2 cells may indicate impaired humoral immunity, leading to delayed antibody production and immunosuppression. Our study is the first to observe PCV3 via TEM and to elucidate its immunosuppressive mechanisms in CD pigs.
This study is of great significance as it successfully rescued naïve PCV3 and, for the first time, observed clear PCV3 viral particles using transmission electron microscopy. The successful infection of CD pigs deepened our understanding of its pathogenic mechanisms. The results revealed key aspects of viral replication, the impact of the virus on immune responses, and associated inflammatory lesions in various tissues. Notably, the study found that the reduction of Th2 cells leads to impaired humoral immunity and delayed antibody production, which may provide valuable insights for vaccine development and swine disease management.
由于病毒分离失败，猪源3型圆环病毒（PCV3）在猪体内的发病机制尚未得到准确阐明。在本研究中，成功拯救了未接触过的PCV3并用于感染剖腹产仔猪（CD猪）。合成、克隆了PCV3基因组，并将其插入pBluescript SK载体。用重组质粒pSK-PCV3转染的PK-15细胞传代培养，通过免疫荧光试验（IFA）和蛋白质免疫印迹法确认Cap蛋白表达。通过透射电子显微镜（TEM）观察到病毒粒子（约20纳米）。绘制病毒生长曲线以确定病毒在细胞内的复制情况。诺考达唑处理表明PCV3复制依赖于细胞中的微管聚合。感染PCV3或PCV3阳性临床样本（wPCV3）的细胞仅显示细胞质荧光。PCV3可成功感染CD猪，导致持续性病毒血症，并观察到组织中病毒载量增加和抗体延迟产生。在感染猪的肺、淋巴结、肝脏和肠道中也观察到炎症性病变。用含有Cap蛋白的病毒样颗粒（VLP）刺激外周血单核细胞（PBMC）可显著抑制细胞增殖。单细胞RNA测序（scRNA-seq）显示，感染后辅助性T细胞2（Th2）显著减少，辅助性T细胞1（Th1）向晚期分化阶段迁移。特别是，Th2细胞的减少可能表明体液免疫受损，导致抗体产生延迟和免疫抑制。我们的研究首次通过TEM观察到PCV3，并阐明了其在CD猪中的免疫抑制机制。
本研究具有重要意义，因为它成功拯救了未接触过的PCV3，并首次使用透射电子显微镜观察到清晰的PCV3病毒粒子。CD猪的成功感染加深了我们对其致病机制的理解。结果揭示了病毒复制的关键方面、病毒对免疫反应的影响以及各种组织中的相关炎症性病变。值得注意的是，该研究发现Th2细胞的减少导致体液免疫受损和抗体产生延迟，这可能为疫苗开发和猪病管理提供有价值的见解。
Zotero 插件
