State Key Laboratory of Biocontrol, Life Sciences School, Sun Yat-Sen University, Guangzhou 510275, China.
Viruses. 2023 Jun 23;15(7):1426. doi: 10.3390/v15071426.
Porcine epidemic diarrhea virus (PEDV) has led to significant economic losses in the global porcine industry since the emergence of variant strains in 2010. The high mutability of coronaviruses endows PEDV with the ability to evade the host immune response, which impairs the effectiveness of vaccines. In our previous study, we generated a highly cell-passaged PEDV strain, CT-P120, which showed promise as a live attenuated vaccine candidate by providing satisfactory protection against variant PEDV infection in piglets. However, the mechanism by which the attenuated CT-P120 adapts to cells during passage, resulting in increased replication efficiency, remains unclear. To address this question, we conducted a comparative transcriptomic analysis of Vero E6 cells infected with either the original parental strain (CT-P10) or the cell-attenuated strain (CT-P120) of PEDV at 6, 12, and 24 h post-infection. Compared to CT-P10, CT-P120 infection resulted in a significant decrease in the number of differentially expressed genes (DEGs) at each time point. Functional enrichment analysis of genes revealed the activation of various innate immune-related pathways by CT-P10, notably attenuated during CT-P120 infection. To validate these results, we selected eight genes (TRAF3, IRF3, IFNL1, ISG15, NFKB1, MAP2K3, IL1A, and CCL2) involved in antiviral processes and confirmed their mRNA expression patterns using RT-qPCR, in line with the transcriptomic data. Subsequent protein-level analysis of selected genes via Western blotting and enzyme-linked immunosorbent assay corroborated these results, reinforcing the robustness of our findings. Collectively, our research elucidates the strategies underpinning PEDV attenuation and immune evasion, providing invaluable insights for the development of effective PEDV vaccines.
自 2010 年变异株出现以来,猪流行性腹泻病毒(PEDV)已导致全球养猪业遭受重大经济损失。冠状病毒的高突变性赋予了 PEDV 逃避宿主免疫反应的能力,从而降低了疫苗的有效性。在我们之前的研究中,我们生成了一株高度细胞适应的 PEDV 株 CT-P120,该病毒株在仔猪中对变异 PEDV 感染提供了令人满意的保护作用,显示出作为活减毒疫苗候选株的潜力。然而,减毒的 CT-P120 在细胞传代过程中适应细胞从而导致复制效率提高的机制尚不清楚。为了解决这个问题,我们对 Vero E6 细胞感染原始亲本株(CT-P10)或细胞减毒株(CT-P120)后的转录组进行了比较分析,感染后分别在 6、12 和 24 小时进行检测。与 CT-P10 相比,CT-P120 感染在每个时间点的差异表达基因(DEGs)数量显著减少。对基因的功能富集分析表明,CT-P10 激活了多种先天免疫相关途径,而在 CT-P120 感染过程中这些途径显著减弱。为了验证这些结果,我们选择了八个参与抗病毒过程的基因(TRAF3、IRF3、IFNL1、ISG15、NFKB1、MAP2K3、IL1A 和 CCL2),并使用 RT-qPCR 验证了它们的 mRNA 表达模式,与转录组数据一致。通过 Western blot 和酶联免疫吸附试验对选定基因的蛋白水平分析进一步证实了这些结果,增强了我们研究结果的可信度。总的来说,我们的研究阐明了 PEDV 减毒和免疫逃逸的策略,为有效 PEDV 疫苗的开发提供了宝贵的见解。