Luo Huaye, Lv Lilei, Yi Jingxuan, Zhou Yanjun, Liu Changlong
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonosis, Yangzhou University, Yangzhou 225009, China.
Microorganisms. 2023 Aug 12;11(8):2075. doi: 10.3390/microorganisms11082075.
The porcine epidemic diarrhea virus (PEDV) is a highly contagious and virulent enteric coronavirus that causes severe enteric disease in pigs worldwide. PEDV infection causes profound diarrhea, vomiting, and dehydration in pigs of all ages, resulting in high mortality rates, particularly among neonatal piglets. The spike glycoprotein (S) of PEDV plays a crucial role in binding to the host cell receptor and facilitating fusion between the viral and host membranes. Pseudotyped viral particles featuring the PEDV S protein are valuable tools for investigating virus entry, identifying neutralizing antibodies, and developing small molecules to impede virus replication. In this study, we used a codon-optimized PEDV S protein to generate recombinant pseudotyped vesicular stomatitis virus (VSV) particles (rVSV-ΔG-EGFP-S). The full-length S protein was efficiently incorporated into VSV particles. The S protein pseudotyped VSV exhibited infectivity towards permissive cell lines of PEDV. Moreover, we identified a new permissive cell line, JHH7, which showed robust support for PEDV replication. In contrast to the SARS-CoV-2 spike protein, the removal of amino acids from the cytoplasmic tail resulted in reduced efficiency of viral pseudotyping. Furthermore, we demonstrated that 25-hydroxycholesterol inhibited rVSV-ΔG-EGFP-S entry, while human APN facilitated rVSV-ΔG-EGFP-S entry through the use of ANPEP knockout Huh7 cells. Finally, by transducing swine intestinal organoids with the rVSV-ΔG-EGFP-S virus, we observed efficient infection of the swine intestinal organoids by the PEDV spike-pseudotyped VSV. Our work offers valuable tools for studying the cellular entry of PEDV and developing interventions to curb its transmission.
猪流行性腹泻病毒（PEDV）是一种具有高度传染性和致病性的肠道冠状病毒，可在全球范围内导致猪发生严重的肠道疾病。PEDV感染可引起各年龄段猪的严重腹泻、呕吐和脱水，导致高死亡率，尤其是新生仔猪。PEDV的刺突糖蛋白（S）在与宿主细胞受体结合以及促进病毒膜与宿主膜融合方面起着关键作用。携带PEDV S蛋白的假型病毒颗粒是研究病毒进入、鉴定中和抗体以及开发阻碍病毒复制的小分子的有价值工具。在本研究中，我们使用密码子优化的PEDV S蛋白来生成重组假型水疱性口炎病毒（VSV）颗粒（rVSV-ΔG-EGFP-S）。全长S蛋白有效地整合到VSV颗粒中。S蛋白假型化的VSV对PEDV的允许细胞系具有感染性。此外，我们鉴定出一种新的允许细胞系JHH7，它对PEDV复制表现出强大的支持作用。与严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白不同，从细胞质尾巴去除氨基酸会导致病毒假型化效率降低。此外，我们证明25-羟基胆固醇抑制rVSV-ΔG-EGFP-S进入，而人氨肽酶N（APN）通过使用ANPEP基因敲除的Huh7细胞促进rVSV-ΔG-EGFP-S进入。最后，通过用rVSV-ΔG-EGFP-S病毒转导猪肠道类器官，我们观察到PEDV刺突假型化的VSV对猪肠道类器官的有效感染。我们的工作为研究PEDV的细胞进入以及开发抑制其传播的干预措施提供了有价值的工具。
