Comparison of viral inactivation methods on the characteristics of extracellular vesicles from SARS-CoV-2 infected human lung epithelial cells.
Comparison of viral inactivation methods on the characteristics of extracellular vesicles from SARS-CoV-2 infected human lung epithelial cells.
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DOI：
10.1002/jev2.12291
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发表时间：
2022-12
影响因子：
16
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中科院分区：
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SARS-CoV-2感染与细胞外囊泡（EV）的相互作用目前特别令人感兴趣。在生物安全等级3（BSL-3）环境之外的仪器中研究SARS-CoV-2污染的EV分离株需要了解病毒灭活方法如何影响细胞外囊泡（EV）的结构和功能。因此，对确定的EV颗粒及其蛋白质、RNA和功能进行了三种常见的病毒灭活方法，紫外线-C（UVC; 1350 mJ/cm 2）、β-丙内酯（BPL; 0.005%）、加热（56 ℃，45 min）。通过逐步离心、超滤和qEV尺寸排阻色谱法从SARS-CoV-2-感染的人肺上皮Calu-3细胞的上清液中分离小EV。EV分离株含有SARS-CoV-2。UVC、BPL和高温完全消除了被污染电动汽车的SARS-CoV-2感染性。通过电子显微镜和纳米粒子跟踪的粒子检测受UVC和BPL的影响小于热处理。EV标志物的Western印迹分析不受这三种方法中任何一种的影响。UVC通过定量RT-PCR降低了SARS-CoV-2峰的可检测性，并轻微改变了EV衍生的β-肌动蛋白检测。成纤维细胞迁移-SARS-CoV-2污染的EV分离株的伤口愈合活性仅在UVC处理后保留。总之，特定的病毒灭活方法与SARS-CoV-2污染EV分离株的特定措施相容。UVC治疗似乎更适合研究从SARS-CoV-2感染细胞释放的EV的功能。
The interaction of SARS‐CoV‐2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS‐CoV‐2 contaminated‐EV isolates in instruments located outside of the biosafety level‐3 (BSL‐3) environment requires knowing how viral inactivation methods affect the structure and function of extracellular vesicles (EVs). Therefore, three common viral inactivation methods, ultraviolet‐C (UVC; 1350 mJ/cm2), β‐propiolactone (BPL; 0.005%), heat (56°C, 45 min) were performed on defined EV particles and their proteins, RNAs, and function. Small EVs were isolated from the supernatant of SARS‐CoV‐2‐infected human lung epithelial Calu‐3 cells by stepwise centrifugation, ultrafiltration and qEV size‐exclusion chromatography. The EV isolates contained SARS‐CoV‐2. UVC, BPL and heat completely abolished SARS‐CoV‐2 infectivity of the contaminated EVs. Particle detection by electron microscopy and nanoparticle tracking was less affected by UVC and BPL than heat treatment. Western blot analysis of EV markers was not affected by any of these three methods. UVC reduced SARS‐CoV‐2 spike detectability by quantitative RT‐PCR and slightly altered EV‐derived β‐actin detection. Fibroblast migration‐wound healing activity of the SARS‐CoV‐2 contaminated‐EV isolate was only retained after UVC treatment. In conclusion, specific viral inactivation methods are compatible with specific measures in SARS‐CoV‐2 contaminated‐EV isolates. UVC treatment seems preferable for studying functions of EVs released from SARS‐CoV‐2 infected cells.