Comparison of viral inactivation methods on the characteristics of extracellular vesicles from SARS‐CoV‐2 infected human lung epithelial cells

Abstract

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/cm 2 ), 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.

The Role of Extracellular Vesicles in SARS-CoV-2-Induced Acute Kidney Injury: An Overview

“…Isolated EVs from SARS-CoV-2 infected Calu-3 cells were found to contain the SARS-CoV-2 nucleoprotein, while EVs from the control were absent of viral components. These results demonstrate that EVs derived from human lung epithelial cells are capable of housing SARS-CoV-2 viral cargo in vitro, suggesting a potential EV-facilitated mechanism of SARS-CoV-2 infection [51].…”

Prenatal SARS-CoV-2 infection alters postpartum human milk-derived extracellular vesicles

“…A combination of stepwise centrifugation, ultrafiltration and qEV-size exclusion chromatography (SC-UF-qEV) has been successfully employed in our previous studies to isolate EVs from the cell culture media and human plasma, resulting in an acceptable recovery yield and high specificity. [34][35][36] This combined method has never been applied to human milk. Noted that human milk matrix is more complex than cell culture media and plasma, containing numerous milk fat and casein micelles which could interfere downstream EV analysis.…”

“…In close collaboration with the IMPRINT cohort, the aim of this study was to ascertain the influence of prenatal SARS-CoV-2 infection on HMEV molecular cargos. Following the optimization of our previously established method [34][35][36] for the successful isolation of EVs from the complex matrix of human milk, we performed a multi-Omic analysis. This incorporated proteomics, surfaceomics (or the proteomic analysis of surface EV proteins), and miRNA sequencing on HMEVs isolated from 9 mothers with prenatal SARS-CoV-2 infection, compared against 9 healthy control subjects.…”

“…HMEV surfaceomics. Of 366 surface EV proteins, top 10% increased(36) and decreased(36) proteins were analyzed by STRING protein-protein interaction with functional enrichment analysis. Color codes represent proteins involved in predicted functions.…”

β-Propiolactone (BPL)-inactivation of SARS-Co-V-2: In vitro validation with focus on saliva from COVID-19 patients for scent dog training

“…The hitherto published inactivation protocols of SARS-CoV and SARS-CoV-2 with BPL vary widely. Successful virus inactivation demonstrated by absence of infectious virus in cell culture including the initial virus titre (10^7 to 10^6 PFU/ml) was only proven in six studies (16% of 37 studies) ( de Castro Barbosa et al, 2022 , Gao et al, 2020 , Jureka et al, 2020 , Kongsomros et al, 2022 , Petráš et al, 2020 , Saleh et al, 2021 ) (supplemental Table 1 ). Similarly, we demonstrated BPL-inactivation of SARS-CoV-2 by a titre reduction of up to 10^4 TCID 50 /ml verified by virus titration and isolation, respectively ( Table 1 , Table 2 ).…”