Virion-Independent Extracellular Vesicle (EV)-Dependent Transmission of SARS-CoV-2 as a Potential New Mechanism of Viral RNA Spread in Human Cells

DOI record: { "DOI": "10.3390/v18010145", "ISSN": [ "1999-4915" ], "URL": "http://dx.doi.org/10.3390/v18010145", "abstract": "<jats:p>The concentration of extracellular vesicles (EVs) in the peripheral blood of COVID-19 patients is increased. Nevertheless, their potential role in the transmission of infection remains unclear. This study was performed to determine whether EVs produced by the sub-genomic replicon system developed in Baby Hamster Kidney (BHK-21) cells could transfer SARS-CoV-2 replicon RNA, leading to the establishment of a viral replication system in human cells. Purified EVs from the SARS-CoV-2 sub-genomic replicon cell line BHK-21 were cultured with a naive human cell line. The success of EV-mediated transfer of SARS-CoV-2 replicon RNA and its productive replication was assessed using G-418 selection, a luciferase assay, immunostaining, and Western blot. We found that the A549 cell line cultured with EVs isolated from SARS-CoV-2 BHK-21 replicon cells developed G-418-resistant cell colonies. SARS-COV-2 RNA replication in A549 cells was confirmed by nano luciferase, Nsp1 protein. SARS-CoV-2 RNA replication causes massive morphological changes. Treatment of cells with the FDA-approved Paxlovid demonstrated a dose-dependent inhibition of viral replication. We isolated two human epithelial cell lines (gastrointestinal and neuroblastoma) and one vascular endothelial cell line that stably support high-level replication of SARS-CoV-2 sub-genomic RNA. Viral elimination did not revert the abnormal cellular shape, vesicle accumulation, syncytia formation, or EV release. Our study’s findings highlight the potential implications of EV-mediated transfer of replicon RNA to permissive cells. The replicon model is a valuable tool for studying virus-induced reversible and irreversible cellular reprogramming, as well as for testing novel therapeutic strategies for SARS-CoV-2.</jats:p>", "alternative-id": [ "v18010145" ], "author": [ { "ORCID": "https://orcid.org/0000-0002-7921-3169", "affiliation": [ { "name": "Division of Gastroenterology and Hepatology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA" } ], "authenticated-orcid": false, "family": "Ekmen", "given": "Nergiz", "sequence": "first" }, { "ORCID": "https://orcid.org/0000-0002-5693-5951", "affiliation": [ { "name": "Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA" } ], "authenticated-orcid": false, "family": "Koksal", "given": "Ali Riza", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA" } ], "family": "Lin", "given": "Dong", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA" } ], "family": "Tian", "given": "Di", "sequence": "additional" }, { "ORCID": "https://orcid.org/0000-0002-6483-6259", "affiliation": [ { "name": "Department of Gastroenterology and Hepatology, Institute of Translational Research, Ochsner Health, New Orleans, LA 70121, USA" } ], "authenticated-orcid": false, "family": "Thevenot", "given": "Paul", "sequence": "additional" }, { "affiliation": [ { "name": "Division of Gastroenterology and Hepatology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA" } ], "family": "Glover", "given": "Sarah", "sequence": "additional" }, { "affiliation": [ { "name": "Division of Gastroenterology and Hepatology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA" }, { "name": "Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA" }, { "name": "Southeast Louisiana Veterans Health Care System, 2400 Canal Street, New Orleans, LA 70119, USA" } ], "family": "Dash", "given": "Srikanta", "sequence": "additional" } ], "container-title": "Viruses", "container-title-short": "Viruses", "content-domain": { "crossmark-restriction": false, "domain": [] }, "created": { "date-parts": [ [ 2026, 1, 23 ] ], "date-time": "2026-01-23T10:41:34Z", "timestamp": 1769164894000 }, "deposited": { "date-parts": [ [ 2026, 1, 23 ] ], "date-time": "2026-01-23T10:47:26Z", "timestamp": 1769165246000 }, "funder": [ { "award": [ "1101 BX004516-01A1" ], "award-info": [ { "award-number": [ "1101 BX004516-01A1" ] } ], "name": "Veterans Affairs Merit Review" }, { "name": "Tulane University Health Sciences Center" }, { "name": "Seed Grant from the Louisiana Cancer Research Center" }, { "name": "Akdamar Fellowship Program" }, { "name": "Department of Gastroenterology and Hepatology, Tulane University Health Science Center" } ], "indexed": { "date-parts": [ [ 2026, 1, 24 ] ], "date-time": "2026-01-24T20:10:42Z", "timestamp": 1769285442680, "version": "3.49.0" }, "is-referenced-by-count": 0, "issue": "1", "issued": { "date-parts": [ [ 2026, 1, 22 ] ] }, "journal-issue": { "issue": "1", "published-online": { "date-parts": [ [ 2026, 1 ] ] } }, "language": "en", "license": [ { "URL": "https://creativecommons.org/licenses/by/4.0/", "content-version": "vor", "delay-in-days": 0, "start": { "date-parts": [ [ 2026, 1, 22 ] ], "date-time": "2026-01-22T00:00:00Z", "timestamp": 1769040000000 } } ], "link": [ { "URL": "https://www.mdpi.com/1999-4915/18/1/145/pdf", "content-type": "unspecified", "content-version": "vor", "intended-application": "similarity-checking" } ], "member": "1968", "original-title": [], "page": "145", "prefix": "10.3390", "published": { "date-parts": [ [ 2026, 1, 22 ] ] }, "published-online": { "date-parts": [ [ 2026, 1, 22 ] ] }, "publisher": "MDPI AG", "reference": [ { "DOI": "10.1056/NEJMoa2001017", "article-title": "A novel coronavirus from patients with pneumonia in China, 2019", "author": "Zhu", "doi-asserted-by": "crossref", "first-page": "727", "journal-title": "N. 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