{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T14:40:42Z","timestamp":1762353642813,"version":"build-2065373602"},"reference-count":45,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,6,15]],"date-time":"2025-06-15T00:00:00Z","timestamp":1749945600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Medical Scientific Fund of the Mayor of the City of Vienna","award":["21122"],"award-info":[{"award-number":["21122"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Polyomaviruses have the potential to cause significant morbidity not only in transplant medicine, but also in other forms of disease or variants of immunosuppression. In kidney transplant recipients or recipients of human stem cell transplants, the BK-Virus is the major proponent of manifestations such as BKPyV-associated nephropathy or hemorrhagic cystitis. As no polyomavirus-specific drug with proven in vivo effects has been developed so far, methods to screen for such drugs are important. This work describes the establishment of a virus-secreting cell line. By infecting a pre-established monkey kidney cell line (COS-1) with a non-rearranged human BK polyomavirus isolated from a kidney transplant patient suffering from BKPyV-associated nephropathy, a continuously replicating cell type with consistent virus secretion could be established and was termed COSSA. Measurements of BKPyV replication, virion production, and secretion were performed both intracellularly and in the cell supernatant. Viral proteins such as VP1 and LTAg were accurately tracked by confocal microscopy, as well as by immunoblot and qPCR. An intracellular flow cytometry (FACS) assay detecting VP1 protein was established and revealed an expanded range of positive intracellular signals. The viruses produced proved to be infectious in human tubular epithelial cell lines. Long-range sequencing of the COSSA genome using Oxford Nanopore Technology revealed a total of five distinct BKPyV integration events. One integration of a partial BKPyV genome was located upstream of the epidermal growth factor receptor gene. The second and third, both truncated forms of integration, were close to histocompatibility gene locuses, while the fourth was characterized by a ninefold and the fifth by a fourfold tandem repeat of the BKPyV genome. From both of the repeat forms, virus replicates were derived showing deletions\/duplications on early and late genes and inversions within the non-coding control region (NCCR). This pattern of repetitive viral genome integration is a potential key driver of enhanced viral replication and increased virion assembly, ultimately supporting efficient virus egress. Quantitative PCR analysis confirmed the release of approximately 108\/mL viral units per 48 h from 2 \u00d7 105 COSSA cells into the culture supernatant. Notably, the NCCR region of the most frequent copies of circular virus and the integrated tetrameric tandem repeat exhibited a rearranged configuration, which may contribute to the observed high replication dynamics. The establishment of a consistent methodology to generate and secrete BKPyV from a cell line is expected to significantly facilitate antiviral drug development.<\/jats:p>","DOI":"10.3390\/ijms26125745","type":"journal-article","created":{"date-parts":[[2025,6,16]],"date-time":"2025-06-16T10:47:22Z","timestamp":1750070842000},"page":"5745","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Establishment of a Stable BK Polyomavirus-Secreting Cell Line: Characterization of Viral Genome Integration and Replication Dynamics Through Comprehensive Analysis"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-9196-2097","authenticated-orcid":false,"given":"Tamara","family":"L\u00f6wenstern","sequence":"first","affiliation":[{"name":"Institute of Medical Genetics, Center for Pathobiochemistry and Genetics, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Vecsei","sequence":"additional","affiliation":[{"name":"Division of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Horner","sequence":"additional","affiliation":[{"name":"Institute of Medical Genetics, Center for Pathobiochemistry and Genetics, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2157-0382","authenticated-orcid":false,"given":"Robert","family":"Strassl","sequence":"additional","affiliation":[{"name":"Division of Clinical Virology, Department of Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anil","family":"Bozdogan","sequence":"additional","affiliation":[{"name":"Division of Clinical Virology, Department of Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6341-1275","authenticated-orcid":false,"given":"Michael","family":"Eder","sequence":"additional","affiliation":[{"name":"Division of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9466-6441","authenticated-orcid":false,"given":"Franco","family":"Laccone","sequence":"additional","affiliation":[{"name":"Institute of Medical Genetics, Center for Pathobiochemistry and Genetics, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Markus","family":"Hengstschl\u00e4ger","sequence":"additional","affiliation":[{"name":"Institute of Medical Genetics, Center for Pathobiochemistry and Genetics, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Farsad","family":"Eskandary","sequence":"additional","affiliation":[{"name":"Division of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ludwig","family":"Wagner","sequence":"additional","affiliation":[{"name":"Division of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1097\/01.tp.0000269706.59977.a5","article-title":"Polyomavirus BK versus JC replication and nephropathy in renal transplant recipients: A prospective evaluation","volume":"84","author":"Drachenberg","year":"2007","journal-title":"Transplantation"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1730","DOI":"10.1016\/j.ekir.2024.04.003","article-title":"Polyomavirus Nephropathy in ABO Blood Group-Incompatible Kidney Transplantation: Torque Teno Virus and Immunosuppressive Burden as an Approximation to the Problem","volume":"9","author":"Eder","year":"2024","journal-title":"Kidney Int. 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