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The EBOV-encoded major matrix protein VP40 is essential for viral assembly, budding, and particle release, making it a promising target for antiviral drug development. However, no approved drugs currently target the viral particle formation process. In this study, we established a simple and highly sensitive screening system to evaluate VP40-mediated virus-like particle (VLP) formation under biosafety level \u22122 conditions. The system uses the HiBiT luminescence-based reporter fused to VP40, allowing for the detection of VP40 release. Our results demonstrate that the HiBiT sequence fused at the N-terminus [HiBiT-VP40 (N)] retains VP40\u2032s ability to form VLPs, supporting its use as a functional reporter. Furthermore, we validated the system by assessing the role of Rab11-dependent trafficking in VP40-mediated budding and by evaluating the effect of nocodazole, a microtubule depolymerizer, on VLP release. This novel screening system provides a convenient and reliable platform for screening potential inhibitors targeting the late stages of EBOV infection, including viral particle formation and release. Additionally, its potential adaptability to other filoviruses suggests wide applicability in the discovery and development of additional novel therapeutic agents.<\/jats:p>","DOI":"10.3390\/v17071016","type":"journal-article","created":{"date-parts":[[2025,7,21]],"date-time":"2025-07-21T07:56:14Z","timestamp":1753084574000},"page":"1016","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development of a Novel, Highly Sensitive System for Evaluating Ebola Virus Particle Formation"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7163-6960","authenticated-orcid":false,"given":"Wakako","family":"Furuyama","sequence":"first","affiliation":[{"name":"National Research Center for the Control and Prevention of Infectious Diseases, Nagasaki University, Nagasaki 852-8523, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5105-3100","authenticated-orcid":false,"given":"Miako","family":"Sakaguchi","sequence":"additional","affiliation":[{"name":"Central Laboratory, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki 852-8523, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hanako","family":"Ariyoshi","sequence":"additional","affiliation":[{"name":"National Research Center for the Control and Prevention of Infectious Diseases, Nagasaki University, Nagasaki 852-8523, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8764-1350","authenticated-orcid":false,"given":"Asuka","family":"Nanbo","sequence":"additional","affiliation":[{"name":"National Research Center for the Control and Prevention of Infectious Diseases, Nagasaki University, Nagasaki 852-8523, Japan"},{"name":"Department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki 852-8523, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1038\/s41572-020-0147-3","article-title":"Ebola virus disease","volume":"6","author":"Jacob","year":"2020","journal-title":"Nat. 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