{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T06:07:24Z","timestamp":1771049244431,"version":"3.50.1"},"reference-count":31,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,11]],"date-time":"2026-02-11T00:00:00Z","timestamp":1770768000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"King Faisal University","award":["KFU#260761"],"award-info":[{"award-number":["KFU#260761"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Background\/Objectives: Coxsackievirus B4 (CVB4), a member of the Enterovirus genus and the Picornaviridae family, is a significant pathogen causing several human diseases such as pancreatitis, myocarditis, cardiomyopathy and type 1 diabetes. Despite its clinical impact, no vaccines or specific antiviral therapies are currently available. This study investigates the attenuation of CVB4 virulence through targeted mutations in the domain V of the IRES (Internal Ribosome Entry Segment) sequence present in the 5\u2032 UTR (Untranslated Region) of the viral genome. Materials and Methods: We engineered six CVB4E2 mutants by introducing single nucleotide mutations in domain V of the IRES sequence using PCR-based site-directed mutagenesis assays. Mutants were rigorously evaluated in vitro for their replicative capacities on HeLa cell culture and for their in vitro translation efficiencies in standard rabbit reticulocyte lysates supplemented with HeLa cell S10 extracts. Using different strategies of immunization and lethal challenges in a Balb\/c mice model, we evaluated the immune responses elicited by the most attenuated C488A mutant strain. Results: The obtained results demonstrated that the live-attenuated C488A mutant with the single mutation C to A at nucleotide position 488 of the viral IRES sequence exhibited a significant reduction in vitro of both viral productivity and translation efficiency. The oral immunization with the live-attenuated C488A mutant induced a potent immune response and protected Balb\/c mice against lethal infection challenge with a pathogenic strain. Conclusions: These findings underscored the critical role of IRES in CVB4 virulence and highlighted the use of the live-attenuated C488A mutant strain as a promising candidate for developing a live-attenuated vaccine against CVB4 infections.<\/jats:p>","DOI":"10.3390\/v18020228","type":"journal-article","created":{"date-parts":[[2026,2,11]],"date-time":"2026-02-11T17:45:36Z","timestamp":1770831936000},"page":"228","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Oral Immunization with the C488A Live-Attenuated Mutant of Coxsackievirus B4E2 (CVB4E2) Induces Potent Immune Response and Protects Balb\/c Mice Against Lethal Infection"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9170-3403","authenticated-orcid":false,"given":"Jawhar","family":"Gharbi","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa 31982, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1992-7976","authenticated-orcid":false,"given":"Ikbel","family":"Hadj Hassine","sequence":"additional","affiliation":[{"name":"Department of Virology, Health Sciences College, Western University, 1393 Western Road, London, ON N6G 3K7, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mouna","family":"Hassine","sequence":"additional","affiliation":[{"name":"Virology and Antiviral Strategies Unit UR17ES30, Higher Institute of Biotechnology, University of Monastir, B.P 74, Tahar Haddad Street, Monastir 5000, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anwar","family":"Al-Bashir","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa 31982, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Reem","family":"Al-Chahri","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa 31982, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ameera","family":"Al-Yami","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa 31982, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0746-488X","authenticated-orcid":false,"given":"Mohamed","family":"Al-Malki","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa 31982, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Noureddine","family":"Chatti","sequence":"additional","affiliation":[{"name":"Virology and Antiviral Strategies Unit UR17ES30, Higher Institute of Biotechnology, University of Monastir, B.P 74, Tahar Haddad Street, Monastir 5000, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4301-2074","authenticated-orcid":false,"given":"Didier","family":"Hober","sequence":"additional","affiliation":[{"name":"Laboratoire de Virologie ULR3610, University of Lille, CHU Lille, 59120 Loos, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Manel Ben","family":"M\u2019hadheb","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa 31982, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,11]]},"reference":[{"key":"ref_1","first-page":"1615","article-title":"Enteroviruses: Classification, diseases they cause, and approaches to development of antiviral drugs","volume":"82","author":"Nikonov","year":"2017","journal-title":"Biochemistry"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1173","DOI":"10.1056\/NEJM197905243002102","article-title":"Isolation of a virus from the pancreas of a child with diabetic ketoacidosis","volume":"21","author":"Yoon","year":"1979","journal-title":"N. Engl. J. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1038\/nrendo.2010.27","article-title":"Pathogenesis of type 1 diabetes mellitus: Interplay between enterovirus and host","volume":"5","author":"Hober","year":"2010","journal-title":"Nat. Rev. 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