{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:29:08Z","timestamp":1785421748608,"version":"3.56.0"},"reference-count":57,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T00:00:00Z","timestamp":1783296000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["22fk0108121h0603"],"award-info":[{"award-number":["22fk0108121h0603"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["23fk0108669h0401"],"award-info":[{"award-number":["23fk0108669h0401"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["24fk0108669h0402"],"award-info":[{"award-number":["24fk0108669h0402"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"award":["22fk0108121h0603"],"award-info":[{"award-number":["22fk0108121h0603"]}],"id":[{"id":"https:\/\/ror.org\/004rtk039","id-type":"ROR","asserted-by":"publisher"}]},{"award":["23fk0108669h0401"],"award-info":[{"award-number":["23fk0108669h0401"]}],"id":[{"id":"https:\/\/ror.org\/004rtk039","id-type":"ROR","asserted-by":"publisher"}]},{"award":["24fk0108669h0402"],"award-info":[{"award-number":["24fk0108669h0402"]}],"id":[{"id":"https:\/\/ror.org\/004rtk039","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["21K08498"],"award-info":[{"award-number":["21K08498"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["24K10235"],"award-info":[{"award-number":["24K10235"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["21K07057"],"award-info":[{"award-number":["21K07057"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["24K11806"],"award-info":[{"award-number":["24K11806"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"award":["21K08498"],"award-info":[{"award-number":["21K08498"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"publisher"}]},{"award":["24K10235"],"award-info":[{"award-number":["24K10235"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"publisher"}]},{"award":["21K07057"],"award-info":[{"award-number":["21K07057"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"publisher"}]},{"award":["24K11806"],"award-info":[{"award-number":["24K11806"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Kaketsuken","award":["Kaketusken"],"award-info":[{"award-number":["Kaketusken"]}]},{"award":["Kaketusken"],"award-info":[{"award-number":["Kaketusken"]}],"id":[{"id":"https:\/\/ror.org\/037x16a12","id-type":"ROR","asserted-by":"publisher"}]},{"name":"GlaxoSmithKline","award":["2021"],"award-info":[{"award-number":["2021"]}]},{"award":["2021"],"award-info":[{"award-number":["2021"]}],"id":[{"id":"https:\/\/ror.org\/01j6sxy67","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005865","name":"Mochida Memorial Foundation For Medical And Pharmaceutical Research","doi-asserted-by":"publisher","award":["Mochida Memorial Foundation for Medical and Pharmaceutical Research"],"award-info":[{"award-number":["Mochida Memorial Foundation for Medical and Pharmaceutical Research"]}],"id":[{"id":"10.13039\/501100005865","id-type":"DOI","asserted-by":"publisher"}]},{"award":["Mochida Memorial Foundation for Medical and Pharmaceutical Research"],"award-info":[{"award-number":["Mochida Memorial Foundation for Medical and Pharmaceutical Research"]}],"id":[{"id":"https:\/\/ror.org\/050rsje66","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007449","name":"Takeda Science Foundation","doi-asserted-by":"publisher","award":["Takeda Science Foundation"],"award-info":[{"award-number":["Takeda Science Foundation"]}],"id":[{"id":"10.13039\/100007449","id-type":"DOI","asserted-by":"publisher"}]},{"award":["Takeda Science Foundation"],"award-info":[{"award-number":["Takeda Science Foundation"]}],"id":[{"id":"https:\/\/ror.org\/02y123g31","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Live-attenuated rotavirus (RV) vaccines are the most effective interventions for preventing RV gastroenteritis (RVGE) in young children. However, the molecular basis of attenuation remains not well understood. Here, we describe a compact but comprehensive strategy to identify RV virulence determinants by combining low-passage bidirectional selection, sequence analysis, and segment-level phenotype testing via a reverse genetics infectious system. Using the virulent murine RV strain EW, virulence was quantified by diarrhea severity\/duration and body-weight gain. Serial passaging in cell culture selected an attenuated population, which regained virulence after passaging in suckling mice. Sequence comparison of the virulent and attenuated EW populations revealed only seven amino acid differences. We summarized literature describing attenuation\/virulence-associated mutations in various RV group A (RVA) strains and found previous findings identical or similar to four of the seven mutations: NSP4-T45M, VP4-S470L, VP4-T612A, and VP7-T75P. Virulent- and attenuated-type EW variants of VP2, VP4, VP7, and NSP4 were introduced individually, or as NSP4\/VP7 or VP4\/VP7 pairs, into a simian SA11-L2 backbone using an 11-plasmid reverse genetics system. Phenotyping of rescued viruses consistently linked cell-culture\u2013adapted VP4 to enhanced replication in vitro and reduced virulence in suckling mice. In vivo passaging strongly favored VP4 residue S470 over cell-culture-selected L470. More generally, our findings (i) underscore VP4 and VP7 as key determinants of EW virulence, (ii) provide a practical framework for identifying driver mutations underlying RVA attenuation, and (iii) highlight attenuation-associated substitutions shared across diverse RVAs.<\/jats:p>","DOI":"10.3390\/v18070747","type":"journal-article","created":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T14:01:08Z","timestamp":1783346468000},"page":"747","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Identification of Murine Rotavirus Virulence Determinants Using Bidirectional Selective Passaging and a Reverse Genetics System"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0841-880X","authenticated-orcid":false,"given":"Saori","family":"Fukuda","sequence":"first","affiliation":[{"name":"Department of Virology, Fujita Health University School of Medicine, Toyoake 470-1192, Aichi, Japan"},{"name":"Center for Infectious Disease Research, Research Promotion Headquarters, Fujita Health University, Toyoake 470-1192, Aichi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masanori","family":"Kugita","sequence":"additional","affiliation":[{"name":"Advanced Medical Research Center for Animal Models of Human Disease, Research Promotion Headquarters, Fujita Health University, Toyoake 470-1192, Aichi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuki","family":"Akari","sequence":"additional","affiliation":[{"name":"Division of One Health, Research Center for GLOBAL and LOCAL Infectious Diseases (RCGLID), Oita University, Yufu 879-5593, Oita, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Johannes M.","family":"Dijkstra","sequence":"additional","affiliation":[{"name":"Office of Research Administration, Fujita Health University, Toyoake 470-1192, Aichi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3942-7098","authenticated-orcid":false,"given":"Yoshiki","family":"Kawamura","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Fujita Health University School of Medicine, Toyoake 470-1192, Aichi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2014-9411","authenticated-orcid":false,"given":"Shizuko","family":"Nagao","sequence":"additional","affiliation":[{"name":"Advanced Medical Research Center for Animal Models of Human Disease, Research Promotion Headquarters, Fujita Health University, Toyoake 470-1192, Aichi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2847-7682","authenticated-orcid":false,"given":"Tetsushi","family":"Yoshikawa","sequence":"additional","affiliation":[{"name":"Center for Infectious Disease Research, Research Promotion Headquarters, Fujita Health University, Toyoake 470-1192, Aichi, Japan"},{"name":"Department of Pediatrics, Fujita Health University School of Medicine, Toyoake 470-1192, Aichi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7228-0839","authenticated-orcid":false,"given":"Takayuki","family":"Murata","sequence":"additional","affiliation":[{"name":"Department of Virology, Fujita Health University School of Medicine, Toyoake 470-1192, Aichi, Japan"},{"name":"Center for Infectious Disease Research, Research Promotion Headquarters, Fujita Health University, Toyoake 470-1192, Aichi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2810-8163","authenticated-orcid":false,"given":"Satoshi","family":"Komoto","sequence":"additional","affiliation":[{"name":"Division of One Health, Research Center for GLOBAL and LOCAL Infectious Diseases (RCGLID), Oita University, Yufu 879-5593, Oita, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"S96","DOI":"10.1093\/cid\/civ1013","article-title":"Global, regional, and national estimates of rotavirus mortality in children <5 years of age, 2000\u20132013","volume":"62","author":"Tate","year":"2016","journal-title":"Clin. Infect. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"958","DOI":"10.1001\/jamapediatrics.2018.1960","article-title":"Rotavirus vaccination and the global burden of rotavirus diarrhea among children younger than 5 years","volume":"172","author":"Troeger","year":"2018","journal-title":"JAMA Pediatr."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1731","DOI":"10.1093\/infdis\/jiaa081","article-title":"Global impact of rotavirus vaccination on diarrhea hospitalizations and deaths among children <5 years old: 2006\u20132019","volume":"222","author":"Burnett","year":"2020","journal-title":"J. Infect. Dis."},{"key":"ref_4","unstructured":"Knipe, D.M., Howley, P.M., Cohen, J.I., Griffin, D.E., Lamb, R.A., Martin, M.A., Racaniello, V.R., and Roizman, B. (2013). Rotaviruses. Fields Virology, Wolters Kluwer Health\/Lippincott Williams & Wilkins. [6th ed.]."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1002\/rmv.448","article-title":"Global distribution of rotavirus serotypes\/genotypes and its implication for the development and implementation of an effective rotavirus vaccine","volume":"15","author":"Santos","year":"2005","journal-title":"Rev. Med. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2431","DOI":"10.1093\/molbev\/msq137","article-title":"Phylodynamic analyses of rotavirus genotypes G9 and G12 underscore their potential for swift global spread","volume":"27","author":"Matthijnssens","year":"2010","journal-title":"Mol. Biol. Evol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"105507","DOI":"10.1016\/j.meegid.2023.105507","article-title":"Full genome-based characterization of an Asian G3P[6] human rotavirus strain found in a diarrheic child in Japan: Evidence for porcine-to-human zoonotic transmission","volume":"115","author":"Akari","year":"2023","journal-title":"Infect. Genet. Evol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1186\/s41182-025-00759-9","article-title":"Full genome characterization of a Kenyan G8P[14] rotavirus strain suggests artiodactyl-to-human zoonotic transmission","volume":"53","author":"Wandera","year":"2025","journal-title":"Trop. Med. Health"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1056\/NEJMoa052664","article-title":"Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine","volume":"354","author":"Vesikari","year":"2006","journal-title":"N. Engl. J. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1617","DOI":"10.1086\/652403","article-title":"Reduction in acute gastroenteritis hospitalizations among US children after introduction of rotavirus vaccine: Analysis of hospital discharge data from 18 US states","volume":"201","author":"Curns","year":"2010","journal-title":"J. Infect. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1108","DOI":"10.1056\/NEJMoa1000446","article-title":"Rotavirus vaccine and health care utilization for diarrhea in U.S. children","volume":"365","author":"Cortes","year":"2011","journal-title":"N. Engl. J. Med."},{"key":"ref_12","unstructured":"Rotavirus vaccines (2013). WHO position paper\u2014January. Wkly. Epidemiol. Rec., 88, 49\u201364."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2448","DOI":"10.1128\/jvi.67.5.2448-2455.1993","article-title":"Murine rotavirus genes encoding outer capsid proteins VP4 and VP7 are not major determinants of host range restriction and virulence","volume":"67","author":"Broome","year":"1993","journal-title":"J. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1006\/viro.1995.1255","article-title":"Identification of group A rotavirus genes associated with virulence of a porcine rotavirus and host range restriction of a human rotavirus in the gnotobiotic piglet model","volume":"209","author":"Hoshino","year":"1995","journal-title":"Virology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1128\/jvi.57.2.585-590.1986","article-title":"Cultivation and Characterization of Three Strains of Murine Rotavirus","volume":"57","author":"Greenberg","year":"1986","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1006\/viro.1995.1060","article-title":"Analyses of homologous rotavirus infection in the mouse model","volume":"207","author":"Burns","year":"1995","journal-title":"Virology"},{"key":"ref_17","first-page":"635","article-title":"The double-edged sword: How evolution can make or break a live-attenuated virus vaccine","volume":"4","author":"Hanley","year":"2011","journal-title":"Evolution"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"767","DOI":"10.3390\/v16050767","article-title":"Reverse Genetics of Murine Rotavirus: A Comparative Analysis of the Wild-Type and Cell-Culture-Adapted Murine Rotavirus VP4 in Replication and Virulence in Neonatal Mice","volume":"16","author":"Kawagishi","year":"2024","journal-title":"Viruses"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1111\/j.1348-0421.2003.tb03424.x","article-title":"Improved recovery of rabies virus from cloned cDNA using a vaccinia virus-free reverse genetics system","volume":"47","author":"Ito","year":"2003","journal-title":"Microbiol. Immunol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00588-18","DOI":"10.1128\/JVI.00588-18","article-title":"Generation of recombinant rotaviruses expressing fluorescent proteins by using an optimized reverse genetics system","volume":"92","author":"Komoto","year":"2018","journal-title":"J. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1006\/viro.1994.1035","article-title":"Differences in plaque size and VP4 sequence found in SA11 virus clones having simian authentic VP4","volume":"198","author":"Taniguchi","year":"1994","journal-title":"Virology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1128\/jvi.51.1.233-236.1984","article-title":"A murine model for oral infection with a primate rotavirus (simian SA11)","volume":"51","author":"Offit","year":"1984","journal-title":"J. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Kawahara, T., Makizaki, Y., Oikawa, Y., Tanaka, Y., Maeda, A., Shimakawa, M., Komoto, S., Moriguchi, K., Ohno, H., and Taniguchi, K. (2017). Oral administration of Bifidobacterium bifidum G9-1 alleviates rotavirus gastroenteritis through regulation of intestinal homeostasis by inducing mucosal protective factors. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0173979"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"001745","DOI":"10.1099\/jgv.0.001745","article-title":"Rotavirus incapable of NSP6 expression can cause diarrhea in suckling mice","volume":"103","author":"Fukuda","year":"2022","journal-title":"J. Gen. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Fukuda, S., Kugita, M., Kumamoto, K., Akari, Y., Higashimoto, Y., Nagao, S., Murata, T., Yoshikawa, T., Taniguchi, K., and Komoto, S. (2024). Generation of Recombinant Authentic Live Attenuated Human Rotavirus Vaccine Strain RIX4414 (Rotarix\u00ae) from Cloned cDNAs Using Reverse Genetics. Viruses, 16.","DOI":"10.3390\/v16081198"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1111\/1348-0421.13107","article-title":"Development of recombinant rotavirus carrying herpes simplex virus 2 glycoprotein D gene based on reverse genetics technology","volume":"68","author":"Kawamura","year":"2024","journal-title":"Microbiol. Immunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3689","DOI":"10.1128\/JVI.01770-07","article-title":"Rearrangements of rotavirus genomic segment 11 are generated during acute infection of immunocompetent children and do not occur at random","volume":"82","author":"Schnepf","year":"2008","journal-title":"J. Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"5543","DOI":"10.1128\/JVI.00041-14","article-title":"Virulence-associated genome mutations of murine rotavirus identified by alternating serial passages in mice and cell cultures","volume":"88","author":"Tsugawa","year":"2014","journal-title":"J. Virol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e02207-18","DOI":"10.1128\/JVI.02207-18","article-title":"Generation of infec-tious recombinant human rotaviruses from just 11 cloned cDNAs encoding the rotavirus genome","volume":"93","author":"Komoto","year":"2019","journal-title":"J. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"806","DOI":"10.1099\/jgv.0.001443","article-title":"Rapid generation of rotavirus single-gene reassortants by means of eleven plasmid-only based reverse genetics","volume":"101","author":"Fukuda","year":"2020","journal-title":"J. Gen. Virol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Dennis, F.E., Fujii, Y., Haga, K., Damanka, S., Lartey, B., Agbemabiese, C.A., Ohta, N., Armah, G.E., and Katayama, K. (2014). Identifi-cation of novel Ghanaian G8P[6] human-bovine reassortant rotavirus strain by next generation sequencing. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0100699"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1016\/j.meegid.2016.06.023","article-title":"Whole genomic analysis of human and bovine G8P[1] rotavirus strains isolated in Nigeria provides evidence for direct bovine-to-human interspecies transmission","volume":"43","author":"Komoto","year":"2016","journal-title":"Infect. Genet. Evol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1439","DOI":"10.1128\/JVI.02730-15","article-title":"Generation of an avian-mammalian rotavirus reassortant by using a helper vi-rus-dependent reverse genetics system","volume":"90","author":"Johne","year":"2016","journal-title":"J. Virol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1025","DOI":"10.1111\/j.1348-0421.1981.tb00109.x","article-title":"Sequential passages of human rotavirus in MA-104 cells","volume":"25","author":"Urasawa","year":"1981","journal-title":"Microbiol. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1726","DOI":"10.1128\/jvi.61.5.1726-1730.1987","article-title":"Cross-reactive neutralization epitopes on VP3 of human rotavirus: Analysis with monoclonal antibodies and antigenic variants","volume":"61","author":"Taniguchi","year":"1987","journal-title":"J. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4646","DOI":"10.1073\/pnas.0509385103","article-title":"Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus","volume":"103","author":"Komoto","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"781","DOI":"10.1128\/iai.38.2.781-784.1982","article-title":"Three human rotavirus serotypes demonstrated by plaque neutralization of isolated strains","volume":"38","author":"Urasawa","year":"1982","journal-title":"Infect. Immun."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"5070","DOI":"10.1128\/jvi.64.10.5070-5075.1990","article-title":"Development of an adult mouse model for studies on protection against rotavirus","volume":"64","author":"Ward","year":"1990","journal-title":"J. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"6267","DOI":"10.1128\/jvi.71.8.6267-6270.1997","article-title":"Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene","volume":"71","author":"Ward","year":"1997","journal-title":"J. Virol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1729","DOI":"10.1086\/509623","article-title":"Reductions in cross-neutralizing antibody responses in infants after attenuation of the human rotavirus vaccine candidate 89-12","volume":"194","author":"Ward","year":"2006","journal-title":"J. Infect. Dis."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"408","DOI":"10.3390\/v12040408","article-title":"Amino acid substitutions in positions 385 and 393 of the hydrophobic region of VP4 may be associated with rotavirus attenuation and cell culture adap-tation","volume":"12","author":"Guo","year":"2020","journal-title":"Viruses"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e00889-20","DOI":"10.1128\/JVI.00889-20","article-title":"Serial Passaging of the Human Rotavirus CDC-9 Strain in Cell Culture Leads to Atten-uation: Characterization from In vitro and In vivo Studies","volume":"94","author":"Resch","year":"2020","journal-title":"J. Virol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"e0055022","DOI":"10.1128\/jvi.00550-22","article-title":"The Role of the VP4 Attachment Protein in Rotavirus Host Range Restriction in an In Vivo Suckling Mouse Model","volume":"96","author":"Kawagishi","year":"2022","journal-title":"J. Virol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"e00510-17","DOI":"10.1128\/JVI.00510-17","article-title":"A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model","volume":"91","author":"Mohanty","year":"2017","journal-title":"J. Virol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Valusenko-Mehrkens, R., Schilling-Loeffler, K., Johne, R., and Falkenhagen, A. (2024). VP4 Mutation Boosts Replication of Recombinant Human\/Simian Rotavirus in Cell Culture. Viruses, 16.","DOI":"10.3390\/v16040565"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e0106725","DOI":"10.1128\/jvi.01067-25","article-title":"Mutations of two amino acids in VP5 mediate the attenuation of human rotavirus vaccine: Evidence from in vitro and in vivo studies","volume":"99","author":"Bessey","year":"2025","journal-title":"J. Virol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"e00265-10","DOI":"10.1128\/mBio.00265-10","article-title":"Rotavirus disrupts calcium homeostasis by NSP4 viroporin activity","volume":"1","author":"Hyser","year":"2010","journal-title":"mBio"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"666","DOI":"10.1038\/s41586-020-03124-4","article-title":"Functional refolding of the pen-etration protein on a non-enveloped virus","volume":"590","author":"Herrmann","year":"2021","journal-title":"Nature"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"5114","DOI":"10.1016\/j.vaccine.2022.06.082","article-title":"Rotavirus vaccine-derived cases in Belgium: Evidence for reversion of attenuating mutations and alternative causes of gastroenteritis","volume":"40","author":"Simsek","year":"2022","journal-title":"Vaccine"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1086\/517374","article-title":"Studies of the role for NSP4 in the pathogenesis of homologous murine rotavirus diarrhea","volume":"177","author":"Angel","year":"1998","journal-title":"J. Infect. Dis."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1016\/j.meegid.2016.08.015","article-title":"Genomic changes detected after serial passages in cell culture of virulent human G1P[8] rotaviruses","volume":"45","author":"Tsugawa","year":"2016","journal-title":"Infect. Genet. Evol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1023\/A:1008068218966","article-title":"Comparisons of nucleotide and deduced amino acid sequences of NSP4 genes of virulent and attenuated pairs of group A and C rotaviruses","volume":"18","author":"Chang","year":"1999","journal-title":"Virus Genes"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1111\/j.1348-0421.2001.tb01277.x","article-title":"A lack of consistent amino acid substitutions in NSP4 between rotaviruses derived from diarrheal and asymptomatically-infected kittens","volume":"45","author":"Oka","year":"2001","journal-title":"Microbiol. Immunol."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Kaneko, M., Takanashi, S., Thongprachum, A., Hanaoka, N., Fujimoto, T., Nagasawa, K., Kimura, H., Okitsu, S., Mizuguchi, M., and Ushijima, H. (2017). Identification of vaccine-derived rotavirus strains in children with acute gastroenteritis in Japan, 2012\u20132015. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0184067"},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Carter, M.H., Gribble, J., Diller, J.R., Denison, M.R., Mirza, S.A., Chappell, J.D., Halasa, N.B., and Ogden, K.M. (2024). Human Rotaviruses of Multiple Genotypes Acquire Conserved VP4 Mutations during Serial Passage. Viruses, 16.","DOI":"10.3390\/v16060978"},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Agbemabiese, C.A., Dennis, F.E., Lartey, B.L., Damanka, S.A., Nakagomi, T., Nakagomi, O., and Armah, G.E. (2024). Whole Genome Sequences of the Wildtype AU-1 Rotavirus A Strain: The Prototype of the AU-1-like Genotype Constellation. Viruses, 16.","DOI":"10.3390\/v16101529"},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Silva, M.F., Silva, B.V., Ramalho, E., Pimenta, Y.C., Silva, L.L., Vieira, L.S., Xavier, M.P., Olivares, A.I., Leite, J.P., and Moraes, M.T. (2026). Examination of In Vivo Mutations in VP4 (VP8*) of the Rotarix\u00ae Vaccine from Shedding of Children Living in the Amazon Region. Viruses, 18.","DOI":"10.3390\/v18010070"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/7\/747\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T04:22:20Z","timestamp":1784002940000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/7\/747"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,6]]},"references-count":57,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["v18070747"],"URL":"https:\/\/doi.org\/10.3390\/v18070747","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,6]]}}}