{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:28:16Z","timestamp":1785432496909,"version":"3.56.0"},"reference-count":33,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2026,5,24]],"date-time":"2026-05-24T00:00:00Z","timestamp":1779580800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008725","name":"Agencia Nacional de Investigaci\u00f3n e Innovaci\u00f3n","doi-asserted-by":"crossref","award":["FMV_1_2021_1_167704"],"award-info":[{"award-number":["FMV_1_2021_1_167704"]}],"id":[{"id":"10.13039\/100008725","id-type":"DOI","asserted-by":"crossref"}]},{"award":["FMV_1_2021_1_167704"],"award-info":[{"award-number":["FMV_1_2021_1_167704"]}],"id":[{"id":"https:\/\/ror.org\/03egaj678","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objectives: Serial passage in embryonated eggs is widely used to attenuate the infectious bronchitis virus (IBV) for vaccine production; however, the evolutionary processes underlying attenuation and immunogenicity remain incompletely understood. Here, we analyzed genome-wide viral evolution during serial passages to investigate how mutations emerge, persist, are lost, or become fixed over time and how these dynamics relate to changes in pathogenicity and immunogenicity. Methods: Deep sequencing was performed on 11 representative serial passages (P2\u2013P79) of the UY\/11\/CA\/18 strain, including two derivative lineages: P7 VIR (virulent) and P53 VAC (attenuated and immunogenic). Results: This study identified an early adaptive phase characterized by a limited set of mutations potentially associated with genome replication, viral RNA processing, and virion assembly, including a key change in non-structural protein 14 and variants in M and 3c (E). This phase was followed by a broader expansion of the variant spectrum across replicase genes and delayed accumulation of Spike protein variants. Most Spike changes emerged during later passages and exhibited transient dynamics, and only a subset reached a high frequency after the establishment of early replicase- and structural-associated changes. Consistent with these dynamics, P7 VIR diverged before the late accumulation of Spike variants and retained a pathogenic phenotype, whereas P53 VAC diverged after the emergence of early high-frequency variants but before the extensive late-stage Spike variation observed in P79, which was associated with reduced immunogenicity. Conclusions: These findings support a multi-step model of IBV attenuation in which progressive filtering of genome-wide variation shapes distinct evolutionary outcomes during serial passages. This evolutionary framework provides insight into the relationship between attenuation and immunogenicity and may help guide the rational design of live attenuated vaccines.<\/jats:p>","DOI":"10.3390\/vaccines14060467","type":"journal-article","created":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T01:45:08Z","timestamp":1779673508000},"page":"467","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Genome-Wide Analysis of Serial Passage of the Infectious Bronchitis Virus Reveals Evolutionary Dynamics Underlying Attenuation and Immunogenicity"],"prefix":"10.3390","volume":"14","author":[{"given":"Joaqu\u00edn","family":"Williman","sequence":"first","affiliation":[{"name":"Secci\u00f3n Gen\u00e9tica Evolutiva, Departamento de Biolog\u00eda Animal, Instituto de Biolog\u00eda, Facultad de Ciencias, Universidad de la Rep\u00fablica, Igu\u00e1 4225, Montevideo 11400, Uruguay"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0530-2046","authenticated-orcid":false,"given":"Gonzalo","family":"Tomas","sequence":"additional","affiliation":[{"name":"Secci\u00f3n Gen\u00e9tica Evolutiva, Departamento de Biolog\u00eda Animal, Instituto de Biolog\u00eda, Facultad de Ciencias, Universidad de la Rep\u00fablica, Igu\u00e1 4225, Montevideo 11400, Uruguay"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5575-6214","authenticated-orcid":false,"given":"Ariel","family":"Vagnozzi","sequence":"additional","affiliation":[{"name":"Instituto de Virolog\u00eda e Innovaciones Tecnol\u00f3gicas, Centro de Investigaciones en Ciencias Veterinarias y Agron\u00f3micas (CICVyA), Instituto Nacional de Tecnolog\u00eda Agropecuaria-Consejo Nacional de Investigaciones T\u00e9cnicas (INTA-CONICET), Hurlingham 1686, Buenos Aires, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudia","family":"Techera","sequence":"additional","affiliation":[{"name":"Secci\u00f3n Gen\u00e9tica Evolutiva, Departamento de Biolog\u00eda Animal, Instituto de Biolog\u00eda, Facultad de Ciencias, Universidad de la Rep\u00fablica, Igu\u00e1 4225, Montevideo 11400, Uruguay"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5728-8517","authenticated-orcid":false,"given":"Sebasti\u00e1n","family":"Brambillasca","sequence":"additional","affiliation":[{"name":"Unidad Acad\u00e9mica de Avicultura, Departamento de Producci\u00f3n Animal y Salud de los Sistemas Productivos, Facultad de Veterinaria, Universidad de la Rep\u00fablica, Ruta 8 y Ruta 102, Montevideo 13000, Uruguay"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4961-4743","authenticated-orcid":false,"given":"Ruben","family":"P\u00e9rez","sequence":"additional","affiliation":[{"name":"Secci\u00f3n Gen\u00e9tica Evolutiva, Departamento de Biolog\u00eda Animal, Instituto de Biolog\u00eda, Facultad de Ciencias, Universidad de la Rep\u00fablica, Igu\u00e1 4225, Montevideo 11400, Uruguay"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana","family":"Marandino","sequence":"additional","affiliation":[{"name":"Secci\u00f3n Gen\u00e9tica Evolutiva, Departamento de Biolog\u00eda Animal, Instituto de Biolog\u00eda, Facultad de Ciencias, Universidad de la Rep\u00fablica, Igu\u00e1 4225, Montevideo 11400, Uruguay"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"199","DOI":"10.3382\/ps.0150199","article-title":"A Filterable Virus, Distinct from That of Laryngotracheitis, the Cause of a Respiratory Disease of Chicks","volume":"15","author":"Beach","year":"1936","journal-title":"Poult. Sci."},{"key":"ref_2","first-page":"413","article-title":"An Apparently New Respiratory Disease of Baby Chicks","volume":"78","author":"Schalk","year":"1931","journal-title":"J. Am. Vet. Med. Assoc."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1051\/vetres:2006055","article-title":"Coronavirus Avian Infectious Bronchitis Virus","volume":"38","author":"Cavanagh","year":"2007","journal-title":"Vet. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1080\/03079457.2012.680432","article-title":"The Long View: 40 Years of Infectious Bronchitis Research","volume":"41","author":"Cook","year":"2012","journal-title":"Avian Pathol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1016\/j.meegid.2016.02.015","article-title":"S1 Gene-Based Phylogeny of Infectious Bronchitis Virus: An Attempt to Harmonize Virus Classification","volume":"39","author":"Valastro","year":"2016","journal-title":"Infect. Genet. Evol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1080\/03079450400013154","article-title":"Development and Use of the H Strain of Avian Infectious Bronchitis Virus from the Netherlands as a Vaccine: A Review","volume":"33","author":"Bijlenga","year":"2004","journal-title":"Avian Pathol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"114","DOI":"10.4161\/bbug.18983","article-title":"Modification of the Avian Coronavirus Infectious Bronchitis Virus for Vaccine Development","volume":"3","author":"Britton","year":"2012","journal-title":"Bioengineered"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"9084","DOI":"10.1128\/JVI.77.16.9084-9089.2003","article-title":"Recombinant Avian Infectious Bronchitis Virus Expressing a Heterologous Spike Gene Demonstrates That the Spike Protein Is a Determinant of Cell Tropism","volume":"77","author":"Casais","year":"2003","journal-title":"J. Virol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Armesto, M., Cavanagh, D., and Britton, P. (2009). The Replicase Gene of Avian Coronavirus Infectious Bronchitis Virus Is a Determinant of Pathogenicity. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0007384"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"13804","DOI":"10.1128\/JVI.78.24.13804-13811.2004","article-title":"Recombinant Infectious Bronchitis Coronavirus Beaudette with the Spike Protein Gene of the Pathogenic M41 Strain Remains Attenuated but Induces Protective Immunity","volume":"78","author":"Hodgson","year":"2004","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.virol.2020.08.009","article-title":"Replicase 1a Gene Plays a Critical Role in Pathogenesis of Avian Coronavirus Infectious Bronchitis Virus","volume":"550","author":"Zhao","year":"2020","journal-title":"Virology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1016\/j.vaccine.2018.01.017","article-title":"Recombinant Live Attenuated Avian Coronavirus Vaccines with Deletions in the Accessory Genes 3ab and\/or 5ab Protect against Infectious Bronchitis in Chickens","volume":"36","author":"Berends","year":"2018","journal-title":"Vaccine"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.virol.2019.04.014","article-title":"S Gene and 5a Accessory Gene Are Responsible for the Attenuation of Virulent Infectious Bronchitis Coronavirus","volume":"533","author":"Zhao","year":"2019","journal-title":"Virology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1274","DOI":"10.1016\/j.vaccine.2008.01.006","article-title":"Avian Coronavirus Infectious Bronchitis Attenuated Live Vaccines Undergo Selection of Subpopulations and Mutations Following Vaccination","volume":"26","author":"McKinley","year":"2008","journal-title":"Vaccine"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1080\/03079450802043783","article-title":"Rapid Selection in Chickens of Subpopulations within ArkDPI-Derived Infectious Bronchitis Virus Vaccines","volume":"37","author":"Toro","year":"2008","journal-title":"Avian Pathol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1637\/10947-093014-Reg","article-title":"Effects of Adaptation of Infectious Bronchitis Virus Arkansas Attenuated Vaccine to Embryonic Kidney Cells","volume":"59","author":"Ghetas","year":"2015","journal-title":"Avian Dis."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1093\/oxfordjournals.aje.a118408","article-title":"A simple method of estimating fifty per cent endpoints","volume":"27","author":"Reed","year":"1938","journal-title":"Am. J. Epidemiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.jviromet.2006.07.018","article-title":"Development and Evaluation of a Real-Time Taqman RT-PCR Assay for the Detection of Infectious Bronchitis Virus from Infected Chickens","volume":"138","author":"Callison","year":"2006","journal-title":"J. Virol. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3094","DOI":"10.1093\/bioinformatics\/bty191","article-title":"Minimap2: Pairwise Alignment for Nucleotide Sequences","volume":"34","author":"Li","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1647","DOI":"10.1093\/bioinformatics\/bts199","article-title":"Geneious Basic: An Integrated and Extendable Desktop Software Platform for the Organization and Analysis of Sequence Data","volume":"28","author":"Kearse","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1007\/s00705-009-0325-9","article-title":"Identification of Sequence Changes Responsible for the Attenuation of Avian Infectious Bronchitis Virus Strain Arkansas DPI","volume":"154","author":"Ammayappan","year":"2009","journal-title":"Arch. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.1016\/j.vaccine.2015.01.036","article-title":"Development and Efficacy of a Novel Live-Attenuated QX-like Nephropathogenic Infectious Bronchitis Virus Vaccine in China","volume":"33","author":"Feng","year":"2015","journal-title":"Vaccine"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1080\/03079457.2010.542742","article-title":"Efficacy and Safety of an Attenuated Live QX-like Infectious Bronchitis Virus Strain as a Vaccine for Chickens","volume":"40","author":"Geerligs","year":"2011","journal-title":"Avian Pathol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4630","DOI":"10.1016\/j.vaccine.2009.05.072","article-title":"Altered Pathogenicity, Immunogenicity, Tissue Tropism and 3\u2032-7kb Region Sequence of an Avian Infectious Bronchitis Coronavirus Strain after Serial Passage in Embryos","volume":"27","author":"Liu","year":"2009","journal-title":"Vaccine"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1007\/s11262-011-0668-7","article-title":"Changes in Nonstructural Protein 3 Are Associated with Attenuation in Avian Coronavirus Infectious Bronchitis Virus","volume":"44","author":"Phillips","year":"2012","journal-title":"Virus Genes"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"785","DOI":"10.1016\/j.vaccine.2005.08.081","article-title":"Development of Attenuated Vaccines from Taiwanese Infectious Bronchitis Virus Strains","volume":"24","author":"Huang","year":"2006","journal-title":"Vaccine"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"5531","DOI":"10.1016\/j.vaccine.2017.06.021","article-title":"Identification of IBV QX Vaccine Markers: Should Vaccine Acceptance by Authorities Require Similar Identifications for All Live IBV Vaccines?","volume":"35","author":"Listorti","year":"2017","journal-title":"Vaccine"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Wang, M., Bo, Z., Zhang, C., Guo, M., Wu, Y., and Zhang, X. (2024). Deciphering the Genetic Variation: A Comparative Analysis of Parental and Attenuated Strains of the QXL87 Vaccine for Infectious Bronchitis. Animals, 14.","DOI":"10.3390\/ani14121784"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e02059-21","DOI":"10.1128\/jvi.02059-21","article-title":"Identification of Amino Acids within Nonstructural Proteins 10 and 14 of the Avian Coronavirus Infectious Bronchitis Virus That Result in Attenuation In Vivo and In Ovo","volume":"96","author":"Keep","year":"2022","journal-title":"J. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.virol.2015.03.022","article-title":"Viral Quasispecies","volume":"479\u2013480","author":"Andino","year":"2015","journal-title":"Virology"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"199647","DOI":"10.1016\/j.virusres.2025.199647","article-title":"Attenuation and Altered Replication of IBV Strain Tl\/CH\/LDT3\/03 after Serial Passage in Chicken Embryo Fibroblasts and Vero Cells","volume":"361","author":"Li","year":"2025","journal-title":"Virus Res."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Liang, R., Liu, K., Li, Y., Zhang, X., Duan, L., Huang, M., Sun, L., Yuan, F., Zhao, J., and Zhao, Y. (2024). Adaptive Truncation of the S Gene in IBV during Chicken Embryo Passaging Plays a Crucial Role in Its Attenuation. PLoS Pathog., 20.","DOI":"10.1371\/journal.ppat.1012415"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e00492-19","DOI":"10.1128\/JVI.00492-19","article-title":"Attenuation of Infectious Bronchitis Virus in Eggs Results in Different Patterns of Genomic Variation across Multiple Replicates","volume":"93","author":"Oade","year":"2019","journal-title":"J. Virol."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/6\/467\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T04:12:30Z","timestamp":1779768750000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/6\/467"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,24]]},"references-count":33,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["vaccines14060467"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14060467","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,24]]}}}