{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T01:08:43Z","timestamp":1788570523510,"version":"build-2803163510"},"reference-count":55,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2022,9,28]],"date-time":"2022-09-28T00:00:00Z","timestamp":1664323200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Oral vaccination of wildlife has shown to be a very effective management tool in rabies control. Evaluation of the genetic stability of vaccine viruses before distributing vaccine baits in the environment is essential because all available oral rabies vaccines, including the genetically engineered rabies virus vaccine strain SPBN GASGAS (Rabitec), are based on replication-competent viruses. To evaluate the genetic stability of this vaccine strain, five serial passages of the Master Seed Virus (MSV) in the production cell line BHK21 Cl13 were performed. Furthermore, to test possible reversion to virulence, a back-passage study in suckling mouse brain (SMB) was performed. Subsequently, the pooled 5th SMB passage was inoculated intracerebrally (i.c.) in adult and suckling mice. The full genome sequences of the isolated 5th passage, in vivo and in vitro, were compared at both the consensus and the quasispecies level with the MSV. Additionally, the full genome sequence of the 6th SMB passage from the individual animals was determined and compared. Full-length integration of the double glycoprotein and modified base substitutions at amino acid position 194 and 333 of the glycoprotein could be verified in all 5th and 6th passage samples. Overall, 11 single nucleotide polymorphisms (SNPs) were detected in the 5th pooled SMB passage, 4 with frequency between 10 and 20%, and 7 with between 2.5 and 10%. SNPs that resulted in amino acid exchange were found in genes: N (one SNP), G (four SNPs), and L (three SNPs). However, none of these SNPs were associated with reversion to virulence since all adult mice inoculated i.c. with this material survived. In the individual samples of the 6th SMB passage 24 additional SNPs (&gt;2.5%) were found, of which only 1 SNP (L-gene, position 6969) had a prevalence of &gt;50% in 3 of 17 samples. The obtained results confirmed the stable expression of genetic modifications and the genetic stability of the consensus strain after serial in vivo and in vitro passaging.<\/jats:p>","DOI":"10.3390\/v14102136","type":"journal-article","created":{"date-parts":[[2022,9,29]],"date-time":"2022-09-29T04:09:36Z","timestamp":1664424576000},"page":"2136","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Oral Rabies Vaccine Strain SPBN GASGAS: Genetic Stability after Serial In Vitro and In Vivo Passaging"],"prefix":"10.3390","volume":"14","author":[{"given":"Stefan","family":"Borutzki","sequence":"first","affiliation":[{"name":"IDT Biologika GmbH, Am Pharmapark, 06861 Dessau-Rosslau, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin","family":"Richter","sequence":"additional","affiliation":[{"name":"IDT Biologika GmbH, Am Pharmapark, 06861 Dessau-Rosslau, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthias","family":"Proemmel","sequence":"additional","affiliation":[{"name":"Ceva Innovation Center GmbH, Am Pharmapark, 06861 Dessau-Rosslau, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Izabela","family":"Fabianska","sequence":"additional","affiliation":[{"name":"IDT Biologika GmbH, Am Pharmapark, 06861 Dessau-Rosslau, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hon Quang","family":"Tran","sequence":"additional","affiliation":[{"name":"IDT Biologika GmbH, Am Pharmapark, 06861 Dessau-Rosslau, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Boris","family":"Hundt","sequence":"additional","affiliation":[{"name":"Ceva Innovation Center GmbH, Am Pharmapark, 06861 Dessau-Rosslau, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dietmar","family":"Mayer","sequence":"additional","affiliation":[{"name":"IDT Biologika GmbH, Am Pharmapark, 06861 Dessau-Rosslau, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Kaiser","sequence":"additional","affiliation":[{"name":"TEW Servicegesellschaft mbH, Am Pharmapark 15, 06861 Dessau-Rosslau, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andreas","family":"Neubert","sequence":"additional","affiliation":[{"name":"Klocke Holding GmbH, Max-Becker-Str. 6, 76356 Weingarten, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ad","family":"Vos","sequence":"additional","affiliation":[{"name":"Ceva Innovation Center GmbH, Am Pharmapark, 06861 Dessau-Rosslau, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Hampson, K., Coudeville, L., Lembo, T., Sambo, M., Kieffer, A., Attlan, M., Barrat, J., Blanton, J.D., Briggs, D.J., and Cleaveland, S. (2015). Estimating the global burden of endemic canine rabies. PLoS Neglect. Trop. Dis., 9.","DOI":"10.1371\/journal.pntd.0003786"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/B978-0-12-387040-7.00019-6","article-title":"Evolution of rabies wildlife control tactics","volume":"79","author":"Rosatte","year":"2011","journal-title":"Adv. Virus Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"20120142","DOI":"10.1098\/rstb.2012.0142","article-title":"The elimination of fox rabies in Europe: Determinants of success and lessons for the future","volume":"368","author":"Freuling","year":"2013","journal-title":"Philos. Trans. R. Soc. Lond. B Biol. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"M\u00fcller, T.F., Schr\u00f6der, R., Wysocki, P., Mettenleiter, T.C., and Freuling, C.M. (2015). Spatio-temporal use of oral rabies vaccines in fox rabies elimination programs in Europe. PLoS Negl. Trop. Dis., 9.","DOI":"10.1371\/journal.pntd.0003953"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"71","DOI":"10.7589\/0090-3558-44.1.71","article-title":"ERA vaccine-derived cases of rabies in wildlife and domestic animals in Ontario, Canada, 1989\u20132004","volume":"44","author":"Armstrong","year":"2008","journal-title":"J. Wildl. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4758","DOI":"10.1016\/j.vaccine.2018.01.083","article-title":"In-depth genome analyses of viruses from vaccine-derived rabies cases and corresponding live-attenuated oral rabies vaccines","volume":"37","author":"Pfaff","year":"2019","journal-title":"Vaccine"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.tvjl.2014.10.026","article-title":"Terrestrial rabies control in the European Union: Historical achievements and challenges ahead","volume":"203","author":"Freuling","year":"2015","journal-title":"Vet. J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1186\/s13567-017-0459-9","article-title":"Oral vaccination of wildlife using a vaccinia-rabies-glycoprotein recombinant virus vaccine (RABORAL V-RG\u00ae): A global review","volume":"48","author":"Maki","year":"2017","journal-title":"Vet. Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1017\/S0950268899002666","article-title":"An update on safety studies of SAD B19 rabies virus vaccine in target and nontarget species","volume":"123","author":"Vos","year":"1999","journal-title":"Epidemiol. Infect."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"524","DOI":"10.1016\/0264-410X(92)90351-J","article-title":"Potential pathogenicity for rodents of vaccines intended for oral vaccination against rabies: A comparison","volume":"10","author":"Artois","year":"1992","journal-title":"Vaccine"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"99","DOI":"10.7589\/0090-3558-26.1.99","article-title":"Ineffectiveness and comparative pathogenicity of attenuated rabies virus vaccines for the striped skunk (Mephitis mephitis)","volume":"26","author":"Rupprecht","year":"1990","journal-title":"J. Wildl. Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1136\/vr.131.3.55","article-title":"Pathogenicity of SAD rabies vaccine given orally in chacma baboons (Papio ursinus)","volume":"131","author":"Bingham","year":"1992","journal-title":"Vet. Rec."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1081","DOI":"10.1007\/s00705-009-0408-7","article-title":"Analysis of vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) after oral rabies vaccination campaigns in Germany and Austria","volume":"154","author":"Beckert","year":"2009","journal-title":"Arch. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"397","DOI":"10.7589\/2013-07-183","article-title":"Vaccine-induced rabies in a red fox (Vulpes vulpes): Isolation of vaccine virus in brain tissue and salivary glands","volume":"50","author":"Hostnik","year":"2014","journal-title":"J. Wildl. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Robardet, E., Picard-Meyer, E., Dobro\u0161tana, M., Jaceviciene, I., M\u00e4har, K., Mui\u017eniece, Z., Pridotkas, G., Masiulis, M., Niin, E., and Ol\u0161evskis, E. (2016). Rabies in the baltic states: Decoding a process of control and elimination. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0004432"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5021","DOI":"10.1016\/j.vaccine.2016.08.013","article-title":"Vaccine-induced rabies case in a cow (Bos taurus): Molecular characterization of vaccine strain in brain tissue","volume":"34","author":"Vuta","year":"2016","journal-title":"Vaccine"},{"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","unstructured":"Cliquet, F., Picard-Meyer, E., Mojzis, M., Dirbakova, Z., Muizniece, Z., Jaceviciene, I., Mutinelli, F., Matulova, M., Frolichova, J., and Rychlik, I. (2015). In-Depth Characterization of Live Vaccines Used in Europe for Oral Rabies Vaccination of Wildlife. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0141537"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"5829","DOI":"10.1016\/j.vaccine.2015.08.091","article-title":"High definition viral vaccine strain identity and stability testing using full-genome population data\u2013The next generation of vaccine quality control","volume":"33","author":"Freuling","year":"2015","journal-title":"Vaccine"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1257","DOI":"10.1016\/S0264-410X(96)00012-6","article-title":"Human adenovirus type 5 vectors expressing rabies glycoprotein","volume":"14","author":"Yarosh","year":"1996","journal-title":"Vaccine"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1186\/s13567-014-0077-8","article-title":"Twenty year experience of the oral rabies vaccine SAG2 in wildlife: A global review","volume":"45","author":"Cliquet","year":"2014","journal-title":"Vet. Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1586\/14760584.2.3.399","article-title":"New approaches to the prevention and eradication of rabies","volume":"2","author":"Dietzschold","year":"2003","journal-title":"Expert. Rev. Vaccines"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1111\/j.1863-2378.2008.01215.x","article-title":"Immunogenicity and safety of recombinant rabies viruses used for oral vaccination of stray dogs and wildlife","volume":"56","author":"Faber","year":"2009","journal-title":"Zoonoses Public Health"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3435","DOI":"10.1016\/j.vaccine.2016.05.013","article-title":"Emergency deployment of genetically engineered veterinary vaccines in Europe","volume":"34","author":"Ramezanpour","year":"2016","journal-title":"Vaccine"},{"key":"ref_25","unstructured":"(2021, August 08). European Medicines Agency Rabitec\u2014Rabies Vaccine (Live, Oral) for Foxes and Raccoon Dogs. Available online: https:\/\/www.ema.europa.eu\/en\/medicines\/veterinary\/EPAR\/rabitec#overview-section."},{"key":"ref_26","unstructured":"USDA (2018). Veterinary Memorandum No. 800.201, USDA\/APHIS\/VS."},{"key":"ref_27","unstructured":"Council of Europe (2013). \u00a75.2.6\u2013Evaluation of safety of veterinary vaccine and immunosera. European Pharmacopoeia 7.7, EDQM."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3938","DOI":"10.1016\/j.vaccine.2017.06.022","article-title":"Oral vaccination of wildlife against rabies: Differences among host species in vaccine uptake efficiency","volume":"35","author":"Vos","year":"2017","journal-title":"Vaccine"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"811","DOI":"10.1016\/j.vaccine.2017.12.076","article-title":"An assessment of shedding with the oral rabies virus vaccine strain SPBN GASGAS in target and non-target species","volume":"36","author":"Vos","year":"2018","journal-title":"Vaccine"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Ortmann, S., Vos, A., Kretzschmar, A., Walther, N., Kaiser, C., Freuling, C., Lojkic, I., and M\u00fcller, T. (2018). Safety studies with the oral rabies virus vaccine SPBN GASGAS in the small Indian mongoose (Herpestes auropunctatus). BMC Vet. Res., 14.","DOI":"10.1186\/s12917-018-1417-0"},{"key":"ref_31","unstructured":"Council of Europe (2014). Rabies vaccine (live, oral) for foxes and raccoon dogs-0746. European Pharmacopoeia 8.0, EDQM."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.virusres.2016.09.016","article-title":"Recent Advances in Inferring Viral Diversity from High-throughput Sequencing Data","volume":"239","author":"Seifert","year":"2017","journal-title":"Virus Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"20063","DOI":"10.1073\/pnas.1012537107","article-title":"Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines","volume":"107","author":"Neverov","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1016\/0042-6822(89)90122-0","article-title":"Arginine or lysine in position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice","volume":"172","author":"Tuffereau","year":"1989","journal-title":"Virology"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1073\/pnas.80.1.70","article-title":"Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus","volume":"80","author":"Dietzschold","year":"1983","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"14141","DOI":"10.1128\/JVI.79.22.14141-14148.2005","article-title":"A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity","volume":"79","author":"Faber","year":"2005","journal-title":"J. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"7041","DOI":"10.1128\/JVI.00357-07","article-title":"Dominance of a nonpathogenic glycoprotein gene over a pathogenic glycoprotein gene in rabies virus","volume":"81","author":"Faber","year":"2007","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3374","DOI":"10.1128\/JVI.76.7.3374-3381.2002","article-title":"Overexpression of the Rabies Virus Glycoprotein Results in Enhancement of Apoptosis and Antiviral Immune Response","volume":"76","author":"Faber","year":"2002","journal-title":"JVI"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1754","DOI":"10.1093\/bioinformatics\/btp324","article-title":"Fast and accurate short read alignment with Burrows-Wheeler transform","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2987","DOI":"10.1093\/bioinformatics\/btr509","article-title":"A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data","volume":"27","author":"Li","year":"2011","journal-title":"Bioinformatics"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e102","DOI":"10.1093\/nar\/gkab576","article-title":"Accurate assembly of minority viral haplotypes from next-generation sequencing through efficient noise reduction","volume":"49","author":"Knyazev","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1007\/978-1-62703-646-7_6","article-title":"Clustal Omega, accurate alignment of very large numbers of sequences","volume":"1079","author":"Sievers","year":"2014","journal-title":"Methods Mol. Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"4350","DOI":"10.1093\/bioinformatics\/btaa213","article-title":"DNA Features Viewer: A sequence annotation formatting and plotting library for Python","volume":"36","author":"Zulkower","year":"2020","journal-title":"Bioinformatics"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"11555","DOI":"10.1128\/JVI.79.18.11555-11558.2005","article-title":"Adaptive value of high mutation rates of RNA viruses: Separating causes from consequences","volume":"79","author":"Elena","year":"2005","journal-title":"J. Virol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2072","DOI":"10.1128\/jvi.63.5.2072-2080.1989","article-title":"Extreme heterogeneity in populations of vesicular stomatitis virus","volume":"63","author":"Steinhauer","year":"1989","journal-title":"J. Virol."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Eggerbauer, E., Pfaff, F., Finke, S., H\u00f6per, D., Beer, M., Mettenleiter, T.C., Nolden, T., Teifke, J.P., M\u00fcller, T., and Freuling, C.M. (2017). Comparative analysis of European bat lyssavirus 1 pathogenicity in the mouse model. PLoS Negl. Trop. Dis., 11.","DOI":"10.1371\/journal.pntd.0005668"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"vex030","DOI":"10.1093\/ve\/vex030","article-title":"On the effective depth of viral sequence data","volume":"3","author":"Illingworth","year":"2017","journal-title":"Virus Evol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"e00561-17","DOI":"10.1128\/JVI.00561-17","article-title":"The Number of Target Molecules of the Amplification Step Limits Accuracy and Sensitivity in Ultradeep-Sequencing Viral Population Studies","volume":"91","author":"Gallet","year":"2017","journal-title":"J. Virol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1038\/nmeth0307-192","article-title":"Primer-site SNPs mask mutations","volume":"4","author":"Quinlan","year":"2007","journal-title":"Nat. Methods"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"vex038","DOI":"10.1093\/ve\/vex038","article-title":"Genetic analysis of a rabies virus host shift event reveals within-host viral dynamics in a new host","volume":"3","author":"Marston","year":"2017","journal-title":"Virus Evol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"3121","DOI":"10.1007\/s00705-015-2611-z","article-title":"Classical swine fever virus marker vaccine strain CP7_E2alf: Genetic stability in vitro and in vivo","volume":"160","author":"Goller","year":"2015","journal-title":"Arch. Virol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"e00171-17","DOI":"10.1128\/JVI.00171-17","article-title":"Transmission Bottleneck Size Estimation from Pathogen Deep-Sequencing Data, with an Application to Human Influenza A Virus","volume":"91","author":"Weissman","year":"2017","journal-title":"J. Virol."},{"key":"ref_54","first-page":"2919","article-title":"Oral rabies vaccination of wildlife: What drives vaccine uptake efficiency in different reservoir species?","volume":"10","author":"Freuling","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1038\/nature19790","article-title":"The evolution of Ebola virus: Insights from the 2013\u20132016 epidemic","volume":"528","author":"Holmes","year":"2016","journal-title":"Nature"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/10\/2136\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:40:55Z","timestamp":1760143255000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/10\/2136"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,28]]},"references-count":55,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2022,10]]}},"alternative-id":["v14102136"],"URL":"https:\/\/doi.org\/10.3390\/v14102136","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,9,28]]}}}