{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T22:14:46Z","timestamp":1779228886314,"version":"3.51.4"},"reference-count":39,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2024,5,8]],"date-time":"2024-05-08T00:00:00Z","timestamp":1715126400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Clinical Research Priority Program \u201cComprehensive Genomic Pathogen Detection\u201d of the University of Zurich"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Enteroviruses are among the most common viruses pathogenic to humans. They are associated with various forms of disease, ranging from mild respiratory illness to severe neurological diseases. In recent years, an increasing number of isolated cases of children developing meningitis or encephalitis as a result of enterovirus infection have been reported, as well as discrete enterovirus D68 outbreaks in North America in 2014 and 2016. We developed an assay to rapidly genotype enteroviruses by sequencing a region within the VP1 gene using nanopore Flongles. We retrospectively analyzed enterovirus-\/rhinovirus-positive clinical samples from the Zurich, Switzerland area mainly collected during two seasons in 2019\/2020 and 2021\/2022. Respiratory, cerebrospinal fluid, and stool samples were analyzed. Whole-genome sequencing was performed on samples with ambiguous genotyping results and enterovirus D68-positive samples. Out of 255 isolates, a total of 95 different genotypes were found. A difference in the prevalence of enterovirus and rhinovirus infections was observed for both sample type and age group. In particular, children aged 0\u20134 years showed a higher frequency of enterovirus infections. Comparing the respiratory seasons, a higher prevalence was found, especially for enterovirus A and rhinovirus A after the SARS-CoV-2 pandemic. The enterovirus genotyping workflow provides a rapid diagnostic tool for individual analysis and continuous enterovirus surveillance.<\/jats:p>","DOI":"10.3390\/pathogens13050390","type":"journal-article","created":{"date-parts":[[2024,5,8]],"date-time":"2024-05-08T12:27:11Z","timestamp":1715171231000},"page":"390","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Retrospective Genotyping of Enteroviruses Using a Diagnostic Nanopore Sequencing Workflow"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-4222-9966","authenticated-orcid":false,"given":"Vanessa","family":"van Ackeren","sequence":"first","affiliation":[{"name":"Institute of Medical Virology, University of Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1955-7007","authenticated-orcid":false,"given":"Stefan","family":"Schmutz","sequence":"additional","affiliation":[{"name":"Institute of Medical Virology, University of Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-6303-1544","authenticated-orcid":false,"given":"Ian","family":"Pichler","sequence":"additional","affiliation":[{"name":"Institute of Medical Virology, University of Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gabriela","family":"Ziltener","sequence":"additional","affiliation":[{"name":"Institute of Medical Virology, University of Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2777-835X","authenticated-orcid":false,"given":"Maryam","family":"Zaheri","sequence":"additional","affiliation":[{"name":"Institute of Medical Virology, University of Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4233-5952","authenticated-orcid":false,"given":"Verena","family":"Kufner","sequence":"additional","affiliation":[{"name":"Institute of Medical Virology, University of Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0384-0000","authenticated-orcid":false,"given":"Michael","family":"Huber","sequence":"additional","affiliation":[{"name":"Institute of Medical Virology, University of Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,5,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1097\/INF.0000000000000227","article-title":"Three-year study of viral etiology and features of febrile respiratory tract infections in Japanese pediatric outpatients","volume":"33","author":"Hara","year":"2014","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1016\/j.jpeds.2007.10.043","article-title":"Respiratory viruses in laryngeal croup of young children","volume":"152","author":"Rihkanen","year":"2008","journal-title":"J. Pediatr."},{"key":"ref_3","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_4","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1097\/INF.0000000000001344","article-title":"A 4-year Study on Epidemiologic and Molecular Characteristics of Human Parechoviruses and Enteroviruses Circulating in Children Younger Than 5 Years in Northern Italy","volume":"36","author":"Bubba","year":"2017","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"S19","DOI":"10.1016\/S1386-6532(02)00030-6","article-title":"The impact of an enteroviral RT-PCR assay on the diagnosis of aseptic meningitis and patient management","volume":"25","author":"Stellrecht","year":"2002","journal-title":"J. Clin. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Brouwer, L., Moreni, G., and Wolthers, K.C. (2021). World-Wide Prevalence and Genotype Distribution of Enteroviruses. Viruses, 13.","DOI":"10.3390\/v13030434"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.jviromet.2015.02.011","article-title":"A generic assay for whole-genome amplification and deep sequencing of enterovirus A71","volume":"215\u2013216","author":"Tuyen","year":"2015","journal-title":"J. Virol. Methods"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"17-00310","DOI":"10.2807\/1560-7917.ES.2018.23.3.17-00310","article-title":"The association between acute flaccid myelitis (AFM) and Enterovirus D68 (EV-D68)\u2014What is the evidence for causation?","volume":"23","author":"Dyda","year":"2018","journal-title":"Euro Surveill."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e239","DOI":"10.1016\/S1473-3099(18)30094-X","article-title":"Enterovirus D68 and acute flaccid myelitis-evaluating the evidence for causality","volume":"18","author":"Messacar","year":"2018","journal-title":"Lancet Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"20964","DOI":"10.2807\/1560-7917.ES2014.19.46.20964","article-title":"Presence of human non-polio enterovirus and parechovirus genotypes in an Amsterdam hospital in 2007 to 2011 compared to national and international published surveillance data: A comprehensive review","volume":"19","author":"Janes","year":"2014","journal-title":"Euro Surveill."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"40045","DOI":"10.57187\/smw.2023.40045","article-title":"Acute flaccid myelitis in Switzerland\u2014Association with enterovirus D68","volume":"153","author":"Bigi","year":"2023","journal-title":"Swiss Med. Wkly"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1016\/S1473-3099(19)30566-3","article-title":"Circulation of non-polio enteroviruses in 24 EU and EEA countries between 2015 and 2017: A retrospective surveillance study","volume":"20","author":"Bubba","year":"2020","journal-title":"Lancet Infect Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2200157","DOI":"10.2807\/1560-7917.ES.2022.27.42.2200157","article-title":"Epidemiology of acute flaccid myelitis in children in the Netherlands, 2014 to 2019","volume":"27","author":"Helfferich","year":"2022","journal-title":"Euro Surveill."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1097\/INF.0000000000002188","article-title":"Twenty-nine cases of Enterovirus-D68\u2013associated acute flaccid myelitis in Europe 2016: A case series and epidemiologic overview","volume":"38","author":"Knoester","year":"2019","journal-title":"Pediatr. Infect Dis. J."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e35","DOI":"10.1016\/S1473-3099(20)30852-5","article-title":"The importance of enterovirus surveillance in a post-polio world","volume":"22","author":"Fischer","year":"2022","journal-title":"Lancet Infect. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Graedel, C., Ireddy, N.R., Koch, M.C., Baumann, C., Miani, M.A.T., Barbani, M.T., Steinlin-Schopfer, J., Suter-Riniker, F., and Leib, S.L. (2022). Genome Sequences of Rare Human Enterovirus Genotypes Recovered from Clinical Respiratory Samples in Bern, Switzerland. Microbiol. Resour. Announc., 11.","DOI":"10.1128\/mra.00276-22"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1268","DOI":"10.3201\/eid1908.130496","article-title":"Accuracy of diagnostic methods and surveillance sensitivity for human enterovirus, South Korea, 1999\u20132011","volume":"19","author":"Hyeon","year":"2013","journal-title":"Emerg. Infect Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1707","DOI":"10.1007\/s00705-010-0755-4","article-title":"Molecular characterization of enteroviruses detected in Gyeong-Ju and Po-Hang provinces of Korea in 2003","volume":"155","author":"Jeong","year":"2010","journal-title":"Arch Virol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"475","DOI":"10.3855\/jidc.2413","article-title":"High frequency of enterovirus serotype circulation in a densely populated area of India","volume":"7","author":"Kumar","year":"2013","journal-title":"J. Infect. Dev. Ctries."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Lizasoain, A., Mir, D., Masachessi, G., Farias, A., Rodriguez-Osorio, N., Victoria, M., and Nates, S. (2021). Environmental Surveillance through Next-Generation Sequencing to Unveil the Diversity of Human Enteroviruses beyond the Reported Clinical Cases. Viruses, 13.","DOI":"10.3390\/v13010120"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Majumdar, M., Celma, C., Pegg, E., Polra, K., and Dunning, J. (2021). Detection and Typing of Human Enteroviruses from Clinical Samples by Entire-Capsid Next Generation Sequencing. Viruses, 13.","DOI":"10.3390\/v13040641"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"606","DOI":"10.1128\/JCM.02121-16","article-title":"High-Throughput Next-Generation Sequencing of Polioviruses","volume":"55","author":"Montmayeur","year":"2017","journal-title":"J. Clin. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"231","DOI":"10.5604\/17331331.1204485","article-title":"Enteroviruses Associated with Aseptic Meningitis in Poland, 2011\u20132014","volume":"65","author":"Wieczorek","year":"2016","journal-title":"Pol. J. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1939","DOI":"10.1128\/JCM.00702-09","article-title":"Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues","volume":"47","author":"Kilpatrick","year":"2009","journal-title":"J. Clin. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1128\/JCM.01739-07","article-title":"Real- time reverse transcription-PCR assay for comprehensive detection of human rhinoviruses","volume":"46","author":"Lu","year":"2018","journal-title":"J. Clin. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2698","DOI":"10.1128\/JCM.00542-06","article-title":"Sensitive, semi-nested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens","volume":"44","author":"Nix","year":"2006","journal-title":"J. Clin. Microbiol."},{"key":"ref_27","unstructured":"WHO, Centers for Disease Control and Prevention & World Health Organization, and Regional Office for Europe (2015). Enterovirus Surveillance Guidelines: Guidelines for Enterovirus Surveillance in Support of the Polio Eradication Initiative, WHO."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Lewandowska, D.W., Zagordi, O., Geissberger, F.D., Kufner, V., Schmutz, S., Boni, J., Metzner, K.J., and Trkola, A. (2017). Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples. Microbiome, 5.","DOI":"10.1186\/s40168-017-0317-z"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Kufner, V., Plate, A., Schmutz, S., Braun, D.L., G\u00fcnthard, H.F., Capaul, R., Zbinden, A., Mueller, N.J., Trkola, A., and Huber, M. (2019). Two Years of Viral Metagenomics in a Tertiary Diagnostics Unit: Evaluation of the First 105 Cases. Genes, 10.","DOI":"10.3390\/genes10090661"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.jcv.2011.03.006","article-title":"An automated genotyping tool for enteroviruses and noroviruses","volume":"51","author":"Kroneman","year":"2011","journal-title":"J. Clin. Virol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1098","DOI":"10.1111\/ecog.05625","article-title":"Estimating the number of species shared by incompletely sampled communities","volume":"44","author":"Zou","year":"2021","journal-title":"Ecography"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e4363","DOI":"10.1002\/ecs2.4363","article-title":"Estimating total species richness: Fitting rarefaction by asymptotic approximation","volume":"14","author":"Zou","year":"2023","journal-title":"Ecosphere"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"8264","DOI":"10.1021\/bi201284u","article-title":"The original Michaelis constant: Translation of the 1913 Michaelis-Menten paper","volume":"50","author":"Michaelis","year":"2011","journal-title":"Biochemistry"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1007\/s00705-019-04520-6","article-title":"Recommendations for the nomenclature of enteroviruses and rhinoviruses","volume":"165","author":"Simmonds","year":"2020","journal-title":"Arch. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1815","DOI":"10.1038\/s41591-018-0211-7","article-title":"Expanded skin virome in DOCK-8-deficient patients","volume":"24","author":"Tirosh","year":"2018","journal-title":"Nat. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4055","DOI":"10.1073\/pnas.1701410114","article-title":"Emerging infectious diseases: A proactive approach","volume":"114","author":"Bloom","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e015470","DOI":"10.1136\/bmjopen-2016-015470","article-title":"A systematic review of the worldwide prevalence of survivors of poliomyelitis reported in 31 studies","volume":"7","author":"Jones","year":"2017","journal-title":"BMJ Open"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Kufner, V., Frey, A.C., Burkhard, S.H., Schmutz, S., Ziltener, G., Zaheri, M., Wiedmer, C.V., Plate, A., Trkola, A., and Huber, M. (2024). Exploring viral etiology in upper respiratory tract infections: Insights from mNGS in Swiss outpatients before and during the SARS-CoV-2 pandemic. Swiss Med. Weekly, in press.","DOI":"10.57187\/s.3797"},{"key":"ref_39","first-page":"001001","article-title":"The Swiss Pathogen Surveillance Platform\u2014Towards a nation-wide One Health data exchange platform for bacterial, viral and fungal genomics and associated metadata","volume":"9","author":"Neves","year":"2023","journal-title":"Microb. Genom."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/13\/5\/390\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:41:32Z","timestamp":1760107292000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/13\/5\/390"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,5,8]]},"references-count":39,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2024,5]]}},"alternative-id":["pathogens13050390"],"URL":"https:\/\/doi.org\/10.3390\/pathogens13050390","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,5,8]]}}}