{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T08:59:20Z","timestamp":1785401960699,"version":"3.56.0"},"posted":{"date-parts":[[2026,1,25]]},"group-title":"Microbiology","reference-count":22,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,1,25]],"date-time":"2026-01-25T00:00:00Z","timestamp":1769299200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"id":[{"id":"https:\/\/ror.org\/03qevzg44","id-type":"ROR","asserted-by":"publisher"}]},{"award":["K08AI171177"],"award-info":[{"award-number":["K08AI171177"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R21AI193384"],"award-info":[{"award-number":["R21AI193384"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,1,25]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>Enterovirus D68 (EV-D68) is a non-polio enterovirus that can cause a polio-like paralysis condition, acute flaccid myelitis (AFM). EV-D68 associated AFM cases waned in the US after 2018 and the reasons for this are unknown. It has recently been demonstrated that EV-D68 containing point mutations in viral structural proteins VP1 and VP3 resulted in decreased paralysis in different neonatal mouse models. However, phenotypes of these mutations in a human multicellular central nervous system (CNS) model are unknown. We hypothesized that mutations in VP1 and VP3 will similarly direct neurotropism in human spinal cord organoids (hSCO). To investigate this, we recreated viruses with mutations in VP3 (I88V) or VP1 (L1I\/N2D\/T98A\/E283K or L1P\/V148A\/K282R) and infected hSCOs. We found that VP3 I88V and VP1 L1I\/N2D\/T98A\/E283K resulted in decreased titer and viral protein staining, consistent with attenuated neurovirulence in previously published murine models. When these mutations were combined, their effects on neurotropism were not additive. Sequence analysis of recently circulating EV-D68 strains reveals that VP3 I88 and VP1 E283 have remained the dominant amino acid residues since 2014, whereas VP1 sites 1, 2, and 98 have higher population diversity, indicating that these residues may be contributing to newly reduced neurovirulence after 2018.<\/jats:p>","DOI":"10.64898\/2026.01.25.701582","type":"posted-content","created":{"date-parts":[[2026,1,26]],"date-time":"2026-01-26T04:10:12Z","timestamp":1769400612000},"source":"Crossref","is-referenced-by-count":1,"title":["Enterovirus D68 VP1 and VP3 determine neurotropism in human spinal cord organoids"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1548-6854","authenticated-orcid":false,"given":"Jessica E","family":"Packard","sequence":"first","affiliation":[{"name":"Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9970-1063","authenticated-orcid":false,"given":"Jennifer E","family":"Jones","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gal","family":"Yovel","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3389-6731","authenticated-orcid":false,"given":"Megan Culler","family":"Freeman","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA"},{"name":"Institute of Infection, Inflammation, and Immunity in Children (i4Kids), Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026020502113806000_2026.01.25.701582v1.1","doi-asserted-by":"publisher","DOI":"10.1093\/oxfordjournals.aje.a120693"},{"issue":"9","key":"2026020502113806000_2026.01.25.701582v1.2","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1001\/jama.2019.1016","article-title":"Enterovirus D68-Associated Acute Flaccid Myelitis: Rising to the Clinical and Research Challenges","volume":"321","year":"2019","journal-title":"Jama"},{"key":"2026020502113806000_2026.01.25.701582v1.3","doi-asserted-by":"crossref","unstructured":"Yeh, M.T. , et al., Mapping Attenuation Determinants in Enterovirus-D68. Viruses, 2020. 12(8).","DOI":"10.3390\/v12080867"},{"key":"2026020502113806000_2026.01.25.701582v1.4","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.00397-24"},{"key":"2026020502113806000_2026.01.25.701582v1.5","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.01058-23"},{"key":"2026020502113806000_2026.01.25.701582v1.6","doi-asserted-by":"crossref","unstructured":"Jones, J.E. , et al., Recombinant B3 clade enterovirus D68 strains are efficiently rescued in 293T cells and infect human spinal cord organoids. bioRxiv, 2024: p. 2024.12.20.629498.","DOI":"10.1101\/2024.12.20.629498"},{"key":"2026020502113806000_2026.01.25.701582v1.7","doi-asserted-by":"publisher","DOI":"10.1016\/S1386-6532(03)00004-0"},{"key":"2026020502113806000_2026.01.25.701582v1.8","doi-asserted-by":"crossref","unstructured":"Ivan Aksamentov, C.R. , Emma B Hodcroft , Richard A. Neher , Nextclade: clade assignment, mutation calling and quality control for viral genomes. 2021, Zenodo: Journal of Open Source Software.","DOI":"10.21105\/joss.03773"},{"key":"2026020502113806000_2026.01.25.701582v1.9","unstructured":"Neuner-Jehle, N. , Gonz\u00e1lez-S\u00e1nchez, Alejandra , Hodcroft, Emma , and European Non-Polio Enterovirus Network (ENPEN), enterovirus-phylo\/nextclade_d68: Enterovirus D68 Nextclade Dataset v1.0.1. 2025, Zenodo."},{"key":"2026020502113806000_2026.01.25.701582v1.10","unstructured":"Melisa M. Shah, M.A.P., MPH ; Joana Y. Lively, MPH ; Vasanthi Avadhanula, PhD ; Julie A. Boom, MD ; James Chappell, MD, PhD ; Janet A. Englund, MD ; Wende Fregoe ; Natasha B. Halasa, MD ; Christopher J. Harrison, MD ; Robert W. Hickey, MD ; Eileen J. Klein, MD ; Monica M. McNeal, MS ; Marian G. Michaels, MD ; Mary E. Moffatt, MD ; Catherine Otten, MD ; Leila C. Sahni, PhD ; Elizabeth Schlaudecker, MD ; Jennifer E. Schuster, MD ; Rangaraj Selvarangan, PhD ; Mary A. Staat, MD ; Laura S. Stewart, PhD ; Geoffrey A. Weinberg, MD ; John V. Williams, MD ; Terry Fan Fei Ng ; Janell A. Routh, MD ; Susan I. Gerber, MD ; Meredith L. McMorrow, MD ; Brian Rha, MD ; Claire M. Midgley, PhD , Enterovirus D68-Associated Acute Respiratory Illness \u2500 New Vaccine Surveillance Network, United States, July\u2013November 2018\u20132020. 2021: Morbidity and Mortality Weekly Report (MMWR)."},{"key":"2026020502113806000_2026.01.25.701582v1.11","unstructured":"Sonja J. Olsen, P.A.K.W., MPH ; Alicia P. Budd, MPH ; Mila M. Prill, MSPH ; John Steel, PhD ; Claire M. Midgley, PhD ; Krista Kniss, MPH ; Erin Burns ; Thomas Rowe, MS ; Angela Foust ; Gabriela Jasso ; Angiezel Merced-Morales, MPH ; C. Todd Davis, PhD ; Yunho Jang, PhD ; Joyce Jones, MS ; Peter Daly, MPH ; Larisa Gubareva, PhD ; John Barnes, PhD ; Rebecca Kondor, PhD ; Wendy Sessions, MPH ; Catherine Smith, MS ; David E. Wentworth, PhD ; Shikha Garg, MD ; Fiona P. Havers, MD ; Alicia M. Fry, MD ; Aron J. Hall, DVM ; Lynnette Brammer, MPH ; Benjamin J. Silk, PhD , Changes in Influenza and Other Resporatory Virus Activity During the COVID-19 Pandemic - United States, 2020-2021. 2021: Morbidity and Mortality Weekly Report (MMWR)."},{"key":"2026020502113806000_2026.01.25.701582v1.12","unstructured":"AFM Cases and Outbreaks. 2025 December 3, 2025 [cited 2025 December 11]; Available from: https:\/\/www.cdc.gov\/acute-flaccid-myelitis\/cases\/index.html."},{"key":"2026020502113806000_2026.01.25.701582v1.13","unstructured":"Kevin C. Ma, P.A.W., MPH ; Heidi L. Moline, MD ; Heather M. Scobie, PhD ; Claire M. Midgley, PhD ; Hannah L. Kirking, MD ; Jennifer Adjemian, PhD ; Kathleen P. Hartnett, PhD ; Dylan Johns, MS ; Jefferson M. Jones, MD ; Adriana Lopez, MHS ; Xiaoyan Lu, MS ; Ariana Perez, MPH ; Cria G. Perrine, PhD ; Andzelika E. Rzucidlo, MPH ; Meredith L. McMorrow, MD ; Benjamin J. Silk, PhD ; Zachary Stein, MPH ; Everardo Vega, PhD ; New Vaccine Surveillance Network Collaborators; Aron J. Hall, DVM1 , Increase in Acute Respiratory Illnesses Among Children and Adolescents Associated with Rhinoviruses and Enteroviruses, Including Enterovirus D68 \u2014 United States, July\u2013September 2022. Morbidity and Mortality Weekly Report, 2022."},{"key":"2026020502113806000_2026.01.25.701582v1.14","doi-asserted-by":"crossref","unstructured":"Tosolini, A.P. and J.N. Sleigh , Intramuscular Delivery of Gene Therapy for Targeting the Nervous System. Frontiers in Molecular Neuroscience, 2020. 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