{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T15:29:41Z","timestamp":1788449381607,"version":"build-2803163510"},"reference-count":38,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T00:00:00Z","timestamp":1778284800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"MCDC to Battelle Memorial Institute","award":["MCDC2001-010"],"award-info":[{"award-number":["MCDC2001-010"]}]},{"name":"MCDC to Battelle Memorial Institute","award":["MCDC2001-010"],"award-info":[{"award-number":["MCDC2001-010"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Infection by neurotropic alphaviruses such as the Eastern equine encephalitis virus (EEEV) causes extensive inflammation in the central nervous system and tissue damage, including disruption of the blood\u2013brain barrier (BBB). Neuroinflammation and BBB disruption following infection are critical pathological considerations for the development of robust countermeasure strategies. Encephalitic disease resulting from EEEV infection currently lacks FDA-approved therapeutic intervention strategies, thus exposing a major capability gap in the ability to address the global health burden that could result from alphavirus infections. In this manuscript, we present a gravity-flow Neurovascular Unit (gNVU) model of the human BBB that may be used for modeling EEEV-induced neuropathology and evaluating countermeasures. The data generated using this model show that EEEV infection causes a time-dependent disruption of BBB integrity and increases the inflammatory load in a manner that correlates with an increase in the viral load. The data also show that the route of introduction of the pathogen has an impact on the pathology measured, with infection through the brain side eliciting a greater inflammatory outcome than infection through the vascular route. Overall, the included data support the utility of this organ-on-a-chip (OOC) platform of the human BBB in understanding encephalitic disease caused by neurotropic viruses and evaluation of therapeutic intervention strategies.<\/jats:p>","DOI":"10.3390\/v18050548","type":"journal-article","created":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T16:07:26Z","timestamp":1779206846000},"page":"548","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Application of a Blood\u2013Brain Barrier Organ-on-a-Chip Model for Assessment of Countermeasure Efficiency Against Eastern Equine Encephalitis Virus"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2678-6481","authenticated-orcid":false,"given":"Niloufar A.","family":"Boghdeh-Olson","sequence":"first","affiliation":[{"name":"Biomedical Research Laboratory, Institute for Biohealth Innovation, George Mason University, Manassas, VA 20110, USA"},{"name":"School of Systems Biology, College of Science, George Mason University, Manassas, VA 20110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael D.","family":"Barrera","sequence":"additional","affiliation":[{"name":"Biomedical Research Laboratory, Institute for Biohealth Innovation, George Mason University, Manassas, VA 20110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clayton M.","family":"Britt","sequence":"additional","affiliation":[{"name":"Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN 37212, USA"},{"name":"Department of Physics and Astronomy, Vanderbilt University, Nashville, TN 37235, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David K.","family":"Schaffer","sequence":"additional","affiliation":[{"name":"Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN 37212, USA"},{"name":"Department of Physics and Astronomy, Vanderbilt University, Nashville, TN 37235, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2002-3010","authenticated-orcid":false,"given":"Jacquelyn A.","family":"Brown","sequence":"additional","affiliation":[{"name":"Organ Pathobiology and Therapeutics Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John P.","family":"Wikswo","sequence":"additional","affiliation":[{"name":"Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN 37212, USA"},{"name":"Department of Physics and Astronomy, Vanderbilt University, Nashville, TN 37235, USA"},{"name":"Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37235, USA"},{"name":"Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aarthi","family":"Narayanan","sequence":"additional","affiliation":[{"name":"Department of Biology, College of Science, George Mason University, Fairfax, VA 22030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1093\/jme\/tjab079","article-title":"Eastern Equine Encephalitis Virus Taxonomy, Genomics, and Evolution","volume":"59","author":"Ciota","year":"2022","journal-title":"J. Med. Entomol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1989","DOI":"10.1056\/NEJMp1914328","article-title":"Eastern Equine Encephalitis Virus\u2014Another Emergent Arbovirus in the United States","volume":"381","author":"Morens","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"100167","DOI":"10.1016\/j.imj.2025.100167","article-title":"Eastern Equine Encephalitis Virus: Pathogenesis, Immune Response, and Clinical Manifestations","volume":"4","author":"Parashar","year":"2025","journal-title":"Infect. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"100184","DOI":"10.1016\/j.imj.2025.100184","article-title":"Occupational Zoonoses, Neurological Diseases, and Public Health: A One Health Approach","volume":"4","author":"Stufano","year":"2025","journal-title":"Infect. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1146\/annurev-immunol-101220-014952","article-title":"Distinct Cellular Tropism and Immune Responses to Alphavirus Infection","volume":"40","author":"Kafai","year":"2022","journal-title":"Annu. Rev. Immunol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"790","DOI":"10.1177\/0300985810372508","article-title":"REVIEW PAPER: Pathology of Animal Models of Alphavirus Encephalitis","volume":"47","author":"Steele","year":"2010","journal-title":"Vet. Pathol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Woodson, C.M., Carney, S.K., and Kehn-Hall, K. (2025). Neuropathogenesis of Encephalitic Alphaviruses in Non-Human Primate and Mouse Models of Infection. Pathogens, 14.","DOI":"10.3390\/pathogens14020193"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1038\/s41392-023-01481-w","article-title":"The Blood\u2013Brain Barrier: Structure, Regulation and Drug Delivery","volume":"8","author":"Wu","year":"2023","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1840913","DOI":"10.1080\/21688370.2020.1840913","article-title":"Basic Physiology of the Blood-Brain Barrier in Health and Disease: A Brief Overview","volume":"9","author":"Kaya","year":"2021","journal-title":"Tissue Barriers"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"790","DOI":"10.1111\/apha.12250","article-title":"The Neurovascular Unit\u2014Concept Review","volume":"210","author":"Muoio","year":"2014","journal-title":"Acta Physiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1038\/nbt.4226","article-title":"A Linked Organ-on-Chip Model of the Human Neurovascular Unit Reveals the Metabolic Coupling of Endothelial and Neuronal Cells","volume":"36","author":"Maoz","year":"2018","journal-title":"Nat. Biotechnol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Xu, S., Jin, T., and Weng, J. (2022). Endothelial Cells as a Key Cell Type for Innate Immunity: A Focused Review on RIG-I Signaling Pathway. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.951614"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1186\/s12974-025-03360-3","article-title":"Innate Immune Sensors and Regulators at the Blood Brain Barrier: Focus on Toll-like Receptors and Inflammasomes as Mediators of Neuro-Immune Crosstalk and Inflammation","volume":"22","author":"Acioglu","year":"2025","journal-title":"J. Neuroinflamm."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"26580","DOI":"10.1038\/s41598-024-78204-w","article-title":"Activation of Glial Cells Induces Proinflammatory Properties in Brain Capillary Endothelial Cells In Vitro","volume":"14","author":"Burkhart","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1403","DOI":"10.1080\/21505594.2018.1509668","article-title":"Direct and Indirect Pro-Inflammatory Cytokine Response Resulting from TC-83 Infection of Glial Cells","volume":"9","author":"Keck","year":"2018","journal-title":"Virulence"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"250","DOI":"10.1038\/s41423-020-00620-5","article-title":"The Roles of Microglia in Viral Encephalitis: From Sensome to Therapeutic Targeting","volume":"18","author":"Chhatbar","year":"2021","journal-title":"Cell. Mol. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Boghdeh, N.A., Risner, K.H., Barrera, M.D., Britt, C.M., Schaffer, D.K., Alem, F., Brown, J.A., Wikswo, J.P., and Narayanan, A. (2022). Application of a Human Blood Brain Barrier Organ-on-a-Chip Model to Evaluate Small Molecule Effectiveness against Venezuelan Equine Encephalitis Virus. Viruses, 14.","DOI":"10.3390\/v14122799"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Boghdeh, N.A., McGraw, B., Barrera, M.D., Anderson, C., Baha, H., Risner, K.H., Ogungbe, I.V., Alem, F., and Narayanan, A. (2023). Inhibitors of the Ubiquitin-Mediated Signaling Pathway Exhibit Broad-Spectrum Antiviral Activities Against New World Alphaviruses. Viruses, 15.","DOI":"10.3390\/v15030655"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"10682","DOI":"10.1128\/JVI.05032-11","article-title":"The Role of the Blood-Brain Barrier during Venezuelan Equine Encephalitis Virus Infection","volume":"85","author":"Brooke","year":"2011","journal-title":"J. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"914","DOI":"10.1128\/JVI.74.2.914-922.2000","article-title":"Role of Dendritic Cell Targeting in Venezuelan Equine Encephalitis Virus Pathogenesis","volume":"74","author":"MacDonald","year":"2000","journal-title":"J. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"10634","DOI":"10.1128\/JVI.01323-08","article-title":"Eastern and Venezuelan Equine Encephalitis Viruses Differ in Their Ability To Infect Dendritic Cells and Macrophages: Impact of Altered Cell Tropism on Pathogenesis","volume":"82","author":"Gardner","year":"2008","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Sharma, A., and Knollmann-Ritschel, B. (2019). Current Understanding of the Molecular Basis of Venezuelan Equine Encephalitis Virus Pathogenesis and Vaccine Development. Viruses, 11.","DOI":"10.3390\/v11020164"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1551","DOI":"10.1080\/14787210.2022.2141224","article-title":"Understanding Host Responses to Equine Encephalitis Virus Infection: Implications for Therapeutic Development","volume":"20","author":"Bradfute","year":"2022","journal-title":"Expert Rev. Anti-Infect. Ther."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Phelps, A.L., Salguero, F.J., Hunter, L., Stoll, A.L., Jenner, D.C., O\u2019Brien, L.M., Williamson, E.D., Lever, M.S., and Laws, T.R. (2023). Tumour Necrosis Factor-\u03b1, Chemokines, and Leukocyte Infiltrate Are Biomarkers for Pathology in the Brains of Venezuelan Equine Encephalitis (VEEV)-Infected Mice. Viruses, 15.","DOI":"10.3390\/v15061307"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Liu, Y., Yuan, Y., and Zhang, L. (2022). Innate Immune Evasion by Alphaviruses. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.1005586"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/j.jneuroim.2017.04.008","article-title":"Virus Entry and Replication in the Brain Precedes Blood-Brain Barrier Disruption during Intranasal Alphavirus Infection","volume":"308","author":"Cain","year":"2017","journal-title":"J. Neuroimmunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e02731-19","DOI":"10.1128\/mBio.02731-19","article-title":"Encephalitic Alphaviruses Exploit Caveola-Mediated Transcytosis at the Blood-Brain Barrier for Central Nervous System Entry","volume":"11","author":"Salimi","year":"2020","journal-title":"mBio"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Potokar, M., Zorec, R., and Jorga\u010devski, J. (2023). Astrocytes Are a Key Target for Neurotropic Viral Infection. Cells, 12.","DOI":"10.3390\/cells12182307"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Bengue, M., Ferraris, P., Barthelemy, J., Diagne, C.T., Hamel, R., Li\u00e9geois, F., Nougair\u00e8de, A., de Lamballerie, X., Simonin, Y., and Pompon, J. (2021). Mayaro Virus Infects Human Brain Cells and Induces a Potent Antiviral Response in Human Astrocytes. Viruses, 13.","DOI":"10.3390\/v13030465"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Salimi, H., and Klein, R.S. (2019). Disruption of the Blood-Brain Barrier During Neuroinflammatory and Neuroinfectious Diseases. Neuroimmune Diseases: From Cells to the Living Brain, Springer Nature.","DOI":"10.1007\/978-3-030-19515-1_7"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Hosking, M.P., and Lane, T.E. (2010). The Role of Chemokines during Viral Infection of the CNS. PLoS Pathog., 6.","DOI":"10.1371\/journal.ppat.1000937"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"8562805","DOI":"10.1155\/2016\/8562805","article-title":"Neuroinvasion and Inflammation in Viral Central Nervous System Infections","volume":"2016","author":"Dahm","year":"2016","journal-title":"Mediat. Inflamm."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/S0165-5728(00)00371-4","article-title":"Astrocytes Express Functional Chemokine Receptors","volume":"111","author":"Dorf","year":"2000","journal-title":"J. Neuroimmunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"155252","DOI":"10.1016\/j.cyto.2020.155252","article-title":"Negative Regulation of IL-8 in Human Astrocytes Depends on \u03b2-Catenin While Positive Regulation Is Mediated by TCFs\/LEF\/ATF2 Interaction","volume":"136","author":"Robinson","year":"2020","journal-title":"Cytokine"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"966","DOI":"10.7150\/ijbs.6996","article-title":"Interleukin-8 Regulates Endothelial Permeability by Down-Regulation of Tight Junction but Not Dependent on Integrins Induced Focal Adhesions","volume":"9","author":"Yu","year":"2013","journal-title":"Int. J. Biol. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"280","DOI":"10.1097\/00004647-199703000-00005","article-title":"Interleukin-8 Released into the Cerebrospinal Fluid After Brain Injury Is Associated with Blood\u2013Brain Barrier Dysfunction and Nerve Growth Factor Production","volume":"17","author":"Kossmann","year":"1997","journal-title":"J. Cereb. Blood Flow Metab."},{"key":"ref_37","first-page":"9372182","article-title":"Activators and Inhibitors of NRF2: A Review of Their Potential for Clinical Development","volume":"2019","author":"Manda","year":"2019","journal-title":"Oxid. Med. Cell. Longev."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Herengt, A., Thyrsted, J., and Holm, C.K. (2021). NRF2 in Viral Infection. Antioxidants, 10.","DOI":"10.3390\/antiox10091491"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/5\/548\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T16:11:31Z","timestamp":1779207091000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/5\/548"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,9]]},"references-count":38,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["v18050548"],"URL":"https:\/\/doi.org\/10.3390\/v18050548","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,9]]}}}